Abstract:
There is provided a sensitive multiplex RT-PCR assay for the detection of circulating prostate antigen expressing cells. Multiplex PCR uses multiple sets of primers to concurrently amplify different DNA sequences that can be readily resolved by gel electrophoresis. When applied to blood samples from prostate cancer patients, the nested multiplex RT-PCR can simultaneously detect PSA-expressing cells, PSM-expressing cells, and a ubiquitously expressed internal PCR control gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH), all within a single reaction.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/016,543, filed Jun. 24, 1996 (by Petition To Correct Filing Date filed under separate cover concurrently herewith). 
     BACKGROUND OF THE INVENTION 
     (1) Field of the Invention 
     The present invention relates to a method for detecting prostate cells. More specifically, this invention relates to a method for detecting circulating prostate cells using a nested multiplex reverse transcriptase polymerase chain reaction assay. 
     (2) The Prior Art 
     Adenocarcinoma of the prostate is the most common internal cancer in males with an estimated 244,000 new cases per year and 38,000 deaths. (P. A. Wingo, et al., Ca. Cancer J. Clin. 45: 8-30 (1995)). The diagnostic tools of serum prostate-specific antigen (PSA) level, digital rectal exam, transrectal prostatic ultrasound, sextant needle biopsies, histologic Gleason scoring, computed tomography, and magnetic resonance imaging currently provide the foundation for the clinical staging of prostate cancer. 
     If the disease is truly confined to the prostate, surgical removal of the gland should remove all traces of malignant cells. However, it has been shown that up to 40% of patients diagnosed preoperatively with prostate-confined disease using current modalities actually have disease outside the margins of surgical resection at surgery. (Lu-Yao, McIerran, Wasson, Wennberg. An assessment of radical prostatectomy. Time trends, geographic variation and outcomes. The Prostate Patent Outcome Research Team, JAMA 269:2633-2655 (1993); Voges, et al., Morphologic analysis of surgical margins with positive findings in prostatectomy for adenocarcinoma of the prostate, Cancer 69:520-526 (1992); Catalona et al., Nerve-sparing radical prostatectomy: extraprostatic tumor extension and preservation of erectile function, J. Urol. 134:1149-1151 (1985); Epstein, et al., Is tumor volume an independent predictor of progression following radical prostatectomy? A multivariate analysis of 185 clinical stage B adenocarcinoma of the prostate with 5 years of follow-up, J. Urol. 148:1478-1481 (1993)). This understaging of males exposes them to the morbidity of radical surgery without providing a cure. 
     An RT-PCR assay for PSA has been developed by Katz and co-workers which recognizes PSA-expressing cells. The RT-PCR assay uses PSA primers to detect prostate cells in the peripheral circulation prior to radical prostatectomy. (A. E. Katz, et al, Molecular Staging of prostate Cancer With the Use of an enhanced Reverse Transcriptase-PCR Assay, Urology 43:765-775 (1994)). This PSA assay detects only PSA antigens. 
     Another assay for detecting occult hematogenous micrometastatic prostatic cells is a modification of similar PCR assays uses PCR primers derived from the cDNA sequences of prostate-specific membrane (PSM) antigen. (R. S. Israeli, et al, Sensitive Nested Reverse Transcription Polymerase Chain Reaction Detection Of Circulating Prostatic Tumor Cells: Comparison Of Prostate-Specific Membrane Antigen And Prostate-Specific Antigen-Based Assays, Cancer Research 53:227-230 (1993)). This PSM assay detects only PSM antigens. 
     Even in view of these advances in prostate cancer cell detection, there still remains a need for better identification of prostate tumor antigens. In particular there is need for a single assay to detect the presence of both PSM and PSA antigens. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to provide a method for the detection of one prostatic cell within a population of 1,000,000 lymphocytes. 
     Another object of this invention is to provide a single assay for detecting the mRNA expression of both PSA and PSM genes. 
