Abstract:
Provided in the present invention is a washing-free template-ready PCR detection method for RNA. On the basis of retaining the advantages of the original template-ready PCR method, i.e., there being no need to purify and extract the RNA, no need for a reverse transcription reaction, etc., the method of the present invention designs a probe for the restriction enzymes to thereby integrate the enzyme digestion reaction, so as to eliminate the interference of various pollution sources of double-stranded DNA with no need for a washing step.

Description:
TECHNICAL FIELD 
       [0001]    The present application relates to a washing-free template-ready PCR process for detecting RNA. 
       BACKGROUND 
       [0002]    RT-PCR (Reverse Transcription Polymerase Chain Reaction) is a method that combines Reverse Transcription (RT) with PCR (Polymerase Chain Reaction). By combining cDNA synthesis with RNA as the template, e.g. RT, with PCR amplification, RT-PCR provides a rapid and sensitive method for the analysis of gene expression. RT-PCR detection or quantitative analysis of RNA is more sensitive and much easier to operate than other RNA analysis techniques such as Northern blotting, RNase protection analysis, hybridization in situ, and S 1  nuclease analysis, and it has developed into a powerful means for detecting and analyzing gene expression levels and pathogens. However, in the application of RT-PCR, we have also encountered some problems, such as: high requirement for the purity of RNA sample—it requires not only the removal of non-nucleic acid substance such as protein, but also the removal of DNA in order to avoid interference of DNA; because it involves multiple operating procedures and long period of operation, it needs special care to prevent RNA degradation in the operation; since the two enzymatic reactions (RT and PCR) have different optimal reaction conditions, it is not easy to find an optimal balance point; all these issues lead to complicated procedure, high operating requirements and render it susceptible to various inhibitory substances and the trouble of non-specific amplification and false positive result. Meanwhile, the cost of reverse transcriptase has been always high and it is difficult to lower the implementation cost of this process. To sum up, the method has troublesome to carry out and the cost is high, resulting in difficulty in its clinical popularity. 
         [0003]    Template-Ready PCR (TRPCR) is a RNA PCR detection method recently reported by the applicant (CN102864233A). In this method, an anchored probe is used to specifically bind with the target RNA, and another template probe that binds with target RNA is adsorbed and immobilized in the reaction tube, after repeated washing steps, unbound RNA and other substances are removed and a PCR reaction system is added for PCR amplification of the template probe, and the result can reflect the situation of presence of target RNA. Compared to traditional RT-PCR method, this process does not need purification and extraction of RNA or reverse transcription reaction, on the other hand, it decreases the previously three hours of PCR pre-treatment process to be 50 minutes. So it is simple, rapid and easy to operate, with good detection sensitivity and specificity for RNA, and is suitable for detecting RNA obtained from the lysate of various sources. While in practical application, the inventor has found that multiple washing steps of TRPCR are still complicated, and there is high requirement for temperature control of binding between template probe and target RNA and the procedure is easily influenced by the environmental temperature; if the room temperature is too low, then non-specific binding occurs, resulting in false positive result and bringing unstable and inconsistent factor to the result. Therefore the inventor has redesigned the TRPCR process and made major innovative improvement thereto by introducing a double-stranded DNA-specific digestion reaction. 
       SUMMARY 
       [0004]    A washing-free template-ready PCR process for detecting RNA is provided, which is easy and quick to operate and has good specificity. 
         [0005]    The technical solution of the present application is carried out as follows: 
         [0006]    a washing-free template-ready PCR process for detecting RNA comprising: 
         [0007]    (1) Design and synthesis of a single-stranded oligonucleotide DNA with a length of 25-50 mer complementary to any section of a target RNA sequence, marked as probe A; wherein the probe A has a GC content of 40% to 60%, and a melting temperature (Tm value) of 75 to 85° C.; the probe A being anchored into a PCR tube to give an anchored PCR tube; wherein the probe A can be immobilized with a conventional method in the art, and the solid medium used to bind to the anchored probe A can be a plastic centrifuge tube, or a magnetic bead, a gel particle or any other solid medium that can be connected to a nucleic acid by absorption; the probe A is preferably a biotinylated probe A, i.e. a biotin is added directly onto the 5′ end by modification in the synthesis process; in particular, the preparation method of the anchored PCR tube can be carried out as follows: 20 ul of TBST buffer containing 5 pmol of biotinylated anchor probe A is added into a 0.2 ml thin-walled PCR tube coated with streptavidin, and incubated at room temperature for 1 hr, the liquid in the tube is removed out, and 100 μl of TBST buffer is added, mixed well, the liquid is removed out, this step is repeated for another two times, then the liquid is removed out; 100 μl of TE buffer is added, then the inner liquid is removed out, stocked at −20° C. before sealing. 
         [0008]    (2) Design and synthesis of another single-stranded oligonucleotide DNA with a length of 70 to 100 mer partially complementary to another section of a target RNA sequence containing a restriction endonuclease recognition site, which has the following construction: in the middle is a sequence of 25 to 50 mer completely complementary to a region containing the restriction endonuclease recognition site (Region 2) of the target RNA, at both ends are PCR primer sequences with a length of 20 to 30 mer respectively, this single-stranded oligonucleotide DNA is marked as probe B; wherein the probe B has a GC content of 40% to 60%, a melting temperature of from 75 to 85° C., and it has no overlap with the region of probe A binding to target RNA; the restriction endonuclease has the following characteristics: it does not cut RNA/DNA hybrid duplex (double strand), it remains specific double-stranded DNA cleavage activity at 37° C. in a PCR system, while losses activity at a temperature ≧55° C., it can be, for example, PstI, BamHI, EcoRI, HindIII, AvaI, MboI or the like. 
         [0009]    (3) Design and synthesis of a single-stranded oligonucleotide DNA of 15 to 30 mer, marked as complementary probe C; wherein this probe is completely complementary to a part (Region 3) of the sequence (Region 2) containing the restriction endonuclease recognition site of the above said probe B; its melting temperature is 50 to 60° C.; the double-stranded DNA fragment formed with this probe binding to the template probe B can be cleaved by the above restriction endonuclease; Region 3 has a length smaller than that of Region 2, and it is totally included into Region 2, so that the binging temperature of the complementary probe C to the template probe B is 20° C. to 25° C. lower than that of the template probe B to the target RNA. 
