Patent Abstract:
A method is provided for fast diagnosis of tubercle bacillus (TB). The method can be used for efficacy test at the same time. 13 specific TB genes and 6 drug-resistance genes are selected. Those genes are formed into a construction for diagnosing tuberculosis and testing drug resistance simultaneously.

Full Description:
TECHNICAL FIELD OF THE INVENTION 
       [0001]    The present invention relates to tuberculosis diagnosis; more particularly, relates to using a chip array construction of specific tubercle bacillus (TB) genes and drug-resistance genes for detecting tubercle bacillus and drug resistance. 
       DESCRIPTION OF THE RELATED ARTS 
       [0002]    TB is an old contagious disease. Although it has been long on developing methods for preventing, controlling and curing this disease, TB is still a key issue in the world, which kills greatest number of people among all contagious diseases. 
         [0003]    A characteristic of TB is that there may be no sign appeared after a person is infected and only 10% of the patients have morbidity. Most of the patients have the thalli lived in their bodies for a long time before the morbidity appears. Thus, the cause that turns a patient of latent TB into one of active TB may be exogenous reinfection or endogenous reactivation. Hence, for diagnosing TB clinically, clinical expression shown on the patients, changes shown on X-ray films and laboratorial experiments are all required for confirmation. 
         [0004]    Regarding laboratorial examination, technologies relating to histopathology, staining of acid-fast bacterium and TB culturing are used. However, they all have their limits. Take staining of acid-fast bacterium as an example. At least 5000 to 10000 bacteria have to be contained in one milli-liter of a specimen. Besides, there exists a high possibility of fake positive for this method. That is because some other bacteria may show positive results too. Regarding TB culturing, although it is the most sensitive diagnosing method, it takes 4 to 8 weeks to obtain the result and is not suitable for clinical use. 
         [0005]    As following development of biological technologies, clinical diagnosis of TB has evolutional progress on molecular diagnostic technologies, like polymerase chain reaction (PCR), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), etc. In the early years, PCR are directly used for detecting molecular marks of TB in patients&#39; specimens, like deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of heat shock proteins 65 (hsp65) and inserted section 6110 (IS6110). In addition, with coordination of restriction fragment length polymorphism (RFLP), the nucleic-acid molecular typing of the detected thallus is identified. However, expression of specific DNA or messenger ribonucleic acid (mRNA) in a patient&#39;s specimen is still not satisfactory clinically. 
         [0006]    Through decoding the TB genes, it is found that genomic deletion is existed between TB and  mycobacterium bovis  BCG, where lost sections are called regions-of-difference (RD). The causes for these RDs may be errors on duplicating DNAs of the genes or on inserting sections, including deletion, insertion, inversion, replication, etc. 
         [0007]    The RD sections have many important genes and pathogenic factors. These sections may differ between pathogens of TB genes. Hence, the RD sections can be used for identifying the TB genes, which identifies specific genes with high sensitivity. 
         [0008]    In 2009, 14 specific target genes were selected as testing targets for constructing a TB gene diagnosis chip. Through using a platform for detecting tiny amount of nucleic acid, multiple gene targets are detected simultaneously, where sensitivity reaches a level for detection with only 5 cells in one milli-liter of blood. Thus, a TB gene detection chip for sputum specimen is constructed. 
         [0009]    This chip can detect 85% of TB complex (TBC), where PCR-RFLP is 62.5%. In an experiment, 52 specimens are picked out by the chip from 56 positive-cultured and positive-dyed sputum specimens, where only 39 are picked out through PCR-RFLP. In another experiment, 16 specimens are picked out by the chip from 24 positive-cultured and negative-dyed sputum specimens, where only 11 are picked out through PCR-RFLP. 
         [0010]    Accordingly, gene chip detection is easily operated without much human labor and time. Moreover, sensitivity of the gene chip detection is far higher than PCR-RFLP. 
         [0011]    In the market, some TB detection sets include Spoligotyping Method (Holland), TB Ag Rapid Test (Taiwan), Amplified MTDR (USA), DR. MTBC Screen Kit (Taiwan) and GenoType MTBDRplus (German). Therein, Spoligotyping Method detects TB oligonucleotide spectrum at first and, then, finds its typing from a database. But, the resolving power is low and drug resistance is not detected. TB Ag Rapid Test uses specific antigen to detect TB directly. But, its cost is high; TB colony has to be cultured; its procedure is complex; it takes time; and, not to mention, drug resistance is not detected. DR. MTBC Screen Kit magnifies specific gene sections through PCR and, then, the chip is processed through hybridization. Although drug-resistance genes against Rifampicin can be found, 100-thousand TB bacteria in one milli-liter of sputum are required for valid detection with a 65% sensitivity only. GenoType MTBDRplus magnifies TB drug-resistance genes through PCR and, then, hybridization is processed with probes. Although drug-resistance genes in TB against Ofloxacin, Streptomycin and Ethambutol can be found, its cost is high; its technology is complex; it takes time for detection; and it only tests efficacy but not TB itself. 
         [0012]    Furthermore, a prior art of detection chip directly detected active TB in a sputum specimen. Yet, it detects Tb only and do not analyzes efficacy on gene cluster. Hence, the prior arts do not fulfill all users&#39; requests on actual use. 
       SUMMARY OF THE INVENTION 
       [0013]    The main purpose of the present invention is to use a construction of specific TB genes and drug-resistance genes for detecting TB and testing drug resistance simultaneously. 
         [0014]    To achieve the above purpose, the present invention is a method of fast tuberculosis diagnosis and efficacy test, comprising steps of: (a) obtaining a sputum specimen and extracting messenger ribonucleic acids (mRNAs) in the sputum specimen to synthesize a required amount of complementary deoxyribonucleic acids (cDNAs) through reverse transcription; (b) labeling the cDNAs with Biotin to obtain a plurality of bioprobes; (c) synthesizing TB genes and drug-resistance genes in vitro into a specific gene cluster of TB and a drug-resistance gene cluster and obtaining a chip array construction through crosslinking by dotting the specific gene cluster of TB, the drug-resistance gene cluster, positive controls, negative controls and blank controls into array on a nylon membrane, where the specific gene cluster of TB is specified through a specific oligonucleotide design; and where the chip array construction is formed into a plurality of gene-testing points on the nylon membrane; (d) hybridizing the gene-testing points of the chip array construction with biomolecules of the bioprobes and washing out un-hybridized bioprobes; and (e) blocking the bioprobes obtained after hybridization of the chip array construction to form crosslinks with Streptavidin-HRP accompanied with a washing process afterwards and, then, adding a coloring agent of diaminobenzidine (DAB) to process color development for analyzing and interpreting an image thus obtained. Accordingly, a novel method of fast tuberculosis diagnosis and efficacy test is obtained. 
     