     In accordance with the present invention there is described a method for detecting circulating prostate cells in a human. In one embodiment, the invention is directed to obtaining a blood sample of a human, isolating total cellular RNA from the blood synthesizing a first strand cDNA by reverse transcription, adding a portion of the first strand cDNA product to a master mix containing PSM and PSA outside primers, adding polymerase to the master mix and amplifying, adding a portion of the first strand cDNA product to the master mix containing PSM and PSA inside primers, adding Taq polymerase to the master mix and amplifying the product, then nesting the amplified portion with a second set of primers, and comparing the blood sample to a positive control. 
     The present invention also encompasses kits for in vitro or in vivo applications for diagnosis of prostate carcinoma. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a flow chart of the nested multiplex RT-PCR assay to detect circulating prostate cancer cells; 
     FIG. 2A is a cDNA map for prostate specific antigen; 
     FIG. 2B is a cDNA map for prostate specific membrane antigen; 
     FIG. 2C is a cDNA map of glyceraldehyde 3-phosphate dehydrogenase; 
     FIG. 3 illustrates the sensitivity of the multiplex RT-PCR assay as determined by diluted LNCaP cells. The human PSA and PSM-expressing cell line LNCaP was used as a positive control in the PCR reactions. Total RNA was extracted from serial diluted LNCaP cells in 10 million cultured B-lymphocytes that do not express PSA or PSM (data not shown). The RNA was then assayed by RT-PCR for the presence of prostate specific antigen (PSA) and prostate specific membrane antigen (PSM) using the 5&#39; and 3&#39; outside primers. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was also amplified as an internal control. Amplified products were separated by 2% agarose gel electrophoresis and the results are shown. The number of diluted LNCaP cells appears above the gel, the sizes of the amplified products are indicated in parentheses, and the lane numbers appear below. N=no DNA negative control; M=molecular weight marker. 
     FIG. 4 shows the sensitivity of the nested multiplex PCR assay as determined by diluted LNCaP cells. 1.0 μl of amplified products (including the no DNA negative control) shown in FIG. 2 were used as templates for a second PCR amplification using F1 and R1 primers. The ethidium bromide stained 2- agarose gel is shown. The number of diluted LNCaP cells appears above the gel, the sizes of the amplified products are indicated in parentheses, and the lane numbers appear below. N=no DNA negative control; M=molecular weight marker. 
     FIG. 5 illustrates PSM detected by Southern blot hybridization. Lane 1 from the gel shown in FIG. 3 was Southern blotted and hybridized with PSM (SEQ ID No. 12) and PSA (SEQ ID No. 11) specific probes. This indicates that one prostate cell from within one million B-lymphocytes can be amplified using the nested multiplex RT-PCR assay. 
     FIG. 6 shows a multiplex RT-PCR of prostate patient mRNA. Messenger RNA from prostate cancer patents and controls were assayed using the multiplex RT-PCR assay. Prostate samples were either from metastatic prostate patents or metastatic hormone-refractory patients. Control samples were provided by healthy volunteers. After amplification, the products were resolved on a 2% agarose gel. LNCaP RNA was used as a positive control for PSA and PSM. N=no DNA control; M=molecular weight marker. 
     FIG. 7 is a representative Southern blot of nested multiplex PCR of prostate patient samples. 1.0 μl of each patient sample, including the no DNA control (N), was removed from the Multiplex RT-PCR tube after 14 cycles (see FIG. 1) and used as a template for a nested multiplex PCR reaction. Primers F1 and R1 were used in this nested PCR assay and the products were resolved on a 2% agarose gel. LNCaP cell RNA was used as a positive control for PSA and PSM. M=molecular weight marker. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is directed to an assay for diagnosing circulating prostate cells. 
     A nested multiplex reverse transcriptase PCR assay was developed to simultaneously detect PSA-expressing prostatic cells, PSM-expressing prostatic cells and an internal control (G3PDH). The primers used for this assay are listed in Table 1. 
