A specific fluorescent PCR detection kit for trichomonas vaginalis

Abstract

The application provides a specific Trichomonas vaginalis fluorescent PCR detection kit, which comprises a lysis solution, magnetic beads, a first washing solution, a second washing solution, an elution solution, sterilized purified water, a PCR reaction solution, Trichomonas vaginalis specific primers and probes, and human genome beta-Actin internal standard specific primers and probes. The primers and probes are designed according to the Trichomonas vaginalis Ferredoxin gene and the human genome beta-Actin gene diagnostic sequence, and the PCR reaction system and conditions are optimized to improve the detection sensitivity; and the amplification result is monitored in real time by a fluorescent PCR instrument. The fluorescent PCR detection kit is designed with high precision, is simple to operate, has high sensitivity and strong specificity, can specifically identify Trichomonas vaginalis infection, and the result is objective and accurate.

Description

Technical Field

[0001] This invention belongs to the field of biomedical diagnostics and relates to the detection of sexually transmitted parasites, especially the diagnostic and detection technology of Trichomonas vaginalis. Specifically, it is a specific fluorescent PCR detection kit for Trichomonas vaginalis. Background Technology

[0002] Trichomonas vaginalis is a parasitic protozoan and a major pathogen of nonviral sexually transmitted diseases (STDs) worldwide, primarily causing diseases of the male and female urogenital system. It is estimated that 4-35% of diagnosed vaginitis cases in China are caused by Trichomonas vaginalis. After infection, women often present with increased vaginal discharge, accompanied by inflammation and irritation; approximately 50-80% of infected individuals are asymptomatic. Trichomonas vaginalis infection has been proven to be associated with HIV transmission and control, and may also cause pregnancy complications in pregnant women, such as premature birth, premature rupture of membranes, and low birth weight infants. Furthermore, it may increase the incidence of cancers in women.

[0003] Wet slide microscopy is currently the most commonly used laboratory diagnostic method for Trichomonas vaginalis. It is simple, rapid, and inexpensive, but its sensitivity is relatively low, only 20-60%. Culture is the gold standard for diagnosing Trichomonas vaginalis, but it requires several days of culture time, is slow, and relatively expensive. Immunological methods include enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), but their sensitivity is low, and they are generally not used clinically. Molecular fluorescent PCR has advantages such as high sensitivity and specificity, and is widely used for pathogen detection. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, this invention provides a specific fluorescent PCR detection kit for Trichomonas vaginalis. This specific fluorescent PCR detection kit for Trichomonas vaginalis aims to solve the technical problems of long detection time and low sensitivity in existing methods for detecting Trichomonas vaginalis.

[0005] This invention provides a specific fluorescent PCR detection kit for Trichomonas vaginalis, comprising the following reagents:

[0006] 1) Lysis buffer: A mixed solution containing 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA, and 30% anhydrous ethanol (volume percentage).

[0007] 2) Magnetic beads: A solution containing 20 mg / ml magnetic beads;

[0008] 3) First washing solution: a mixed solution containing 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA, and 50% anhydrous ethanol (volume percentage).

[0009] 4) Second washing solution: a solution of anhydrous ethanol with a volume percentage concentration of 70%;

[0010] 5) Eluent: Sterile purified water;

[0011] 6) First PCR reaction solution: 40mM Tris-HCl solution at pH 8.8, 2-10mM Mg 2+ The solutions contained dATP, dGTP, and dCTP at final concentrations of 500 μM, dTTP at final concentration of 100 μM, dUTP at final concentration of 400 μM, TV-F upstream primer and TV-R downstream primer at final concentrations of 1.5 pmol / μl, TV-P-FAM probe at 0.75 pmol / μl, β-Actin upstream primer IC-F and β-Actin downstream primer IC-R at final concentrations of 1.5 pmol / μl, and β-Actin-VIC probe IC-P-VIC at a final concentration of 0.5 pmol / μl.