    
     
       BRIEF DESCRIPTIONS OF THE DRAWINGS 
         [0015]    The present invention will be better understood from the following detailed description of the preferred embodiment according to the present invention, taken in conjunction with the accompanying drawings, in which 
           [0016]      FIG. 1  is the flow view showing the preferred embodiment according to the present invention; 
           [0017]      FIG. 2  is the view showing the testing areas; 
           [0018]      FIG. 3  is the view showing the gene arrangements; 
           [0019]      FIG. 4  is the view showing the interpretation of the bacillus tuberculosis testing; and 
           [0020]      FIG. 5  is the view showing the interpretation of the drug resistance testing. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0021]    The following description of the preferred embodiment is provided to understand the features and the structures of the present invention. 
         [0022]    Please refer to  FIG. 1 , which is a flow view showing a preferred embodiment according to the present invention. As shown in the figure, the present invention is a method of fast tuberculosis diagnosis and efficacy test, comprising the following steps: 
         [0023]    (a) DNA extraction  11 : A sputum specimen of a patient is collected and messenger ribonucleic acids (mRNAs) in the sputum specimen is extracted for synthesizing a required amount of complementary deoxyribonucleic acids (cDNAs) through reverse transcription. 
         [0024]    (b) Multiple linear amplification and labeling  12 : The cDNAs are labeled with Biotin to form a plurality of bioprobes. 
         [0025]    (c) Fabrication of chip array construction  13 : Tubercle bacillus (TB) genes along with drug-resistance genes are synthesized in vitro into a specific gene cluster of TB along with a drug-resistance gene cluster, where the specific gene cluster of TB is specified through a specific oligonucleotide design. Then, a chip array construction is formed through crosslinking by dotting the specific gene cluster of TB, the drug-resistance gene cluster, positive controls, negative controls and blank controls into array on a nylon membrane. Therein, the specific gene cluster of TB comprises 13 specific TB genes; the drug-resistance gene cluster comprises 6 drug-resistance genes; and the chip array construction is formed into a plurality of gene-testing points on the nylon membrane. 
         [0026]    (d) Hybridization  14 : The bioprobes are hybridized with the chip array construction, where the gene-testing points of the chip array construction are hybridized with biomolecules of the labeled bioprobes. Then, un-hybridized bioprobes are washed out. 
         [0027]    (e) Color development  15 : After hybridization with the chip array construction, the bioprobes are blocked to form crosslinks with Streptavidin-HRP accompanied with a washing process afterwards. Then, a coloring agent of diaminobenzidine (DAB) is added for color development to analyze and interpret an image thus obtained. 
         [0028]    As shown in Table 1, the specific gene cluster of TB comprises specific oligonucleotide sequences selected from the specific TB genes. These 13 specific TB genes comprises hsp65, Rv0577, Rv3120, Rv2073c, Rv1970, Rv3875, Rv3347c, Rv1510, Rv0186, Rv0124, TbD1, mtp40 and mpb83, which are obtained through analysis by Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, Calif.). 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Gene 
                   