     
                                           TABLE 1__________________________________________________________________________Sequences of primers used forNested Multiplex PCR and Southern blotsName         5&#39;-3&#39; Sequence      PCR product__________________________________________________________________________PSA5&#39; (SEQ ID NO.1)        GATGACTCAGCCACGACCT 710 bpPSA3&#39; (SEQ ID NO.2)        CACAGACACCCCATCCTATCPSAF1 (SEQ ID NO.3)        CTGTCAGAGCCTGCCGAGCTC                            634 bpPSAR1 (SEQ ID NO.4)        GATATGTCTCCAGGCATGGCCPSM5&#39; (SEQ ID NO.5)        CAATGAAGCTACTAACATTACTCC                            552 bpPSM3&#39; (SEQ ID NO.6)        TCGGAGTAGAGAATGACTCCTTTGPSMF1 (SEQ ID NO.7)        CAGATACCACATTTAGCAGGAAC                            219 bpPSMR1 (SEQ ID NO.8)        CATATCCTGGAGGAGGTGGTTCG3PDH5&#39; (SEQ ID NO.9)        TGAAGGTCGGAGTCAACGGATTTGGT                            983 bpG3PDH3 (SEQ ID NO.10)        CATGTGGGCCATGAGGTCCACCACPSAF2 (SEQ ID NO.11)        CATGCTGTGTGCTGGACGCTGGACPSMF2 (SEQ ID NO.12)        CTGGATTCTGTTGAGCTAGCAC__________________________________________________________________________ 
    
     Primers utilized in the initial or outside PCR amplification of PSA (PSA5&#39; (SEQ ID No. 1) and PSA3&#39; (SEQ ID No. 2)) were identical to those previously reported by Katz et al. (A. Lundwall and H. Lilja, Molecular cloning of the human prostate specific antigen cDNA, FEBS Letters 214:317-322 (1987)), that are located in exons 3 and 5 and span introns 3 and 4 (see FIGS. 2A-2C). Primers (PSAF1 (SEQ ID No. 3) and PSAR1 (SEQ ID No. 4)) for the nested inside PSA amplification were located 13 base pairs (bp) internal to PSA5&#39; (SEQ ID No. 1) and 22 bp internal to PSA3&#39; (SEQ ID No. 2) according to the published sequence of PSA. (A. Lundwall and H. Lilja, Molecular cloning of human prostate specific antigen cDNA. FEBS Letters 214:317-322 (1987). 
     Primers used in the PSM reactions were chosen for their ability to optimally amplify PSM without interference from the PSA and G3PDH primers contained within the multiplex reaction, and to amplify a fragment that could be easily resolved from either PSA or G3PDH by electrophoresis. Primers used for the initial outside PSM reaction (PSM5&#39; nucleotides 398-422 (SEQ ID No. 5) and PSM3&#39; nucleotides 927-950 (SEQ ID No. 6)) were intentionally chosen upstream of the reported homology with the human transferrin receptor to avoid any possible false amplification. R. S. Israeli, et al, Molecular Cloning of a Complementary DNA encoding a Prostate-Specific Membrane Antigen, Cancer Research 54:6306-6310 (1994). Nested PSM primers (PSMF1 nucleotides (SEQ ID No. 7) 496-518 and PSMR1 nucleotides 694-715 (SEQ ID No. 8)), were located internal to the initial primers 73 bps and 210 bps, respectively. Due to the currently unknown intron/exon structure of the PSM gene, these primer sets are not known to span in intron. However, PCR of human genomic DNA with either PSM primer set was unsuccessful in amplifying the products predicted from the cDNA sequence, thus it is assumed that at least one intron is spanned by these primers. The PCR primers used for the amplification of the internal control, G3PDH, were purchased commercially and are known to span several introns. 