         [0010]    (4) Taking the sample to be tested, the template probe B, complementary probe C and lysis buffer are added, mixed by repeated pipetting, the obtained mixture is transferred to a centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C., the supernatant is taken to give a lysate supernatant; wherein the lysis buffer is a conventional buffer in the art which contains a surfactant (e.g. Tween 20, Triton X-100, NP-40, and the like) for lysing cell or tissue. 
         [0011]    The sample to be tested can be obtained from tissues or cells, or clinical samples such as luggies (sputum), blood and the like. 
         [0012]    When the sample to be tested is obtained from luggies (sputum), the preparation method of lysate supernatant is carried out as follows: prepare 1 to 5 ml sputum+5 to 10 ml sputum lysis buffer, the obtained mixture is vibrated at 37° C. for 10 min, then centrifuged at 4° C., 5000 rpm for 10 min, the supernatant is discarded, 200 ul of lysis buffer (wherein the concentrations of the template probe B and complementary probe C are both 0.1 nmol/L) is added to the precipitate, treated by repeated pipetting and transferred to a 1.5 ml centrifuge tube, it is shaken at room temperature for 10 min, then centrifuged at 4° C., 15000 rpm for 20 min, supernatant is taken to give supernatant of lysate; the composition of the sputum lysis buffer is: PBS+0.1% (w/vol) DTT. 
         [0013]    When the sample to be tested is obtained from cells, the method for obtaining lysate supernatant is carried out as follows: 24-well plate is employed for cell culture, the culture medium is taken out, 200 ul of lysis buffer (where the concentrations of the probe B and the complementary probe C are both 0.1 nmol/L) is added, the obtained mixture is treated by repeated pipetting and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C., 15000 rpm for 20 min, the supernatant is taken to give supernatant of lysate. 
         [0014]    When the sample to be tested is obtained from a tissue, the method for obtaining lysate supernatant is as follows: a tissue of 0.1 cm 3  in size is put into 1.5 ml centrifuge tube, 200 ul of lysis buffer (wherein the concentrations of template probe B and the complementary probe C are both 0.1 nmol/L) is added, the tissue is broken with a tip, then the obtained mixture is shaken at room temperature for 10 min, centrifuged at 4° C., 15000 rpm for 20 min, the supernatant is taken as lysate supernatant. 
         [0015]    (5) 20 uL of the obtained lysate supernatant is added into the anchor PCR tube, incubated at 55 to 65° C. for 10 to 30 min, the liquid in the tube is removed out, 20 uL of RE/PCR reaction solution consisting of PCR buffer, dNTP, Taq enzyme, PCR primers (which are a pair of primers identical to both ends of the probe B, respectively, the solution thereof is 0.2 umol/L), SYBR green I, or TaqMan fluorescent probe, and the above said restriction endonuclease is added to perform PCR amplification, the amplified product is subjected to fluorescent quantitative analysis; the PCR reaction condition is as follows: 37° C. for 5 to 20 min, 92 to 95° C. for 2 to 5 min, then 35 to 40 cycles of 92 to 95° C. for 3 to 30 sec, and 55 to 66° C. for 20 to 60 sec; the buffer is a conventional PCR reaction buffer; the step of taking out (sucking out) liquid can remove such substances that may interfere with the PCR reaction, the integrated cleavage reaction can prevent not only the interference of the template probe B not hybridized to target RNA to the PCR reaction, but also the cross-contamination caused by aerosol diffusion of trace PCR product, so as to ensure the specificity and the success rate of the PCR reaction. 
         [0016]    (6) Taking (blank) lysate as a negative control, it is subjected the treatment and analysis of the above steps (4) and (5), the sample with sample Ct value being smaller than the Ct value of (blank) lysate and the absolute value of the difference between them being equal to or greater than 1 is determined as positive. 
         [0017]    According to the present invention, an anchor probe A is employed to bind to and immobilize the target RNA, another template probe B is used to bind to the target RNA, meanwhile an auxiliary complementary probe C is used to complementarily bind to the remaining template probe B that is not bound to the target RNA, wherein the double-stranded DNA structure formed by the template probe B binding the complementary probe C contains a restriction endonuclease cleavage site, as the restriction endonuclease has a good activity in the PCR system, it does not cleave RNA/DNA hybrid duplexes, after the hybridization reaction is finished and the hybridization reaction solution is removed out, a PCR system containing the restriction endonuclease is added, and incorporate a step of keeping temperature to maintain enzyme cleavage into this process prior to the PCR reaction procedure, in this process the double stranded DNA formed by the remaining template probe B binding to a complementary probe C rather than a target RNA is cleaved by the enzyme, and only the template probe B bound to a target RNA remains intact, and amplified in the subsequent PCR reaction. The fluorescent dye SYBR green I, or TaqMan Fluorescent probe contained in this system can be used for performing the real-time quantitative PCR reaction, the said enzyme cleavage reaction and the PCR reaction are carrier out continuously in a whole system, which does not require additional operation such as changing fluid. 
         [0018]    Preferably, the lysis buffer in step (4) has the following composition: 4.0 mol/L of guanidine isothiocyanate, 350 mmol/L of NaCl, 1% (v/v) of Triton X-100, 10 mmol/L of 2-mercaptoethanol, 0.1 mg/ml of salmon sperm DNA (commercially available), the solvent is 10 mmol/L of Tris-HCl with pH of 7.5. 
         [0019]    Preferably, the RE/PCR reaction solution of the step (5) has the following composition: 50 mmol/L of KCl, 1.5 mmol/L of MgCl 2 , 0.2 mmol/L of dNTP, 0.05% (v/v) of Tween20, 0.4×SYBR Green I (the meaning of “0.4×” concentration is: the final concentration of each component in the reaction mixture is 0.4 times of the final concentration of that in 1×SYBR Green I solution, while the stock solution of SYBR Green I purchased from Microprobe is 10000×, so the final solution thereof is diluted by volume to be 0.4× when used for PCR process), 0.5 U/20 uL of Taq enzyme, 4 U/20 uL of restriction endonuclease, 0.2 umol/L of PCR universal primer, the solvent is 10 mmol/L of Tris-HCl with pH of 9.0. 
         [0020]    The following is specific description of a target gene, restriction endonuclease for primer and probe sequences in specific detection process: 
         [0021]    The general PCR primer sequences are optimized general sequences as follows: 5′-GACTGACTCCTGGCATCCTCGG-3′ and 5′-CCTTCTCTGGACCTGCGACGAC-3′; 
         [0022]    When the inventive process is used for detecting human DKK1 gene, the anchor probe A has the following sequence: 5′-CCGTTCTTGTAGAACACACACATACGTACACACACACAAACC TC-3′, the template probe B has the following sequence: 5′-GACTGACTCCTGGCATCCTC GGCGCTTC  CTGCAG GCGAGACAGATTTGCACGCCTGCGTCGTCGCAGG TCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-TCGC CTGCAG GAAGCG-3′, wherein the underlined sequence is the recognition site of PstI, and the restriction endonuclease is PstI. 