[0012] 7) Trichomonas vaginalis-specific primers and probes:

[0013] TV-F: 5'-cgaatgctctctcaagtttgc-3';

[0014] TV-R: 5'-ccttctcatcatcctcagca-3';

[0015] TV-P-FAM: 5'-FAM-ctttggaacaatcacagccgtcaag-BQ1-3';

[0016] 8) Human genome β-Actin internal standard specific primers and probes:

[0017] IC-F: 5'-tccctggagaagagctacga-3';

[0018] IC-R: 5'-agcactgtgttggcgtacag-3';

[0019] IC-P-VIC: 5'-VIC-cctgtggcatccacgaaactacctt-BQ1-3';

[0020] 9) Second PCR reaction solution: 2 U / μl Taq enzyme, 0.5 U / μl UNG enzyme;

[0021] Furthermore, the kit also includes:

[0022] Positive controls: Positive controls include Trichomonas vaginalis genomic DNA and human genomic DNA, used to monitor the PCR process;

[0023] Negative control: The negative control is human genomic DNA, used to monitor the PCR process.

[0024] The present invention also provides a method for detecting Trichomonas vaginalis using the above-mentioned kit, comprising the following steps:

[0025] 1) Extraction of sample DNA

[0026] (1) Take a 1.5ml centrifuge tube, add 600μl of lysis buffer, 20μl of magnetic beads and 200μl of sample in sequence. The positive control and negative control are treated in the same way. Cover the tube, invert it to mix thoroughly, and let it stand at room temperature for 10min.

[0027] (2) Place the 1.5ml centrifuge tube on the magnetic rack and let it stand for 15 seconds. When the magnetic beads are completely attracted to the magnet, discard the supernatant.

[0028] (3) Add 600 μl of the first washing solution to a 1.5 ml centrifuge tube, invert the tube to mix thoroughly; place it on a magnetic rack and let it stand for 15 s. When the magnetic beads are completely attracted to the magnet, discard the supernatant.

[0029] (4) Add 600 μl of the second washing solution to a 1.5 ml centrifuge tube, invert the tube to mix thoroughly; place it on a magnetic rack and let it stand for 15 s. When the magnetic beads are completely attracted to the magnet, discard the supernatant.

[0030] (5) Let stand at room temperature for 10 minutes;

[0031] (6) Add 40-100 μl of elution buffer to a 1.5 ml centrifuge tube, invert the tube to mix thoroughly; place it on a magnetic rack and let it stand for 30 seconds. When the magnetic beads are completely attracted to the magnet, transfer the elution buffer to a new 1.5 ml centrifuge tube. This is the extracted DNA.

[0032] 2) PCR amplification

[0033] 1) Prepare PCR reaction tubes with the number of samples to be tested plus 2, and add 8 μl of the first PCR reaction solution, 2 μl of the second PCR reaction solution and 10 μl of the extracted DNA sample in sequence;

[0034] 2) Cap the PCR reaction tube, invert it several times to mix, centrifuge for a few seconds, and then place it in a fluorescence PCR instrument. The PCR cycling conditions are: 37℃ for 2 min, 95℃ for 5 min.

[0035] Loop 1:

[0036] 95℃ for 10s, 55℃ for 20s, 72℃ for 20s, 5 cycles;

[0037] Cycle 2: 95℃ for 10s, 60℃ for 45s, 40 cycles;

[0038] Among them, 60℃ for 45s is set to read fluorescence, and the fluorescence channel is selected as TV-FAM, IC-VIC;

[0039] 3) Results Analysis

[0040] After PCR amplification, the results were analyzed using the analysis software of the fluorescence PCR instrument. The presence of Trichomonas vaginalis in the sample was determined by the fluorescence signal value detected by the FAM / VIC channel.