               
               
                 No. 
                 Name 
                 Oligonucleotide Sequence 
               
               
                   
               
             
             
               
                  1 
                 hsp65 
                 CAT CGG TCT TCT TGG CTA CCT CTT TGA CCA GCT CG 
               
               
                   
               
               
                  2 
                 Rv0577 
                 CGT CGT AAC CCC AGC CGA ACA ACG ATG TGT AGA AC 
               
               
                   
               
               
                  3 
                 Rv3120 
                 CGG ATG CCA GAA TAG TCG GCA AAG TAC CAG AGC A 
               
               
                   
               
               
                  4 
                 Rv2073c 
                 GCC GGC TTT GGC CGA TCC GTA GAC ATA GTT G 
               
               
                   
               
               
                  5 
                 Rv1970 
                 GTC ACC GGA CTG GTT GTT GAG GTA TGC GGT G 
               
               
                   
               
               
                  6 
                 Rv3875 
                 CTT CCC CTC GTC AAG GAG GGA ATG AAT GGA CGT G 
               
               
                   
               
               
                  7 
                 Rv3347c 
                 GTG TTG TAG CTG CCC GAG TTG AAT ACC CCG AAG TT 
               
               
                   
               
               
                  8 
                 Rv1510 
                 CCA GAT AGA TGA CCG TGT AGA CGC AGG CAA CGG 
               
               
                   
               
               
                  9 
                 Rv0186 
                 GGT CCT CGG AAA GGT ACT CGA AGT TGC GGC 
               
               
                   
               
               
                 10 
                 Rv0124 
                 CGT CTG CAC GAA CTG CTG ATG AAA CGC CG 
               
               
                   
               
               
                 11 
                 TbD1 
                 TCG GCT GCT CGG TCC CTC TGA TAC TTG AGA TTC TG 
               
               
                   
               
               
                 12 
                 mtp40 
                 ATC CGC AGT GAT GCC AAC TCA GGA AAC CAC AC 
               
               
                   
               
               
                 13 
                 mpb83 
                 GAG GTC AGG GTA CTG AGC ATC GGG TTG TTG GAA G 
               
               
                   
               
             
          
         
       
     
         [0029]    As shown in Table 2, the drug-resistance gene cluster for testing anti-tuberculosis drugs comprises 6 oligonucleotide sequences, which comprises katG, rpoB, gyrA, embB, rpsL and rrs. 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Oligonucleotide 
                   
               
               
                 Drug 
                 Name 
                 Oligonucleotide sequence (5′ to 3′) 
               
               
                   
               
             
             
               
                 Isoniazid 
                 katG-W1 
                 AAC TAG CTG TGA GAC AGT CAA TCC CGA TGC CCG 
               
               
                   
                 katG-W315 
                 CGA TGC CGC TGG TGA TCG CGT CCT TA 
               
               
                   
                 katG-Q315 
                 CGA TGC CGC TGG TGA TCG TGT CCT TA 
               
               
                   
               
               
                 Rifampicin 
                 rpoB-W1 
                 GAC TCG GAC TAG GAC TAG CGG CTG TTT TGC TCT 
               
               
                   
                 rpoB-W450 
                 CCC TCA GGG GTT TCG ATC GGG CAC AT 
               
               
                   
                 rpoB-Q450 
                 CCC TCA GGG GTT TCG ATC GAG CAC AT 
               
               
                   
                 rpoB-W513 
                 TCG ACC ACC TTG CGG TAC GGC GTT TC 
               
               
                   
                 rpoB-Q513 
                 TCG ACC ACC TTG CGG TAC GGA GTT TC 
               
               
                   
                 rpoB-W522 
                 GTA CAC GAT CTC GTC GCT AAC CAC GCC GT 
               
               
                   
                 rpoB-Q522 
                 GTA CAC GAT CTC GTC GCT AAC TAC GCC GT 
               
               
                   
                 rpoB-W526 
                 GTC GGC GGT CAG GTA CAC GAT CTC GT 
               
               
                   
                 rpoB-Q526 
                 GTC GGC GGT CAG GTA CAT GAT CTC GT 
               
               
                   
                 rpoB-W529 
                 TCC TCC TCG TCG GCG CTC AGG TAC A 
               
               
                   
                 rpoB-Q529 
                 TCC TCC TCG TCG GAG CTC AGG TAC A 
               
               
                   
                 rpoB-W531 
                 CCA CCA CGT GGC GGT CCT C 
               
               
                   
                 rpoB-Q531 
                 CCA CTA CGT GGC GGT CCT C 
               
               
                   