     Experimental 
     Patient selection 
     Patients were recruited from the McKay Department of Urology of the Carolinas Medical Center. Metastatic prostate cancer patients were defined by a serum PSA test level greater than 20, no prior or current hormonal treatment, and bone metastasis as indicated by a positive bone scan. Hormone-refractory prostate cancer patients were defined by a serum PSA level greater than 20, a rising serum PSA level after hormonal treatment has been previously established, and a positive bone scan indicating metastasis. Women volunteers with no specific exclusion or inclusion criteria, and healthy male volunteers under the age of 40 were recruited from hospital employees. 
     RNA extraction 
     Blood (10 ml) was collected from prostate cancer patients and healthy volunteers in ethylene diaminetetraacetic acid (EDTA) tubes and immediately processed. Samples were centrifuged at 2000 rpm for 30 minutes, and the buffy coat cells were removed with a sterile transfer pipette. Total cellular RNA was isolated using TriZOL Reagent (Gibco/BRL). RNA was resuspended in RNAse-free (diethyl pyrocarbonate-treated) H 2  O and quantified by optical density at 260 nm. It should be understood that bone marrow samples may also be used in the process of the present invention. 
     Cell Culture 
     The human prostate cell line, LNCaP (ATCC CRL-1740) and human B-lymphocyte cell line ED1, were cultured in RPMI media containing 10% fetal bovine serum at 5% CO 2  and 37° C. Trypsin (0.5%) solution was used to disperse confluent LNCaP cultures for RNA isolation or subsequent passage. 
     cDNA First Strand Synthesis by Reverse Transcription 
     An aliquot containing 1.0 μg of total RNA was primed by random hexamers (2.5 μM) in the presence of RNAse inhibitor (1.0 U), 1 mM of each deoxynucleotide triphosphate, 2.5 U reverse transcriptase (Perkins Elmer), 1× PCR buffer II (Perkins Elmer), and 5.0 mM MgCl 2  in a final volume of 20 μl. The samples were incubated for 30 minutes at 42° C., followed by 5 minutes at 99° C. to heat inactivate the reverse transcriptase. 
     First Multiplex PCR Reaction 
     The first PCR reaction was done with one-half (10 μl) of the first strand cDNA product which was added to a master mix containing a final concentration of 1× PCR buffer II, 2 mM MgCl 2 , 0.8 mM spermidine, 2.8% dimethyl sulfoxide (DMSO), 0.4 μM of each PSM primer (PSM5&#39; (SEQ ID No. 5), and PSM3&#39; (SEQ ID No. 6)), 0.3 μm of each PSA primer (PSA5&#39; (SEQ ID No. 1) and PSA3&#39; (SEQ ID No. 2), 0.3 μM of each G3PDH primer (G3PDH5 (SEQ ID No. 9) and G3PDH3&#39; (SEQ ID No. 10), Clontech), in a total of 50 μl. 
     The total reaction mixture was then divided between two tubes (25 μl each), heated to 95° C. for 5 minutes after which 2.5 U of AmpliTaq polymerase (Perkin-Elmer) was added, and cycled at 94° C. (1 min.), 62° C. (1 min.), 72° C. (1 min.). One set of tubes was removed after 14 cycles for use as starting template for nested PCR, and the other set was amplified for a total of 35 cycles. Amplified products were visualized on a 2.0% agarose gel. 
     Second Multiplex PCR Reaction to Nest 
     The second multiplex reaction was done with 1.0 μl of amplified product from the initial PCR was removed from the tube amplified for 14 cycles and was added to a master mix containing a final concentration of 1× PCR buffer II, 2.0 mM MgCl 2 , 0.2 mM dNTPs, 0.4 μM, 0.18 μM PSA inside primers (PSAF1 (SEQ ID No. 3) and PSAR1 (SEQ ID No. 4)), and 2.8% DMSO in a final volume of 50 μl. The tubes were heated to 95° C. for 5 minutes, 2.5 U of AmpliTaq polymerase was added, and amplified at 94° C. (1 min.), 62° C. (1 min), 72° C. (1 Min) for 33 cycles. Amplified products were visualized on a 2% agarose gel. 