[0041] This invention designs primers and probes based on the diagnostic sequence of the Trichomonas vaginalis ferredoxin gene and the human β-Actin genome sequence, and optimizes PCR reaction conditions to improve detection sensitivity. Amplification results can be observed in real time through fluorescence signal values. This invention's fluorescent PCR detection kit is rigorously designed, simple to operate, highly sensitive, and highly specific, capable of identifying Trichomonas vaginalis infection with objective and accurate result interpretation. Furthermore, the human genomic housekeeping gene β-Actin is incorporated as an internal standard to monitor the PCR detection process. Attached Figure Description

[0042] Figure 1 Example 2 - FAM fluorescence curve of Trichomonas vaginalis primer and probe;

[0043] Figure 2 Example 2 - Fluorescence curve of internal standard primer probe VIC;

[0044] Figure 3 Example 4 - Optimization results of PCR reaction for Trichomonas vaginalis;

[0045] Figure 4 Example 2 - Results of kit specificity test;

[0046] Figure 5 Experimental Example 3 - Results of reagent kit sensitivity test;

[0047] Figure 6 Example 4 - Results of repeatability test of the kit. Detailed implementation method:

[0048] Example 1

[0049] The sequence for detecting Trichomonas vaginalis is as shown in SEQ ID NO.1: Ferredoxin gene sequence.

[0050] Internal standard detection sequence: The human genome β-Actin gene sequence is shown in SEQ ID NO.2.

[0051] Example 2

[0052] Based on Example 1, the following primers and probes were designed for the specific detection of Trichomonas vaginalis and internal standards using the ferredoxin ferredoxin protein from Trichomonas vaginalis and the human genomic β-Actin gene sequence:

[0053] TV-F: 5'-cgaatgctctctcaagtttgc-3'; as shown in SEQ ID NO.3.

[0054] TV-R: 5'-ccttctcatcatcctcagca-3'; as shown in SEQ ID NO.4.

[0055] TV-P-FAM: 5'-FAM-ctttggaacaatcacagccgtcaag-BQ1-3'; as shown in SEQ ID NO.5.

[0056] IC-F: 5'-tccctggagaagagctacga-3'; as shown in SEQ ID NO.6.

[0057] IC-R: 5'-agcactgtgttggcgtacag-3'; as shown in SEQ ID NO.7.

[0058] IC-P-VIC: 5'-VIC-cctgtggcatccacgaaactacctt-BQ1-3'; as shown in SEQ ID NO.8.

[0059] The above primers and probes detected Trichomonas vaginalis samples. Both the FAM and VIC channels corresponding to Trichomonas vaginalis and the internal standard showed significant amplification signals (see appendix). Figure 1 Appendix Figure 2 This method achieves the dual detection purpose of vaginal trichomoniasis and internal standard.

[0060] Example 3

[0061] The composition and structure of the Trichomonas vaginalis fluorescent PCR detection kit are as follows:

[0062] 1) Lysis buffer: A mixed solution containing 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA, and 30% anhydrous ethanol (volume percentage).

[0063] 2) Magnetic beads: A solution containing 20 mg / ml magnetic beads;

[0064] 3) First washing solution: a mixed solution containing 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA, and 50% anhydrous ethanol (volume percentage concentration).

[0065] 4) Second washing solution: a solution with a volume percentage concentration of 70% anhydrous ethanol;

[0066] 5) Eluent: Sterile purified water;

[0067] 6) First PCR reaction solution: 40mM Tris-HCl (pH 8.8), 2-10mM Mg 2+ Concentrations: dATP, dGTP, and dCTP at a final concentration of 500 μM; dTTP at a final concentration of 100 μM; dUTP at a final concentration of 400 μM; TV upstream primer (TV-F) and TV downstream primer (TV-R) at a final concentration of 1.5 pmol / μl; TV-FAM probe (TV-P-FAM) at a final concentration of 0.75 pmol / μl; β-Actin upstream primer (IC-F) and β-Actin downstream primer (IC-R) at a final concentration of 1.5 pmol / μl; and β-Actin-VIC probe (IC-P-VIC) at a final concentration of 0.5 pmol / μl.

[0068] 7) Second PCR reaction solution: 2 U / μl Taq enzyme, 0.5 U / μl UNG enzyme.