               
               
                 Ofloxacin 
                 gyrA-W1 
                 CGG GAA TCC TCT TCT ACC TCA ACA ACT CCG CGC 
               
               
                   
                 gyrA-W80 
                 CCC ATG GTC TCG GCA ACC GAC CG 
               
               
                   
                 gyrA-Q80 
                 CCC ATG GTC TCG GCA ACT GAC CG 
               
               
                   
                 gyrA-W88-91 
                 CGT AGA TCG ACG CGT CGC CGT GC 
               
               
                   
                 gyrA-Q88-91 
                 CGT ATA TCG ACG CGT CGC CGT GC 
               
               
                   
                 gyrA-W94 
                 GCC ATG CGC ACC AGG CTG TCG TAG AT 
               
               
                   
                 gyrA-Q94 
                 GCC ATG CTC ACC AGG CTG TCG TAG AT 
               
               
                   
               
               
                 Ethambutol 
                 embB-W1 
                 GTG TCC AGC TTC TTA GCC GAG TAG TCC GGT GT 
               
               
                   
                 embB-W306 
                 CGG GCC ATG CCC AGG ATG TAG CC 
               
               
                   
                 embB-Q306 
                 CGG GCC ATG CCC AGG ATA TAG CC 
               
               
                   
                 embB-W319 
                 GGG CTG CCG AAC CAG CGG AAA TAG TTG G 
               
               
                   
                 embB-Q319 
                 GGG CTG TCG AAC CAG CGG AAA TAG TTG G 
               
               
                   
                 embB-W406 
                 CGA GCG CGA TGA TGC CCT CCG 
               
               
                   
                 embB-Q406 
                 CGA GCT CGA TGA TGC CCT CCG 
               
               
                   
               
               
                 Streptomycin 
                 rpsL-W1 
                 GCG GTC TTG ACC TTA CTG ATC TTG TCC CGA 
               
               
                   
                 rpsL-W43 
                 GAA GCG CCG AGT TCG GCT TCT TCG GAG 
               
               
                   
                 rpsL-Q43 
                 GAA GCG TCG AGT TCG GCT TCT TCG GAG 
               
               
                   
                 rpsL-W88 
                 GCA CAC CAG GCA GGT CCT TCA CCC 
               
               
                   
                 rpsL-Q88 
                 GCA CAC TAG GCA GGT CCT TCA CCC 
               
               
                   
               
               
                 Streptomycin 
                 rrs-W1 
                 CGT AGG AGT CTG GGC CGT ATC TCA GTC CCA 
               
               
                   
                 rrs-W513 
                 CCT ACG TAT TAC CGC GGC TGC TGG CA 
               
               
                   
                 rrs-Q513 
                 CCT ACT TAT TAC CGC GGC TGC TGG CA 
               
               
                   
                 rrs-W514 
                 GCA CCC TAC GTA TTA CCG CGG CTG CT 
               
               
                   
                 rrs-Q514 
                 GCA CTC TAC GTA TTA CCG CGG CTG CT 
               
               
                   
                 rrs-W1401 
                 TGA CGT GAC GGG CGG TGT GTA CAA GG 
               
               
                   
                 rrs-Q1401 
                 TGA CGT GAC GGG CGG TAT GTA CAA GG 
               
               
                   
                 rrs-W1484 
                 GAC TTC GTC CCA ATC GCC GAT CCC ACC TTC 
               
               
                   
                 rrs-Q1484 
                 GAC TTC GTC CCA ATC GCC GAT CCT ACC TTC 
               
               
                   
               
               
                 Positive control 
                 rrl 
                 GTG TTA CCA CTG ACT GGT ACG GCT ACC TTC CTG 
               
               
                   
               
             
          
         
       
     