         [0023]    When the said process is used for detecting human TLR2 gene, the anchor probe A has the following sequence: 5′-GTTGGCCCTCTATATCCATGGGTTCTGCATCC ATGAAGTC-3′, the template probe B has the following sequence: 5′-CCTTCTCTGGACCTGCGACGACTATGAATTT GATATCCACGA G GATCC  TGCAACCAATTCCCGAGGATGCCAGGAGTCAGTC-3′, and the complementary probe C has the following sequence: 5′-GTTGCA GGATCC TCGTGGA-3′, wherein the underlined sequence is the recognition site of BamHI, and the restriction endonuclease is BamHI. 
         [0024]    When the said process is used for detecting human TLR7 gene, the anchor probe A has the following sequence: 5′-CCATCTTGGGGGCACATGCTGAAGAGAGTTA CTGTG-3′, the template probe B has the following sequence: 5′-GACTGACT CC TGGCATCCTCGGTGATTTCCTCT GAA TTC CAGAGTTCTA AGAGACTCACTC TC CATGGTCGTCGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-AACTCTG  GA A TTC AGAGGA AAT-3′, wherein the underlined sequence is the recognition site of EcoRI, and the restriction endonuclease is EcoRI. 
         [0025]    When the said process is used for detecting human XRCC6, the anchor probe A has the following sequence: 5′-CCAGCGACTCCTCTGGGTACACGAACAGGGAG GGCC-3′, the template probe B has the following sequence: 5′-CCTTCT C T G GACCTGCGACGACTGGTCAAGCTCTA  GAATTC GTTITGCACCTGGATT ATCCGAGGATGCCAGGAGTCAGTC-3′, and the complementary probe C has the following sequence: 5′-GCAAAAC GAAT T C  T AGAGCTT-3′, wherein the underlined sequence is the recognition site of EcoRI, and the restriction endonuclease is EcoRI. 
         [0026]    When the said process is used for detecting human telomerase reverse transcriptase (hTERT), the anchor probe A has the following sequence: 5′-GGTGCGGGCCTGGGTGTGGGCCGCCCCTCC-3′, the template probe B has the following sequence: 5′-GACTGAC TCCTGGCATCCTCGGATGGAGCCCTG CG GGATCC CCTGGCACTGGACGTCGTCGCA GGT CC AGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-GCCAGG GGATCC CGCAG-3′, wherein the underlined sequence is the recognition site of BamHI, and the restriction endonuclease is BamHI. The sequence of TaqMan probe is as follows: 5′-FAM-C CCTGCGGGATCCCCTGGCACT-BHQ1-3′. 
         [0027]    When the said process is used for detecting human GUSB gene, the anchor probe A has the following sequence: 5′-CACGACCGCGGGGTGGTTCTTGTCCCTAC GCACCAC-3′, the template probe B has the following sequence: 5′-GACTGA CTCCTGGCATCCTCGGCTCTTGGTGAC AGCCACAGTGC GGATCC CCACA GG G A GTGTCGTCGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-TGTGG GGAT CC  GCACT GT-3′ wherein the underlined sequence is the recognition site of BamHI, and the, and the restriction endonuclease is BamHI. The sequence of the TaqMan probe is: 5′-FAM-GCCACAGTGCGGATCCCCACAGG-BHQ1-3′(FAM is the fluorophore, and BHQ1 is the quencher). 
         [0028]    When the said process is used for detecting human Bcl-2 gene, the anchor probe A has the following sequence:5′-GCACCTCTCGCCCC AGCTCCCACCCCACGGCCC-3′, the template probe B has the following sequence:5′-GACTGACTCCTGGCATC CTCGGGGCAGCCGGGGT  CTGCAG CGGCGAGGTCCGTCGTCGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-TCGCCG CTGCAG ACCC-3′, wherein the underlined sequence is the recognition site of PstI, and the restriction endonuclease is PstI. 
         [0029]    When the said process is used for detecting human PAR-2gene, the anchor probe A has the following sequence: 5′-GTGCTAGGATTACAGGCATGAGGCACCGCACCCAGC C-3′, the template probe B has the following sequence:5′-GACTGACTCCTGGCATCCTCGGTTCCT TGG ATGGTGCCA CTGCAG GAGAGAG AGGCT GCGTCGTCGCAGGTCC AGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-TCTCTC CTGCAG TG GCACC-3′, wherein the underlined sequence is the recognition site of PstI, and the restriction endonuclease is PstI. 
         [0030]    When the said process is used for detecting human hRPLP0 gene, the anchor probe A has the following sequence:5′-GCAGGAGCAGCTGTGGTGGCAGCAGCCACAGGGGC-3′, the template probe B has the following sequence: 5′-GACTGACTCCTGGCATCCTCGGGTAGCCAAT  CTGCAG ACAGACACTGGCAACATT GCG GT CGTCGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-GTGTCTGT CTGCAG ATTGG CT-3′, wherein the underlined sequence is the recognition site of PstI, and the restriction endonuclease is PstI. 
         [0031]    When the said process is used for detecting human hGAPDH gene, the anchor probe A has the following sequence: 5′-TGCCAGTGAGCTTCCCGTTCAGCTCAGGGATGACC-3′, the template probe B has the following sequence: 5′-GACTGACTCCTGGCATCCTCGGATTTCCATTG ATGAC AAGCTT CCCGTTCTCAGC CTT G A C GTCGTCGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-AACGGG  AAGCTT GTCAT CA-3′, wherein the underlined sequence is the recognition site of HindIII, and the restriction endonuclease is HindIII. 
         [0032]    When the said process is used for detecting human ZO1gene, the anchor probe A has the following sequence:5′-GGCTCTGACCGCTGG TCA GGAGATCGTGACCGGCTGC-3′, the template probe B has the following sequence:5′-GACTGACTCCTGGCA TCCTCGGTTCTGCCTC ATCATTTCCTCGGGATAT GGATCC TTTC TATACACCTTTGTCGT CGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-GAAA GGATCC  ATATCCCGAGG-3′, wherein the underlined sequence is the recognition site of BamHI, and the restriction endonuclease is BamHI. 
         [0033]    When the said process is used for detecting human hTERC gene, the anchor probe A has the following sequence:5′-CCGAGTCCTGGGT G C A CGTCCCACAGCTCAGGG-3′, the template probe B has the following sequence:5′-CCTTCTCTGGACCTGC GAC GACTCCGGAGAAG C CCCGGG CCGACCGCGGCCTCCGAGGATGCCAGG AGTCA GTC-3′, and the complementary probe C has the following sequence: 5′-GTCGG CCCGGG GCTTC-3′, wherein the underlined sequence is the recognition site of AvaI, and the restriction endonuclease is AvaI. 
         [0034]    When the said process is used for detecting mouse beta-actin gene, the anchor probe A has the following sequence:5′-CCTTCCCCGG GG T GGACTCAGGGCATGGACGCG-3′, the template probe B has the following sequence:5′-GACTGACTCCTG GCA TCCTCGGGGAATACAG  CCCGGGG AGCATCGTCGCCCGCGTCGTCGC AGGTCC AGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-ATGCTC CCCGGG CTGTAT-3′, the underlined sequence is the recognition site of AvaI, and the restriction endonuclease is AvaI. 
         [0035]    When the said process is used for detecting mouse Col4a1 gene, the anchor probe A has the following sequence: 5′-CCGTACCCAAGTCCTGCCCGTGGGCACGCTCGTTGC-3′, the template probe B has the following sequence:5′-CCTTCTCTGGACCTGCGACGACCTGGGGGA CCCAT GGATCC TGGCAGCCCATC GGG GCCGAGGATGCCAGGAGTCAGTC-3′, and the complementary probe C has the following sequence: 5′-TGCCA GGATCC  ATGGGTC-3′, wherein the underlined sequence is the recognition site of BamHI, and the restriction endonuclease is BamHI. 
         [0036]    When the said process is used for detecting mouse Wrn gene, the anchor probe A has the following sequence:5′-TITCTCCTGCAGGATGTCCACAGCAGACAGTAGCTG G-3′, the template probe B has the following sequence:5′-GACTGACTCCTGGCATCCTCGGAAGGA GCAATCACTAGCTTC ATAACTGTAA ACAAT  GGATCC AGGGTCGTCGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-TCCCT  GGATCC ATTGTTTACA-3′, wherein the underlined sequence is the recognition site of BamHI, and the restriction endonuclease is BamHI. 
         [0037]    When the said process is used for detecting the ampicillin resistance gene expressed by  E. coli , the anchor probe A has the following sequence:5′-CCAGCCAGCCGGAAGGGCCGAGCGCAG AAGTGG-3′, the template probe B has the following sequence:5′-GACTGACTCCTGGC ATCCTCGGGGCGAAAACTCTCAAG GATC TTACCGCTGTTG A GATC  CAGTCGTCGCAGGTCCAGAGAAGG-3′, and the complementary probe C has the following sequence: 5′-CTG GATC  TC AACAGCGGTAA GAT CCTT-3′, wherein the underlined sequence is the recognition site of MboI, and the restriction endonuclease is MboI. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Comparison of four RNA detection methods 
               