     Southern Blot Hybridization Assay 
     Agarose gels were blotted to nylon membranes (Zetaprobe, Bio-Rad) by the Southern Blot procedure. (Sambrook et al, Molecular Cloning: A Laboratory Manual (1989), Cold Springs Harbor, N.Y.: Cold Springs Harbor Laboratory Press). After the filters were UV crosslinked, hybridizations were carried out using Rapid-hyp buffer (Amersham) using 30 pmol of τ- 32  P ATP labeled oligonucleotides (PSAF2 (SEQ ID No. 11), PSMAF2 (SEQ ID No: 12)) at 42° C. The filters were washed in 4×SSC, 0.1% SDS at 25° C. and exposed to Kodak XAR film. 
     PCR Fragment Cloning and Sequencing 
     PCR fragments were gel isolated, cloned into the pCRII vector (Invitrogen), and sequenced. Sequencing was performed on an ABI 373A automated DNA sequencer using the Taq dye deoxy cycle sequencing kit (ABI) as described by the manufacturer. DNA sequences obtained were analyzed, and sequence alignments were generated using Geneworks version 2.45 (Intelligenetics). 
     Results 
     Sensitivity of the Multiplex RT-PCR Assay As Determined by Diluted LNCaP Cells 
     To determine the sensitivity of the multiplex RT-PCR assay, cells from the human PSA- and PSM-expressing prostate cell line, LNCaP, were serially diluted and added to 10 million cultured ED1 cells. ED1 cells are cultured B-lymphocytes that do not express either PSA or PSM (data not shown). Although it is likely that the amount of mRNA expression of cells cultured in vitro differ between laboratories and media conditions, all LNCaP cells used in this experiment were from the same cell passage. RNA was extracted from each LNCaP/ED1 mix and 1.0 μg was assayed by the multiplex PCR assay using random hexamers for the synthesis of the first cDNA strand, and the outside (5&#39; and 3&#39;) primers for the subsequent PCR amplification. The amplified products were then resolved on a ethidium bromide stained 2% agarose gel which is shown in FIG. 3. The 983 bp amplified fragment of the positive PCR control, G3PDH, is visible in all lanes. PSA (710 bp) and PSM (552 bp) fragments are visible when the ratio of LNCaP cells to B-lymphocytes is at least 1:100 (lanes 6 and 7, FIG. 3). 
     Nested multiplex PCR using the inside (F1 and R1) primers was then performed using 1.0 μl of amplified product from each outside reaction as the starting template. FIG. 3 shows the results of the nested multiplex assay. The appropriately sized fragment in the lane containing 10 LNCaP cells (lane 2) demonstrated that the multiplex PCR assay could easily detect 1 PSA-expressing cell from a background of 1 million cells. The appropriately sized PSM fragment was visible, albeit faint, in the lane containing 1000 LNCaP cells (lane 4) but not visible by ethidium bromide staining in the lane containing 10 LNCaP cells. The fragment was clearly detected, however, in lane 2 after Southern blot transfer and hybridization with an internal  32  P end-labeled oligonucleotide probe, PSMF2, (SEQ ID No. 12) (FIG. 5, Table 1). This demonstrates that the nested PSM primers were also able to amplify a few as one LNCaP cell in a background of one million lymphocytes. Representative amplified fragments of PSA, PSM, and G3PDH were isolated from the gel and sequenced. The cDNA sequence of each amplified fragment aligned with the corresponding published cDNA sequence thus verifying the PCR products (data not shown). 