         [0030]    Please refer to  FIG. 2  and  FIG. 3 , which are views showing testing areas and gene arrangements. As shown in the figures, a chip array construction  20  comprises a testing area of bacillus tuberculosis  21  and a testing area of drug resistance  22 . In  FIG. 2 , P is a positive control  23 , N is a negative control  24  and B is a blank control  25 . 
         [0031]    The testing area of bacillus tuberculosis  21  comprises a plurality of gene-testing points  2   a  for separately conjugating a specific gene cluster of TB with specific bioprobes to be reacted with specific biomolecules of the specific bioprobes for color development. This specific gene cluster of TB comprises 13 specific TB genes, which are hsp65, Rv0577, Rv3120, Rv2073c, Rv1970, Rv3875, Rv3347c, Rv1510, Rv0186, Rv0124, TbD1, mtp40 and mpb83. 
         [0032]    The testing area of drug resistance  22  has a plurality of gene-testing points conjugated with a drug-resistance gene cluster to be reacted with anti-tuberculosis drugs of Isoniazid, Rifampicin, Ofloxacin, Ethambutol and Streptomycin for color development. The conjugated drug-resistance gene cluster comprises 6 drug-resistance genes, which are katG, rpoB, gyrA, embB, rpsL and rrs. 
         [0033]    The above gene-testing points  2   a.   2   b  are arranged into array. 
         [0034]    Please refer to  FIG. 4 , which is a view showing an interpretation of the bacillus tuberculosis testing. As shown in the figure, 13 specific TB genes and 6 drug-resistance genes are arranged in array on a nylon membrane to form a chip array construction. Therein, a testing area of bacillus tuberculosis  21  is processed through color development. If a color is developed, a specific gene is detected by expression for identification. In the figure, a result of color development for the chip array construction are as follows: hsp65(+), Rv0577(+), Rv31 20(−), Rv2073c(−), TbD1 (+), Rv1970(−), Rv3875(+), Rv3347c(+), Rv1510(−), Rv0186(+), Rv0124(+), mtp40(+) and mpb83(+). For interpretation, the sign (+) means positive reaction. More detailed comparison is shown in the following Table 3. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 hsp65 
                 Rv0577 
                 Rv3120 
                 Rv2073c 
                 TbD1 
                 Rv1970 
                 Rv3875 
                 Rv3347c 
                 Rv1510 
                 Rv0186 
                 Rv0124 
                 mtp40 
                 mpb83 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Organisms other than 
                 − 
                 − 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Mycobacterium 
                 
               
               
                 NTM* 
                 + 
                 − 
               
               
                 
                   M. canettii 
                 
                 + 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 
                   M. tuberculosis 
                 
                 + 
                 + 
                 + 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   M. africamun (lb) 
                 + 
                 + 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 Oryx  bacillus   
                 + 
                 + 
                 + 
                 − 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   M. africamun (lib) 
                 + 
                 + 
                 + 
                 − 
                 +/− 
                 − 
                 + 
                 + 
                 + 
                 − 
                 − 
                 + 
                 + 
               
               
                 Dassiebacillun 
                 + 
                 + 
                 + 
                 − 
                 + 
                 − 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 
                   M. microti 
                 
                 + 
                 + 
                 + 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 
                   M. caprie 
                 
                 + 
                 + 
                 − 
                 − 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 
                   M. bovis 
                 
                 + 
                 + 
                 − 
                 − 
                 + 
                 − 
                 + 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
               
               
                   M. bovis  BCG 
                 + 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
               
               
                   
               
               
                 *NTM: Nontuberculous  Mycobacterium   
               
             
          
         
       
     
         [0035]    Please refer to  FIG. 5 , which is a view showing an interpretation of the drug resistance testing. As shown in the figure, 13 specific TB genes and 6 drug-resistance genes are arranged to form a chip array construction on a nylon membrane. Therein, a testing area of drug resistance  22  is used for testing Ethambutol. EmbB-W1 is set as a positive control to develop color for embB; embB-W306, embB-W319 and embB-W406 are wild-type probes for embB codon 306, 319 and 406; and, embB-Q306, embB-Q319 and embB-Q406 are inner controls for embB codon 306, 319 and 406 in easily-mutating positions., when the gene is mutated and is not connected to the wild-type probe, the color is not developed and, thus, mutation of the drug-resistance gene is analyzed. A result is shown as follows: codon 306 (+), codon 319 (+) and codon 306 (+). Interpretation made for the result is that embB codon 306 is mutated, which shows this gene has drug resistance to Ethambutol. 
         [0036]    To sum up, the present invention is a method of fast tuberculosis diagnosis and efficacy test, where specific TB genes and drug-resistance genes are used as probes to test TB and drug resistance simultaneously through analysis after hybridization; and, thus, the present invention is a fast method with low cost for detecting TB and testing drug resistance simultaneously.

Technology Classification (CPC): 2