             
          
           
               
                   
                 WFTR- 
                   
                 SORT- 
                   
               
               
                   
                 PCR 
                 TRPCR 
                 PCR 
                 RT-PCR 
               
               
                   
                   
               
             
          
           
               
                 Anchor probe/primer 
                 + 
                 + 
                 + 
                 − 
               
               
                 template probe 
                 + 
                 + 
                 − 
                 − 
               
               
                 complementary probe 
                 + 
                 − 
                 − 
                 − 
               
               
                 Cell lysis 
                 + 
                 + 
                 + 
                 + 
               
               
                 RNA Extraction 
                 − 
                 − 
                 − 
                 + 
               
               
                 Use of phenol chloroform or 
                 − 
                 − 
                 − 
                 + 
               
               
                 affinity column 
               
               
                 DNase treatment and 
                 − 
                 − 
                 − 
                 + 
               
               
                 re-extraction 
               
               
                 Reverse Transcriptase enzyme 
                 − 
                 − 
                 + 
                 + 
               
               
                 and RT reaction 
               
               
                 Integration of dsDNA specific 
                 + 
                 − 
                 + 
                 − 
               
               
                 enzyme digestion 
               
               
                 PCR universal primer 
                 + 
                 + 
                 − 
                 − 
               
               
                 PCR optimization 
                 + 
                 + 
                 + 
                 − 
               
               
                 Application of cDNA clone 
                 − 
                 − 
                 + 
                 + 
               
               
                 Difficulty of Operation 
                 Simple 
                 Simple 
                 Simple 
                 Complex 
               
               
                 Steps from cell lysis to the 
                 6 
                 12 
                 8 
                 15 
               
               
                 result 
               
               
                 Total Time (hr) 
                 ~2 
                 ~2.5 
                 ~2.5 
                 ~5 
               
               
                 Comparison of costs 
                 0.5X 
                 0.4X 
                 0.8X 
                 1X 
               
               
                   
               
             
          
         
       
     
         [0038]    Summary: The present invention is developed based on the inventor&#39;s two other inventions, i.e. TRPCR (Template-Ready PCR) and RDIRD (Removal of DNA contamination by Incorporated Restriction Digestion). The present invention integrates TRPCR anchor probe, template probe, and RT-free system, and creatively introduce RE/PCR system similar to the restriction endonuclease of RDIRD by using complementary probe, so as to remove the template probe that is not bound to target RNA with the enzyme digestion, avoiding the 6 steps of repeated washing in the former TR-PCR. This process preserves the advantageous effects of RNA purification free, RT free, high efficient amplification, and it is more convenient, more fast and stable to operate. Compared with the conventional RT-PCR process, it has fewer steps, less time consuming, and greatly reduces the difficulty and it has lower cost. Due to its advantage of simple operation and low cost, it is also a simple PCR process for detecting RNA compared to the inventors&#39; another invention SORT-PCR (one-Step-lysis and One-tube RT-PCR), however, the limitation of this process is that it cannot apply for cDNA clone. 
         [0039]    The method of the present invention does not need any washing step, therefore the operation is more convenient and fast, it has good sensitivity and specificity for RNA detection, and it is suitable for detecting the samples of various sources. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0040]      FIG. 1  is a flowchart of the inventive process; 
       
    
    
     EXPLANATION TO THE MAIN STEPS 
       [0041]    S 0 : The immobilization of the anchor probe A; 
         [0042]    S 1 : Lysing cells to release RNA, wherein the lysate contains the template probe B and complementary probe C, and the probe binding region contains a restriction site (RS); 
         [0043]    S 2 : The anchor probe A binds a target RNA, meanwhile the template probe B bound to the target RNA is also adsorbed; other substances are removed out, and the excess template probe B and complementary probe C bind with each other to form dsDNA; 
         [0044]    S 3 : Digestion reaction, trace amount of dsDNA formed by template probe B/complementary C is cleaved, while the template probe B bound to the target RNA remains intact; 
         [0045]    S 4 : PCR is carried out, wherein the template probe B is amplified. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0046]    The present application will be described with following specific example, however, the protection scope of the present application is not intending to be limited thereby. 
       Example 1 
     Detection of the Expression of Human DKK1 Gene by WFTR-PCR 
       [0047]    For probe and primer sequences, please see the section of Summary. 
         [0048]    Preparation of anchor PCR tube: 50 ul of TBST buffer, anchor probe A containing 5 pmol of biotinylation (directly modified at the 5′end in the synthesis procedure, commercially available) were placed in 0.2 ml thin walled PCR tube coated with streptavidin (PCR tube coated with streptavidin was purchased from Roche), kept at room temperature for 1 hr, the liquid inside the tube was removed out, 100 ul of TBST buffer was added, evenly mixed, dried and washed, this step was performed for three times, and was dried at last; then 100 ul of TE buffer was added, the liquid inside the tube was removed and the tube was sealed and stocked at −20′C for future use. 
         [0049]    RE/PCR reaction solution composition: 50 mmol/L of KCl, 1.5 mmol/L of MgCl 2 , 0.2 mmol/L of dNTP, 0.05% (v/v) of Tween20, 0.4×SYBR Green I, 0.5 U/20 uL of Taq enzyme, 4 U/20 uL of restriction endonuclease, 0.2 umol/L of PCR universal primer, the solvent was 10 mmol/L of Tris-HCl with pH of 9.0. 
         [0050]    Human skin cancer cell strain A431 cells were cultured in a 24-well plate, the density was 1000 cells/well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C added in the lysis buffer were 0.1 nmol/L), pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, supernatant was taken to obtain the cell lysate supernatant. 
         [0051]    The lysis buffer had the following composition: 4.0 mol/L of guanidine isothiocyanate, 350 mmol/L of NaCl, 1% Triton X-100, 10 mmol/L of 2-mercaptoethanol, 0.1 mg/ml of salmon sperm DNA (Sigma), the solvent was 10 mmol/L of Tris-HCl of pH 7.5. 
         [0052]    20 ul of above said cell lysate supernatant was taken and added into an anchored PCR tube, insulated at 60° C. for 30 min, the liquid in the tube was removed out, 20 ul of RE/PCR reaction solution (20 ul of PCR buffer +0.4×SYBR green I, 0.2 umol/L of primer pair+0.2 mmol/L of dNTP +0.5 U of Taq enzyme+4 U of PstI) was added, and the following procedure was carried out in the PCR amplifier (37′C for 10 min, initial denaturation at 95° C. for 3 min, then 40 cycles of: 95° C. for 5 s, 63° C. for 30 s, two-step method), and SYBR green I fluorescence was used for quantitative analysis. Negative control 1 was the lysate containing 0.1 nmol/L of template probe B and 0.1 nmol/L of complementary probe C, and negative control 2 was the lysate supernatant of  E. coli  DH5a (1 ml of culture medium with OD600=1.0, after centrifugation the lysate containing 0.1 nmol/L of template probe B and 0.1 nmol/L of complementary probe C was added, and supernatant was taken after lysis and centrifugation), and the result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sample Group 
                 Ct-cell 
                 Ct- control 1 
                 Ct-control 2 
               