[0069] Example 4

[0070] The PCR reaction optimization process for the kit of this invention is as follows:

[0071] Optimization of Trichomonas vaginalis primer and probe concentrations: The final concentration gradients of Trichomonas vaginalis primers in the first PCR reaction solution were set to 0.5 pmol / μl, 1 pmol / μl, 1.5 pmol / μl, and 2 pmol / μl, with corresponding final probe concentration gradients of 0.25 pmol / μl, 0.5 pmol / μl, 0.75 pmol / μl, and 1 pmol / μl. Simultaneous fluorescent PCR amplification was performed using the above four different primer and probe concentration gradients. When the final primer concentrations were 1.5 pmol / μl and 2 pmol / μl, and the final probe concentrations were 0.75 pmol / μl and 1 pmol / μl, the fluorescence signal value of Trichomonas vaginalis was the highest, the amplification curve was the most aesthetically pleasing, and there was no significant difference between the two. Therefore, the optimal primer and probe concentrations for Trichomonas vaginalis were selected as 1.5 pmol / μl for each primer and 0.75 pmol / μl for the probe.

[0072] Mg 2+ Enzyme concentration optimization: setting Mg for Trichomonas vaginalis 2+ The final concentration gradients in the first PCR reaction solution were 2 mM, 4 mM, 6 mM, and 10 mM; using the above four different Mg... 2+Simultaneous fluorescent PCR amplification was performed using PCR reaction solutions with concentration gradients, when Mg 2+ At a concentration of 6 mM, the detection sensitivity and specificity of Trichomonas vaginalis are the highest.

[0073] Optimization of Taq enzyme concentration: The final concentration gradient of Trichomonas vaginalis Taq enzyme in the second PCR reaction solution was set to 1 U / μl, 2 U / μl, and 3 U / μl. Fluorescent PCR amplification was performed simultaneously using the above three different Taq enzyme concentration gradients. When the Taq enzyme concentration was 2 U / μl and 3 U / μl, the fluorescence signal value of Trichomonas vaginalis detection was the highest and the amplification curve was the most aesthetically pleasing. There was no significant difference between the two. Therefore, the optimal Taq enzyme concentration was selected as 2 U / μl.

[0074] Optimization of PCR reaction conditions: After optimization, the PCR reaction conditions are as follows: First PCR reaction solution: 40 mM Tris-HCl (pH 8.8), 6 mM Mg 2+ The final concentrations of dATP, dGTP, and dCTP were 500 μM each; the final concentration of dTTP was 100 μM; the final concentration of dUTP was 400 μM; the final concentrations of TV upstream primer (TV-F) and TV downstream primer (TV-R) were 1.5 pmol / μl each; the final concentration of TV-FAM probe (TV-P-FAM) was 0.75 pmol / μl; the final concentrations of β-Actin upstream primer (IC-F) and β-Actin downstream primer (IC-R) were 1.5 pmol / μl each; and the final concentration of β-Actin-VIC probe (IC-P-VIC) was 0.5 pmol / μl. The second PCR reaction solution consisted of 2 U / μl Taq enzyme and 0.5 U / μl UNG enzyme (see appendix). Figure 3 ).

[0075] Example 5

[0076] 154 vaginal secretion swab samples were collected from women and tested using wet mount microscopy and fluorescent PCR.

[0077] (I) Wet section microscopy

[0078] Mix vaginal secretions with a small amount of saline solution to form a smear, and observe it under a high-power microscope.

[0079] (II) Fluorescent PCR Method

[0080] 1) Extraction of sample DNA

[0081] (1) Take a 1.5ml centrifuge tube, add 600μl of lysis buffer, 20μl of magnetic beads and 200μl of sample (positive control and negative control are processed in the same way), cap the tube, invert it to mix thoroughly, and let it stand at room temperature for 10min.

[0082] (2) Place the 1.5ml centrifuge tube on the magnetic rack and let it stand for 15 seconds. When the magnetic beads are completely attracted to the magnet, discard the supernatant.