     Nested Multiplex RT-PCR of Prostate Cancer Patient and Control mRNA 
     Messenger RNA (mRNA) was isolated from blood samples collected from prostate cancer patients, healthy young male and female volunteers. Strict criteria for patient selection were used to insure the detection of circulating prostate cells and to help define the multiplex PCR assay conditions. The nested multiplex PCR assay used for the patient samples is summarized in FIG. 6. Briefly, 1.0 μg of total RNA was first synthesized into cDNA by reverse transcriptase in a 20 μl reaction volume. One-half of this RT reaction was placed into a tube containing 40 μl of a master mix that included all necessary PCR reagents. This master mix tube was then divided into two separate reactions, each containing 25 μl which were amplified for 35 and 14 cycles, respectively. Nested PCR was then performed using 1 μl of the 14 cycle amplified product as template, and all products from both initial and nested PCR were resolved on a 2% agarose gel. 
     The results after 35 cycles of the multiplex PCR assay utilizing the outside (5&#39; and 3&#39;) primers on seven patient and six volunteer samples are shown in FIG. 7. Lanes 1-3 show the amplified fragments from the male controls, lanes 4-6 the amplified fragments from the female controls, and lanes 7-13 from the prostate cancer patients. 1.0 μg of RNA isolated from LNCaP cells was used as a positive control (lane 14) and 1 μl of water was used as a negative control. All lanes except the no template negative control were positive for G3PDH expression while PSA and PSM were amplified in the lane containing the LNCaP cells. This result indicates that no reaction failed due to unsuccessful PCR amplification. No PSA or PSM fragments were seen, however, in any patient lane after the initial amplification. 
     FIG. 7 shows the results of the nested multiplex PCR amplification from the seven prostate cancer patients and six volunteers. The lanes in FIG. 7 correspond to the same patient samples and lanes as in FIG. 6 after nested multiplex PCR amplification. Both PSA and PSM amplified fragments are clearly visible in two hormone-refractory patients (lanes 9 and 10), one metastatic patient (lane 13) and the LNCaP positive control (lane 14). PSA-expression alone was seen in one metastatic patient sample (lane 11), while in one metastatic hormone-refractory patient only PSM-expression was observed (lane 12). No amplified fragments were visible by ethidium bromide staining in any of the male or female control patients (lanes 1-6) or in two of the prostate cancer patients (hormone refractory lane 7, untreated metastatic lane 8). An additional 10 control samples (five male, five female) were also negative for PSA and PSM (data not shown). To verify that no amplified fragments were present that were undetectable by ethidium bromide staining alone, the gel in FIG. 7 was Southern blotted, and hybridized to radiolabeled oligonucleotide probes (PSAF2 (SEQ ID No: 11) and PSMF2 (SEQ ID No: 12)). None of the control samples were positive for either PSA or PSM expression by molecular hybridization (data not shown). 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 12(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerPSA5&#39;)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GATGACTCAGCCACGACCT19(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(primerPSA3&#39;)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:CACAGACACCCCATCCTATC20(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerPSAF1)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:CTGTCAGAGCCTGCCGAGCTC21(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerPSAR1)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GATATGTCTCCAGGCATGGCC21(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic Oligonucleotide(PrimerPSM5&#39;)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:CAATGAAGCTACTAACATTACTCC24(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerPSM3&#39;)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TCGGAGTAGAGAATGACTCCTTTG24(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(primerPSMF1)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:CAGATACCACATTTAGCAGGAAC23(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerPSMR1)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CATATCCTGGAGGAGGTGGTTC22(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerG3PDH5&#39;)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:TGAAGGTCGGAGTCAACGGATTTGGT26(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerG3PDH3&#39;)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:CATGTGGGCCATGAGGTCCACCAC24(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerPSAF2)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:CATGCTGTGTGCTGGACGCTGGAC24(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: other nucleic acid(A) DESCRIPTION: /desc = &#34;Synthetic oligonucleotide(PrimerPSMF2)&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:CTGGATTCTGTTGAGCTAGCAC22__________________________________________________________________________