               
                   
                   
               
             
             
               
                   
                 1 
                 27.32 
                 31.36 
                 31.26 
               
               
                   
                 2 
                 27.45 
                 31.20 
                 30.89 
               
               
                   
                 3 
                 27.50 
                 31.42 
                 30.93 
               
               
                   
                 4 
                 27.67 
                 31.55 
                 31.21 
               
               
                   
                 5 
                 27.55 
                 31.39 
                 30.91 
               
               
                   
                 6 
                 27.12 
                 31.68 
                 30.89 
               
               
                   
                   
               
             
          
         
       
     
         [0053]    Ct value of the cell sample was in the range of from 27.67 to 27.12, namely 27.44+/−0.23; Ct value of negative control 1, i.e. lysate, was in the range of from 31.68 to 31.20, namely 31.43+/−0.25; Ct value of negative control 2, i.e.  E. coli  lysate supernatant, was in the range of from 31.26 to 30.89, namely 31.02+/−0.17. Ct of the cell sample was significantly smaller than that of negative control 1 or negative control 2 (significantly was defined as the absolute value of difference was ≧1), indicating that WFTR-PCR can detect DKK1 mRNA of cell sample; Ct value of negative control 2 was slightly smaller than that of negative control 1, but the absolute value of difference was smaller than 1, which was within the negative fluctuation range. 
       Example 2 
     Detection of the Expression of Human TLR2 Gene by WFTR-PCR 
       [0054]    For probe and primer sequences, please see the section of Summary, and the preparation of anchor PCR tube, the composition of RE/PCR reaction solution were the same as Example 1. 
         [0055]    The preparation of the cell lysate supernatant of the cell samples, negative control 1 and negative control 2 were the same as Example 1, and the PstI in RE/PCR reaction solution of Example 1 was replaced with BamHI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sample Group 
                 Ct-cell 
                 Ct-control 1 
                 Ct-control 2 
               
               
                   
                   
               
             
             
               
                   
                 1 
                 26.53 
                 31.29 
                 31.32 
               
               
                   
                 2 
                 26.37 
                 31.58 
                 31.21 
               
               
                   
                 3 
                 26.46 
                 31.22 
                 30.88 
               
               
                   
                 4 
                 26.42 
                 31.49 
                 30.99 
               
               
                   
                 5 
                 26.59 
                 31.63 
                 30.92 
               
               
                   
                 6 
                 26.64 
                 31.71 
                 31.39 
               
               
                   
                   
               
             
          
         
       
     
         [0056]    Ct value of the cell sample was 26.50+/−0.10; Ct value of negative control 1, i.e. lysate, was 31.49+/−0.19; Ct value of negative control 2, i.e.  E. coli  lysate supernatant, was 31.12+/−0.22. Ct of the cell sample was significantly smaller than that of negative control 1 or negative control 2 (significantly was defined as the absolute value of difference was ≧1), indicating that WFTR-PCR can detect TLR2 mRNA of cell sample; Ct value of negative control 2 was slightly smaller than that of negative control 1, but the absolute value of difference was smaller than 1, which was within the negative fluctuation range. 
       Example 3 
     Detection of the Expression of Human TLR7 Gene by WFTR-PCR 
       [0057]    For probe and primer sequences, please see the section of Summary, and the preparation of anchor PCR tube, the composition of RE/PCR reaction solution were the same as Example 1. 
         [0058]    The preparation of the cell lysate supernatant of the cell samples, negative control 1 and negative control 2 were the same as Example 1, and the PstI in RE/PCR reaction solution of Example 1 was replaced with EcoRI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sample Group 
                 Ct-cell 
                 Ct-control 1 
                 Ct-control 2 
               
               
                   
                   
               
             
             
               
                   
                 1 
                 28.33 
                 31.44 
                 31.22 
               
               
                   
                 2 
                 28.41 
                 31.31 
                 31.47 
               
               
                   
                 3 
                 28.56 
                 31.67 
                 31.15 
               
               
                   
                 4 
                 28.22 
                 31.58 
                 31.09 
               
               
                   
                 5 
                 28.49 
                 31.62 
                 30.97 
               
               
                   
                 6 
                 28.71 
                 31.52 
                 31.11 
               
               
                   
                   
               
             
          
         
       
     
         [0059]    Ct value of the cell sample was 28.45+/−0.17; Ct value of negative control 1, i.e. lysate, was 31.52+/−0.13; Ct value of negative control 2, i.e.  E. coli  lysate supernatant, was 31.17+/−0.17. Ct of the cell sample was significantly smaller than that of negative control 1 or negative control 2 (significantly was defined as the absolute value of difference was ≧1), indicating that WFTR-PCR can detect TLR7 mRNA of cell sample; Ct value of negative control 2 was slightly smaller than that of negative control 1, but the absolute value of difference was smaller than 1, which was within the negative fluctuation range. 
       Example 4 
     Detection of the Expression of hTERT mRNA in Human Lung Cancer Cell Strain A549 by WFTR-PCR 
       [0060]    For probe and primer sequences, please see the section of Summary, and the preparation of anchor PCR tube, the composition of RE/PCR reaction solution were the same as Example 1. 
         [0061]    BamHI was used in RE. Human lung cancer cell strain A549 was incubated in a 24-well plate, the number was from 1 to 10 6  cells/well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C contained in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, and the PstI in RE/PCR reaction solution of Example 1 was replaced with BamHI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Determination 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 of the result 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 cell 
                 31.28 
                 30.76 
                 31.02 
                 negative 
               
               
                 10 
                 cells 
                 28.71 
                 28.53 
                 28.62 
                 positive 
               
               
                 100 
                 cells 
                 24.53 
                 24.67 
                 24.60 
                 positive 
               
               
                 1000 
                 cells 
                 21.22 
                 21.58 
                 21.40 
                 positive 
               
               
                 10 5   
                 cells 
                 17.48 
                 17.62 
                 17.55 
                 positive 
               
               
                 10 6   
                 cells 
                 13.51 
                 13.33 
                 13.42 
                 positive 
               
             
          
           
               
                 Negative control 1 
                 31.63 
                 31.33 
                 31.48 
                 negative 
               