[0083] (3) Add 600 μl of the first washing solution to a 1.5 ml centrifuge tube, invert the tube to mix thoroughly; place it on a magnetic rack and let it stand for 15 s. When the magnetic beads are completely attracted to the magnet, discard the supernatant.

[0084] (4) Add 600 μl of the second washing solution to a 1.5 ml centrifuge tube, invert the tube to mix thoroughly; place it on a magnetic rack and let it stand for 15 s. When the magnetic beads are completely attracted to the magnet, discard the supernatant.

[0085] (5) Let stand at room temperature for 10 minutes;

[0086] (6) Add 40-100 μl of elution buffer to a 1.5 ml centrifuge tube, invert the tube to mix thoroughly; place it on a magnetic rack and let it stand for 30 seconds. When the magnetic beads are completely attracted to the magnet, transfer the elution buffer to a new 1.5 ml centrifuge tube. This is the extracted DNA.

[0087] 2) PCR amplification

[0088] (1) Prepare PCR reaction tubes with the number of samples to be tested + 2, and add 8 μl of the first PCR reaction solution, 2 μl of the second PCR reaction solution and 10 μl of the extracted DNA sample in sequence.

[0089] (2) Cap the PCR reaction tube, invert and mix several times, centrifuge for a few seconds, and then place it in a fluorescence PCR instrument. The PCR cycling conditions are: 37℃ for 2 min, 95℃ for 5 min, cycle 1 (95℃ for 10 s, 55℃ for 20 s, 72℃ for 20 s, 5 cycles), cycle 2 (95℃ for 10 s, 60℃ for 45 s, 40 cycles); the 60℃ for 45 s setting is used to read fluorescence (fluorescence channel selected as TV-FAM, IC-VIC).

[0090] Results of wet mount microscopy and fluorescent PCR detection:

[0091] A total of 154 vaginal secretion swab samples were tested using wet mount microscopy and fluorescence PCR. Among them, 22 samples were positive by wet mount microscopy and 46 samples were positive by fluorescence PCR.

[0092] Test Example 1 Kit Composition

[0093] 1) Lysis buffer: a mixed solution of 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA, and 30% anhydrous ethanol;

[0094] 2) Magnetic beads: 20 mg / ml magnetic bead solution;

[0095] 3) First washing solution: a mixed solution of 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA, and 50% anhydrous ethanol;

[0096] 4) Second washing solution: a solution of 70% anhydrous ethanol;

[0097] 5) Eluent: Sterile purified water;

[0098] 6) First PCR reaction solution: 40mM Tris-HCl (pH 8.8), 6mM Mg 2+ Concentrations: dATP, dGTP, and dCTP at a final concentration of 500 μM; dTTP at a final concentration of 100 μM; dUTP at a final concentration of 400 μM; TV upstream primer (TV-F) and TV downstream primer (TV-R) at a final concentration of 1.5 pmol / μl; TV-FAM probe (TV-P-FAM) at a final concentration of 0.75 pmol / μl; β-Actin upstream primer (IC-F) and β-Actin downstream primer (IC-R) at a final concentration of 1.5 pmol / μl; and β-Actin-VIC probe (IC-P-VIC) at a final concentration of 0.5 pmol / μl.

[0099] 7) Second PCR reaction solution: 2 U / μl Taq enzyme, 0.5 U / μl UNG enzyme.

[0100] Test Example 2: Reagent Specificity Test

[0101] This kit is used to amplify and detect samples of Trichomonas vaginalis, Toxoplasma gondii, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, and human genome.

[0102] The results are shown (see appendix). Figure 4 Only Trichomonas vaginalis showed a positive result; other samples showed no amplification signal, meeting the specificity detection criteria.

[0103] Experimental Example 3: Reagent Kit Sensitivity Test

[0104] Take samples of Trichomonas vaginalis determined by digital PCR, and dilute them to sensitivity gradients at concentrations of 50,000, 5,000, 500, and 50 copies / ml. Use this kit for detection, with two replicates for each concentration.