               
                 Negative control 2 
                 31.15 
                 31.27 
                 31.21 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0062]    It can be seen that using the WFTR-PCR process of the present Example, the expression of hTERT in the 10 to 106 lung cancer A549 cells can be detected, and the detection result is related with the number of cells, wherein the more the cells are, the smaller the Ct value is. 
       Example 5 
     WFTR-PCR being Used for Detecting hTERT mRNA in Sputum Specimen of Lung Cancer Patient and of Normal Person as Control 
       [0063]    With respect to probe and primer sequences, please refer to the section of Summary. The preparation of anchor PCR tube, and RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. 
         [0064]    There were 20 pathologically determined lung cancer patients and 15 normal volunteers as control, wherein the normal volunteers had sputum when smoking, had no symptom and their chest X-ray showed negative. The volume of sputum was 1 to 5 ml per person. 10 ml of sputum solution (PBS+0.1% DTT) was added, and vibrated for 10 minutes at the temperature of 37° C., then centrifuged at the speed of 5000 rpm at 4° C. for 10 minutes, the supernatant was removed, 200 ul of lysate was added to the precipitate (the same as Example 1) and pipetted repeatedly, the mixture was transferred into a 1.5 ml centrifugal tube, vibrated for 10 minutes at room temperature, and then it was centrifuged at the speed of 15000 rpm at the temperature of 4° C. for 20 minutes, the supernatant was taken to give lysate supernatant was obtained. The following operation and procedure were the same as Example 1. The lysate supernatant of 1000 A549 cells was used as positive control, and a blank lysate containing template probe B and complementary probe C was used as negative control. The determination standard of negative result was: Ct value of the sample was significantly smaller than that of lysate of negative control (significantly was defined as the absolute value of difference was ≧1), and the other results were all determined to be negative. The result showed that among the 20 lung cancer patients, 17 patients were determined to be positive and 3 patients were determined to be negative, the detection rate was 85%. The test result of 15 normal persons as control were all negative. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                 Pathological 
                 Ct 
                 WFTR result 
               
               
                 No. 
                 Age 
                 Gender 
                 Diagnosis 
                 value 
                 determination 
               
               
                   
               
             
             
               
                 LC001 
                 60 
                 M 
                 lung cancer 
                 30.83 
                 negative 
               
               
                 LC002 
                 59 
                 M 
                 lung cancer 
                 23.72 
                 positive 
               
               
                 LC003 
                 61 
                 F 
                 lung cancer 
                 24.78 
                 positive 
               
               
                 LC004 
                 60 
                 M 
                 lung cancer 
                 23.89 
                 positive 
               
               
                 LC005 
                 76 
                 M 
                 lung cancer 
                 26.54 
                 positive 
               
               
                 LC006 
                 59 
                 M 
                 lung cancer 
                 25.59 
                 positive 
               
               
                 LC007 
                 60 
                 M 
                 lung cancer 
                 31.42 
                 negative 
               
               
                 LC008 
                 58 
                 F 
                 lung cancer 
                 26.10 
                 positive 
               
               
                 LC009 
                 67 
                 M 
                 lung cancer 
                 32.02 
                 negative 
               
               
                 LC010 
                 57 
                 M 
                 lung cancer 
                 25.55 
                 positive 
               
               
                 LC011 
                 74 
                 M 
                 lung cancer 
                 27.64 
                 positive 
               
               
                 LC012 
                 47 
                 F 
                 lung cancer 
                 31.16 
                 negative 
               
               
                 LC013 
                 60 
                 F 
                 lung cancer 
                 26.00 
                 positive 
               
               
                 LC016 
                 49 
                 M 
                 lung cancer 
                 22.72 
                 positive 
               
               
                 LC017 
                 65 
                 M 
                 lung cancer 
                 19.18 
                 positive 
               
               
                 LC018 
                 53 
                 M 
                 lung cancer 
                 15.41 
                 positive 
               
               
                 LC019 
                 64 
                 F 
                 lung cancer 
                 27.36 
                 positive 
               
               
                 LC020 
                 60 
                 F 
                 lung cancer 
                 21.21 
                 positive 
               
               
                 NC001 
                 55 
                 M 
                 normal 
                 31.83 
                 negative 
               
               
                 NC002 
                 30 
                 M 
                 normal 
                 30.72 
                 negative 
               
               
                 NC003 
                 38 
                 F 
                 normal 
                 31.78 
                 negative 
               
               
                 NC004 
                 42 
                 M 
                 normal 
                 30.89 
                 negative 
               
               
                 NC005 
                 46 
                 F 
                 normal 
                 31.54 
                 negative 
               
               
                 NC006 
                 69 
                 M 
                 normal 
                 31.59 
                 negative 
               
               
                 NC007 
                 66 
                 M 
                 normal 
                 30.98 
                 negative 
               
               
                 NC008 
                 61 
                 M 
                 normal 
                 31.10 
                 negative 
               
               
                 NC009 
                 72 
                 M 
                 normal 
                 32.07 
                 negative 
               
               
                 NC010 
                 33 
                 F 
                 normal 
                 31.55 
                 negative 
               
               
                 NC011 
                 41 
                 F 
                 normal 
                 30.64 
                 negative 
               
               
                 NC012 
                 29 
                 M 
                 normal 
                 32.16 
                 negative 
               
               
                 NC013 
                 35 
                 M 
                 normal 
                 32.00 
                 negative 
               
               
                 NC014 
                 36 
                 M 
                 normal 
                 31.49 
                 negative 
               
               
                 NC015 
                 39 
                 M 
                 normal 
                 31.67 
                 negative 
               
               
                 − 
                   
                   
                 Negative 
                 31.54 
                 negative 
               
               
                   
                   
                   
                 control 
               
               
                 + 
                   
                   
                 Positive 
                 26.67 
                 positive 
               
               
                   
                   
                   
                 control 
               
               
                   
               
             
          
         
       
     
       Example 6 
     Detection of hTERT mRNA in Human Lung Cancer Tissue 
       [0065]    For the sequences of probe and primer, please refer to the section of Summary, and the preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. For the above 20 lung cancer patients with confirmed diagnosis, after being cut down in a surgery, the lung cancer tissues were immediately stored in liquid nitrogen. The cancer tissue was subjected to pathological determination. A tissue of about 0.1 cm 3  in size was placed into 1.5 ml centrifuge tube, 200 ul of lysis buffer (same as Example 5) was added, the tissue piece was crushed with a tip, then the mixture was shaken at room temperature for 10 min, then centrifuged at 4° C., and 15000 rpm for 20 min, the supernatant was taken to give a lysate supernatant. The following operation was the same as Example 5. The results showed that 18 cases of lung cancer had Ct values (between 18.11 and 23.23) significantly smaller than the Ct values of the lysate of the control (&gt;30), they were determined as positive for hTERT mRNA, and the detection rate was 90%. 
       Example 7 
     Detection the Expression of Human XRCC6 Gene by WFTR-PCR 
       [0066]    For the sequences of probe and primer, please refer to the section of Summary, and the preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. The preparation of lysate supernatant of the cell sample, negative control 1 and negative control 2 were the same as Example 1, and the PstI in RE/PCR reaction solution of Example 1 was replaced with EcoRI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sample Group 
                 Ct-cell 
                 Ct-control 1 
                 Ct-control 2 
               
               
                   
                   
               
             
             
               
                   
                 1 
                 28.47 
                 31.55 
                 31.36 
               
               
                   
                 2 
                 28.31 
                 31.63 
                 31.52 
               
               
                   
                 3 
                 28.60 
                 31.42 
                 31.35 
               
               
                   
                 4 
                 28.72 
                 31.39 
                 31.14 
               
               
                   
                 5 
                 28.57 
                 31.71 
                 31.27 
               
               
                   
                 6 
                 28.81 
                 31.66 
                 31.44 
               
               
                   
                   
               
             
          
         
       
     
         [0067]    Ct value of the cell sample was 28.58+/−0.18; Ct value of negative control 1, i.e. lysate, was 31.56+/−0.13; Ct value of negative control 2, i.e.  E. coli  lysate supernatant, was 31.35+/−0.13. Ct of the cell sample was significantly smaller than that of negative control 1 or negative control 2 (significantly was defined as the absolute value of difference was ≧1), indicating that WFTR-PCR can detect XRCC6 mRNA of the cell sample; Ct value of negative control 2 was slightly smaller than that of negative control 1, but the absolute value of difference was smaller than 1, which was within the negative fluctuation range. 
       Example 8 
     Detection of hTERT mRNA in Human Lung Cancer Cell Strain H1299 
       [0068]    For the sequences of probe and primer, please refer to the section of Summary, and the preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. Human lung cancer cell strain H1299 was incubated in a 24-well plate, the number was from 1 to 10 6  cells/well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, and the PstI in RE/PCR reaction solution of Example 1 was replaced with BamHI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 cell 
                 30.72 
                 30.58 
                 30.65 
                 negative 
               
               
                 10 
                 cells 
                 27.51 
                 27.63 
                 27.57 
                 positive 
               
               
                 100 
                 cells 
                 23.48 
                 23.36 
                 23.42 
                 positive 
               
               
                 1000 
                 cells 
                 20.34 
                 20.46 
                 20.40 
                 positive 
               
               
                 10 5   
                 cells 
                 16.79 
                 16.71 
                 16.75 
                 positive 
               
               
                 10 6   
                 cells 
                 12.51 
                 12.83 
                 12.67 
                 positive 
               
             
          
           
               
                 Negative Control 1 
                 31.49 
                 31.65 
                 31.57 
                 negative 
               
               
                 Negative Control 2 
                 31.35 
                 31.21 
                 31.28 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0069]    It can be seen that using the WFTR-PCR process of the present Example, the expression of hTERT mRNA in the 10 to 10 6  lung cancer A549 cells can be detected, and the detection result is related with the number of cells, wherein the more the cells are, the smaller the Ct value is. 
       Example 9 
     Detection of hTERT mRNA in Human Skin Cancer Cell Strain A431 
       [0070]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the number was from 1 to 10 6  cells/well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, and the PstI in RE/PCR reaction solution of Example 1 was replaced with BamHI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 cell 
                 31.28 
                 31.44 
                 31.36 
                 negative 
               
               
                 10 
                 cells 
                 29.29 
                 29.43 
                 29.36 
                 positive 
               
               
                 100 
                 cells 
                 25.68 
                 25.44 
                 25.56 
                 positive 
               
               
                 1000 
                 cells 
                 22.23 
                 22.38 
                 22.31 
                 positive 
               
               
                 10 5   
                 cells 
                 18.41 
                 18.34 
                 18.38 
                 positive 
               
               
                 10 6   
                 cells 
                 14.79 
                 14.93 
                 14.86 
                 positive 
               
             
          
           
               
                 Negative Control 1 
                 31.67 
                 31.55 
                 31.61 
                 negative 
               
               
                 Negative Control 2 
                 31.37 
                 31.45 
                 31.41 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0071]    It can be seen that using the WFTR-PCR process of the present Example, the expression of hTERT mRNA in the 10 to 10 6  skin cancer A431 cells can be detected, and the detection result is related with the number of cells, wherein the more the cells are, the smaller the Ct value is. 
       Example 10 
     SYBR Green I Dye being Replaced by TaqMan Probe, which has No Influence on the Test Effect of WFTR-PCR 
       [0072]    For probe and primer sequences please refer to the section of Summary, and the preparation of anchor PCR tube, and RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. Cell strain A549 cell of human lung cancer was cultured in a 24-well plate, and the number is 1 to 10 6  cells in each well. Culture medium was extracted and removed after overnight culturing. And 200 ul of lysis buffer was added into each well (the concentrations of the template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L), pipetted repeatedly, then the mixture was transferred to a 1.5 ml centrifuge tube, vibrated at room temperature for 10 minutes and centrifuged at 15000 rpm, and at 4° C. for 20 minutes, the supernatant was taken to give cell lysate supernatant. For the composition of lysis buffer please refer to the above description. The following procedure, negative control 1, and negative control were the same as Example 1, and the PstI in RE/PCR reaction solution of Example 1 was replaced with BamHI, and the SYBR green I of Example 1 was replaced with 0.5 umol/L of TaqMan probe. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 cell 
                 33.81 
                 33.77 
                 33.79 
                 negative 
               
               
                 10 
                 cells 
                 30.22 
                 30.46 
                 30.34 
                 positive 
               
               
                 100 
                 cells 
                 27.65 
                 27.52 
                 27.59 
                 positive 
               
               
                 1000 
                 cells 
                 23.74 
                 23.83 
                 23.79 
                 positive 
               
               
                 10 5   
                 cells 
                 19.56 
                 19.72 
                 19.64 
                 positive 
               
               
                 10 6   
                 cells 
                 16.49 
                 16.58 
                 16.54 
                 positive 
               
             
          
           
               
                 Negative Control 1 
                 34.15 
                 34.37 
                 34.26 
                 negative 
               
               
                 Negative Control 2 
                 33.95 
                 34.07 
                 34.01 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0073]    It can be seen that using the WFTR-PCR process with TaqMan probe, the expression of hTERT mRNA in 10 to 10 6  lung cancer A549 cells can also be detected, and the detection result is related with the number of cells, wherein the more the cells are, the smaller the Ct value is. 
       Example 11 
     Detection of GUSB mRNA in Human Skin Cancer Cell Strain A431 
       [0074]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the number was from 1 to 10 6  cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, the PstI in RE/PCR reaction solution of Example 1 was replaced with BamHI, and the SYBR green I of Example 1 was replaced with TaqMan probe. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 22.47 
                 22.39 
                 22.43 
                 positive 
               
               
                 Negative Control 1 
                 33.55 
                 33.69 
                 33.62 
                 negative 
               
               
                 Negative Control 2 
                 33.49 
                 33.31 
                 33.40 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0075]    It can be seen that using the WFTR-PCR process of this Example, the expression of GUSB mRNA in 1000 skin cancer A549 cells can be detected. 
       Example 12 
     Detection of BCl-2 mRNA in Human Skin Cancer Cell Strain A431 
       [0076]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. PstI was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 19.77 
                 19.58 
                 19.68 
                 positive 
               
               
                 Negative Control 1 
                 31.66 
                 31.43 
                 31.55 
                 negative 
               