[0105] The test results show (see attached) Figure 5The test results for Trichomonas vaginalis samples at concentrations of 50,000, 5,000, and 500 copies / ml were all positive, indicating that the detection sensitivity of this kit for Trichomonas vaginalis reaches 500 copies / ml.

[0106] Experimental Example 4: Reagent Repeatability Test

[0107] Take vaginal Trichomonas vaginalis samples that have been determined by digital PCR, and dilute them to medium and low concentrations of 5000 and 1500 copies / ml, respectively. Use this kit to detect the samples in 5 replicates.

[0108] The test results show (see attached) Figure 6 The test results for Trichomonas vaginalis samples at concentrations of 5000 and 1500 were positive. The coefficients of variation (CV) of the five repeated tests were 1.07% and 1.15%, respectively, indicating that the kit has good repeatability in the detection of Trichomonas vaginalis. sequence list <110> Shanghai Kehua Bioengineering Co., Ltd. <120> A specific fluorescent PCR detection kit for Trichomonas vaginalis <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 582 <212> DNA <213> Homo sapiens <400> 1 cgatttattg aattttttct ttcttaataa ttatatttaa tataaactct acactattaa 60 aagttaaatg gccgaagata acttgatttg ataaatcaca ttcaattgat tgagctttgt 120 attcaaaata tttacttcac ttctctttag cgaatgctct ctcaagtttg ccgctttgga 180 acaatcacag ccgtcaaggg tggtgtcaag aagcaactca agttcgaaga tgaccagaca 240 ctcttcacag ttcttacaga agccggcctc atgtcagctg atgacacatg ccagggcaac aaggcttgcg gcaagtgcat ctgcaagcac gtttccggca aggtcgctgc tgaggatgat 360 gagaaggaat tcctcgagga tcagccagct aacgctcgcc ttgcttgcgc tatcacactc agtggtgaaa acgatggtgc tgttttcgag ctctaaataa ttgaaagttt attaaattgt ttttgatttt tttccaatac ttagttaca ttcaaaatga atcgctttat tttttgtttt tatgttgtca cagtctatat ttcggatcaa ctgtaataga gg <210> 2 <211> 1812 <212> DNA <213> Homo sapiens <400> 2 accgccgaga ccgcgtccgc cccgcgagca cagagcctcg cctttgccga tccgccgccc gtccacaccc gccgccagct caccatggat gatgatatcg ccgcgctcgt cgtcgacaac ggctccggca tgtgcaaggc cggcttcgcg ggcgacgatg ccccccgggc cgtcttcccc 180 tccatcgtgg ggcgccccag gcaccagggc gtgatggtgg gcatgggtca gaaggattcc 240 tatgtgggcg acgaggccca gagcaagaga ggcatcctca ccctgaagta ccccatcgag 300 cacggcatcg tcaccaactg ggacgacatg gagaaaatct ggcaccacac cttctacaat 360 gagctgcgtg tggctcccga ggagcacccc gtgctgctga ccgaggcccc cctgaacccc 420 aaggccaacc gcgagaagat gacccagatc atgtttgaga ccttcaacac cccagccatg 480 tacgttgcta tccaggctgt gctatccctg tacgctctg gccgtaccac tggcatcgtg 540 atggactccg gtgacggggt caccacact gtgcccatct acgaggggta tgccctcccc 600 catgccatcc tgcgtctgga cctggctggc cgggacctga ctgactacct catgaagatc 660 ctcaccgagc gcggctacag cttcaccacc acggccgagc gggaaatcgt gcgtgacatt 720 aaggaagc tgtgctacgt cgccctggac ttcgagcaag agatggccac ggctgcttcc 780 agctcctccc tggagaagag ctacgagctg cctgacggcc aggtcatcac cattggcaat 840 gagcggttcc gctgccctga ggcactcttc cagccttcct tcctgggcat ggagtcctgt 900 ggcatccacg aaactacctt caactccatc atgaagtgtg acgtggacat ccgcaaagac 960 ctgtacgcca acacagtgct gtctggcggc accaccatgt accctggcat tgccgacagg 1020 atgcagaagg agatcactgc cctggcaccc agcacaatga agatcaagc cattgctcct 1080. cctgagcgca agtactccgt gtggatcggc ggctccatcc tggcctcgct gtccaccttc 1140 cagcagatgt ggatcagcaa gcaggagtat gacgagtccg gccctccat cgtccaccgc aaatgcttct aggcggacta tgacttagtt gcgttacacc ctttcttgac aaaacctaac ttgcgcagaa aacaagatga gattggcatg gctttatttg ttttttttgt tttgttttgg tttttttttt ttttttggct tgactcagga tttaaaaact ggacggtga aggtgacagc agtcggttgg agcgagcatc ccccaaagtt cacaatgtgg ccgaggactt tgattgcaca ttgttgtttt tttaatagtc attccaaata tgagatgcgt tgttacagga agtcccttgc catcctaaaa gccaccccac ttctctctaa ggagaatggc ccagtcctct cccaagtcca cacaggggag gtgatagcat tgctttcgtg taattatgt aatgcaaaat ttttttaatc ttcgccttaa tacttttttta ttttgtttta ttttgaatga tgagccttcg tgccccccct tcccctttt ttgtccccca acttgagatg tatgaaggct tttggtctcc ctgggagtgg 1740. gtggaggcag ccagggctta cctgtacact gacttgagac cagttgaata aaagtgcaca 1800 ccttaaaaat ga 1812 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 cgaatgctct ctcaagtttg c 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 ccttctcatc atcctcagca 20 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <400> 5 ctttggaaca atcacagccg tcaag 25 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tccctggaga agagctacga 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 agcactgtgt tggcgtacag 20 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <400> 8 cctgtggcat ccacgaaact acctt 25