               
                 Negative Control 2 
                 31.37 
                 31.22 
                 31.30 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0077]    It can be seen that using the WFTR-PCR process of this Example, the expression of Bcl-2 mRNA can be detected in 1000 skin cancer A431 cells. 
       Example 13 
     Detection of PAR-2 mRNA in Human Skin Cancer Cell Strain A431 
       [0078]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. PstI was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 23.15 
                 23.28 
                 23.22 
                 positive 
               
               
                 Negative Control 1 
                 31.81 
                 31.62 
                 31.72 
                 negative 
               
               
                 Negative Control 2 
                 31.24 
                 31.19 
                 31.22 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0079]    It can be seen that using the WFTR-PCR process of this Example, the expression of PAR-2 mRNA can be detected in 1000 skin cancer A431 cells. 
       Example 14 
     Detection of RPLP0 mRNA in Human Skin Cancer Cell Strain A431 
       [0080]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. PstI was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 17.59 
                 17.82 
                 17.71 
                 positive 
               
               
                 Negative Control 1 
                 31.51 
                 31.66 
                 31.59 
                 negative 
               
               
                 Negative Control 2 
                 31.27 
                 31.38 
                 31.33 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0081]    It can be seen that using the WFTR-PCR process of this Example, the expression of RPLP0 mRNA can be detected in 1000 skin cancer A431 cells. 
       Example 15 
     Detection of GAPDH mRNA in Human Skin Cancer Cell Strain A431 
       [0082]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. HindIII was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, but only replacing PstI with HindIII. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 18.29 
                 18.41 
                 18.35 
                 positive 
               
               
                 Negative Control 1 
                 31.72 
                 31.57 
                 31.65 
                 negative 
               
               
                 Negative Control 2 
                 31.37 
                 31.20 
                 31.29 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0083]    It can be seen that using the WFTR-PCR process of this Example, the expression of GAPDH mRNA can be detected in 1000 skin cancer A431 cells. 
       Example 16 
     Detection of ZO1 mRNA in Human Skin Cancer Cell Strain A431 
       [0084]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the density of the cells were 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, the PstI in RE/PCR reaction solution of Example 1 was replaced with BamHI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 21.37 
                 21.18 
                 21.28 
                 positive 
               
               
                 Negative Control 1 
                 31.43 
                 31.62 
                 31.53 
                 negative 
               
               
                 Negative Control 2 
                 31.29 
                 31.13 
                 31.21 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0085]    It can be seen that using the WFTR-PCR process of this Example, the expression of ZO1 mRNA can be detected in 1000 skin cancer A431 cells. 
       Example 17 
     Detection of hTERC mRNA in Human Skin Cancer Cell Strain A431 
       [0086]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. AvaI was used for RE. Human skin cancer cell strain A431 was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, but only replacing PstI with AvaI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 24.72 
                 24.63 
                 24.68 
                 positive 
               
               
                 Negative Control 1 
                 31.69 
                 31.53 
                 31.61 
                 negative 
               
               
                 Negative Control 2 
                 31.22 
                 31.34 
                 31.28 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0087]    It can be seen that using the WFTR-PCR process of this Example, the expression of hTERC mRNA can be detected in 1000 skin cancer A431 cells. 
       Example 18 
     Detection of b-Actin mRNA in NIH3T3 Cell of Mouse Fibroblast Tumor 
       [0088]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. AvaI was used for RE. Mouse fibroblast tumor NIH3T3 cell was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, but only replacing PstI with AvaI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 18.89 
                 18.75 
                 18.82 
                 positive 
               
               
                 Negative Control 1 
                 31.57 
                 31.74 
                 31.67 
                 negative 
               
               
                 Negative Control 2 
                 31.41 
                 31.27 
                 31.34 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0089]    It can be seen that using the WFTR-PCR process of this Example, the expression of b-actin mRNA can be detected in 1000 mouse fibroblast tumor NIH3T3 cells. 
       Example 19 
     Detection of Col4a1 mRNA in NIH3T3 Cell of Mouse Fibroblast Tumor 
       [0090]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. Mouse fibroblast tumor NIH3T3 cell was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, but only replacing PstI with BamHI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 28.19 
                 28.88 
                 28.04 
                 positive 
               
               
                 Negative Control 1 
                 31.77 
                 31.62 
                 31.70 
                 negative 
               
               
                 Negative Control 2 
                 31.23 
                 31.40 
                 31.32 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0091]    It can be seen that using the WFTR-PCR process of this Example, the expression of Col4a1 mRNA can be detected in 1000 mouse fibroblast tumor NIH3T3 cells. 
       Example 20 
     Detection of Wrn mRNA in NIH3T3 Cell of Mouse Fibroblast Tumor 
       [0092]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. BamHI was used for RE. Mouse fibroblast tumor NIH3T3 cell was incubated in a 24-well plate, the density was 1000 cells per well. After incubation overnight, the culture medium was removed, and 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) into each well, pipetted repeatedly and transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1, negative control 2 were the same as Example 1, but only replacing PstI with BamHI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 1000 cells 
                 27.76 
                 27.59 
                 27.68 
                 positive 
               
               
                 Negative Control 1 
                 31.47 
                 31.58 
                 31.53 
                 negative 
               
               
                 Negative Control 2 
                 31.44 
                 31.29 
                 31.37 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0093]    It can be seen that using the WFTR-PCR process of this Example, the expression of Wrn mRNA can be detected in 1000 mouse fibroblast tumor NIH3T3 cells. 
       Example 21 
     Detection of Expression of Ampicillin Resistance Gene (Amp r ) in  Escherichia coli    
       [0094]    For probe and primer sequences, please see the section of Summary. The preparation of anchor PCR tube, RE/PCR reaction solution were the same as Example 1. MboI was used for RE. 1 ml of LB culture medium containing 50 ug/ml of ampicillin was used for incubating  Escherichia coli  DH5a transferred with pcDNA3.1 plasmid, the  E. coli  was cultured at the temperature of 37° C. until the OD600 of the medium=1.0, and the bacteria precipitate was collected by centrifugation. 200 ul of lysis buffer was added (concentrations of template probe B and complementary probe C in the lysis buffer were both 0.1 nmol/L) to the bacteria precipitate, pipetted repeatedly then transferred to a 1.5 ml centrifuge tube, shaken at room temperature for 10 min, centrifuged at 4° C. and 15000 rpm for 20 min, the supernatant was taken to obtain the cell lysate supernatant. The composition of the lysis buffer was the same as the above said description. The following procedure and negative control 1 were the same as Example 1, and negative control 2 was the lysate supernatant of A431 cells prepared in Example 1. For RE/PCR PstI was replaced with MboI. The result was as follows: 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Result 
               
               
                 Sample Group 
                 Ct-1 
                 Ct-2 
                 Ct-Average 
                 Determination 
               
               
                   
               
             
             
               
                 
                   E. coli 
                 
                 15.24 
                 15.48 
                 15.36 
                 positive 
               
               
                 Negative Control 1 
                 31.41 
                 31.56 
                 31.49 
                 negative 
               
               
                 Negative Control 2 
                 31.39 
                 31.27 
                 31.33 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0095]    It can be seen that using the WFTR-PCR process of this Example, the expression of Amp r  gene can be detected in the  E. coli  DH5a transferred with pcDNA3.1.