Claims

1. A specific fluorescent PCR detection kit for Trichomonas vaginalis, characterized in that, The following reagents are included: 1) Lysis buffer: A mixed solution containing 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA, and 30% anhydrous ethanol (volume percentage). 2) Magnetic beads: A solution containing 20 mg / ml magnetic beads; 3) First washing solution: a mixed solution containing 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA, and 50% anhydrous ethanol (volume percentage). 4) Second washing solution: 70% anhydrous ethanol solution (by volume); 5) Eluent: Sterile purified water; 6) First PCR reaction solution: 40 mM Tris-HCl solution with pH 8.8, 2-10 mM Mg 2+ solution, final concentration of dATP, dGTP, dCTP is 500 μM respectively, final concentration of dTTP is 100 μM, final concentration of dUTP is 400 μM, final concentration of TV-F upstream primer, TV-R downstream primer is 1.5 pmol / μl respectively, final concentration of TV-P-FAM probe is 0.75 pmol / μl, final concentration of β-Actin upstream primer IC-F, β-Actin downstream primer IC-R is 1.5 pmol / μl respectively, final concentration of β-Actin-VIC probe IC-P-VIC is 0.5 pmol / μl; 7) Trichomonas vaginalis-specific primers and probes: TV-F: 5'-cgaatgctctctcaagtttgc-3'; TV-R: 5'-ccttctcatcatcctcagca-3'; TV-P-FAM: 5'-FAM-ctttggaacaatcacagccgtcaag-BQ1-3'; 8) Human genome β-Actin internal standard specific primers and probes: IC-F: 5'-tccctggagaagagctacga-3'; IC-R: 5'-agcactgtgttggcgtacag-3'; IC-P-VIC: 5'-VIC-cctgtggcatccacgaaactacctt-BQ1-3'; 9) Second PCR reaction solution: 2 U / μl Taq enzyme, 0.5 U / μl UNG enzyme.

2. The specific fluorescent PCR detection kit for Trichomonas vaginalis according to claim 1, characterized in that... Also includes: 1) Positive controls: Positive controls include Trichomonas vaginalis genomic DNA and human genomic DNA, used to monitor the PCR process; 2) Negative control: The negative control is human genomic DNA, used to monitor the PCR process.

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