CN116949196A - Specific trichomonas vaginalis fluorescence PCR detection kit - Google Patents

Abstract

The invention provides a specific trichomonas vaginalis fluorescent PCR detection kit which comprises a lysate, magnetic beads, a first washing liquid, a second washing liquid, an eluent, sterilized purified water, a PCR reaction liquid, trichomonas vaginalis specific primer probes and human genome beta-action internal standard specific primer probes. According to the invention, a primer probe is designed according to the diagnosis sequences of trichomonas vaginalis iron redox protein (Ferredoxin) genes and human genome beta-action genes, and a 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 has the advantages of strict design, simple operation, high sensitivity and strong specificity, can specifically identify trichomonas vaginalis infection, and has objective and accurate result judgment.

Description

Specific trichomonas vaginalis fluorescence PCR detection kit

Technical Field

The invention belongs to the field of biomedical diagnosis, relates to detection of sexually transmitted parasites, in particular to a diagnosis and detection technology of trichomonas vaginalis, and particularly relates to a specific trichomonas vaginalis fluorescent PCR detection kit.

Background

Trichomonas vaginalis is a parasitic protozoa, a major pathogen of non-viral disease (STD) worldwide, mainly causing urogenital diseases in men and women. It is estimated that 4-35% of the vaginitis diagnosed in China is caused by trichomonas vaginalis. After female infection, it is often characterized clinically by increased vaginal secretions, with inflammation and irritation, about 50-80% of infected individuals being asymptomatic. Trichomonas vaginalis infection has been shown to be associated with HIV transmission and control, and may cause pregnancy complications in pregnant women, such as premature birth, premature rupture of membranes, and low body weight infants, and in addition, may increase the incidence of female cancer.

Wet-film microscopy is currently the most commonly used laboratory diagnostic method for trichomonas vaginalis, which is simple, rapid and inexpensive, but has a low sensitivity of only 20-60%. Culture is a gold standard for trichomonas vaginalis diagnosis, but requires several days of culture time, is slow to detect, and is relatively expensive. The immunological method includes enzyme-linked immunosorbent assay (ELISA) method, immunofluorescence assay (IFA) method, etc., but has low sensitivity, and is not generally examined by immunological method in clinic. The molecular fluorescent PCR diagnosis method has the advantages of high sensitivity, good specificity and the like, and has been widely used for pathogen detection.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a specific trichomonas vaginalis fluorescent PCR detection kit, which aims to solve the technical problems of long trichomonas vaginalis detection time and low sensitivity in the method in the prior art.

The invention provides a specific trichomonas vaginalis fluorescence PCR detection kit, which comprises the following reagents:

1) Lysate: a mixed solution containing 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA and 30% absolute ethanol by volume percent concentration;

2) Magnetic beads: a solution containing 20mg/ml magnetic beads;

3) First washing liquid: a mixed solution containing 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA and 50% absolute ethanol by volume percent concentration;

4) And (2) a second washing solution: a solution of 70% by volume absolute ethanol;

5) Eluent: sterilizing and purifying water;

6) First PCR reaction solution: 40mM Tris-HCl solution at pH 8.8, 2-10mM Mg ²⁺ Solutions, 500. Mu.M dATP, dGTP, dCTP, 100. Mu.M dTTP, 400. Mu.M dUTP, 1.5 pmol/. Mu.l TV-F upstream primer, 0.75 pmol/. Mu.l TV-P-FAM probe, 1.5 pmol/. Mu.l beta-action upstream primer IC-F, 1.5 pmol/. Beta-action downstream primer IC-R, and 0.5 pmol/. Mu.l beta-action-VIC probe IC-P-VIC, respectively;

7) Trichomonas vaginalis specific primer probe:

TV-F：5’-cgaatgctctctcaagtttgc-3’；

TV-R：5’-ccttctcatcatcctcagca-3’；

TV-P-FAM：5’-FAM-ctttggaacaatcacagccgtcaag-BQ1-3’；

8) Human genome beta-action internal standard specific primer probe:

IC-F：5’-tccctggagaagagctacga-3’；

IC-R：5’-agcactgtgttggcgtacag-3’；

IC-P-VIC：5’-VIC-cctgtggcatccacgaaactacctt-BQ1-3’；

9) Second PCR reaction solution: 2U/. Mu.l Taq enzyme, 0.5U/. Mu.l UNG enzyme;

further, the kit further comprises:

positive control: positive controls including trichomonas vaginalis genomic DNA and human genomic DNA were used to monitor the PCR process;

negative control: the negative control was human genomic DNA and used to monitor the PCR process.

The invention also provides a method for detecting trichomonas vaginalis by adopting the kit, which comprises the following steps:

1) Extraction of sample DNA

(1) Taking 1.5ml centrifuge tube, sequentially adding 600 μl of lysate, 20 μl of magnetic beads and 200 μl of sample, treating positive control and negative control by the same method, covering the tube cover, reversing upside down, mixing completely, and standing at room temperature for 10min;

(2) Placing 1.5ml centrifuge tube on a magnetic rack, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(3) Adding 600 mu l of first washing liquid into a 1.5ml centrifuge tube, and fully and uniformly mixing the two washing liquids in an upside down manner; placing the magnetic beads on a magnetic frame, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(4) Adding 600 mu l of second washing liquid into a 1.5ml centrifuge tube, and fully and uniformly mixing the two washing liquids in an upside down manner; placing the magnetic beads on a magnetic frame, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(5) Standing at room temperature for 10min;

(6) Adding 40-100 μl of eluent into 1.5ml centrifuge tube, and mixing completely by upside down; placing the magnetic beads on a magnetic rack, standing for 30s, and transferring the eluent to a new 1.5ml centrifuge tube when the magnetic beads are completely adsorbed to the magnet position, thus obtaining the extracted DNA;

2) PCR amplification

1) Preparing a PCR reaction tube with the number of samples to be detected being +2, and sequentially adding 8 mu l of a first PCR reaction liquid, 2 mu l of a second PCR reaction liquid and 10 mu l of extracted DNA samples;

2) Covering a PCR reaction tube cover, mixing the materials for a plurality of times in an upside down way, and placing the materials in a fluorescent PCR instrument after flashing and centrifuging for a plurality of seconds, wherein the PCR circulation conditions are as follows: 2min at 37 ℃ and 5min at 95 ℃,

cycle 1:

95℃for 10s, 5520s, 72℃for 20s,5 cycles;

cycle 2:95 ℃ for 10s, 60 ℃ for 45s and 40 cycles;

wherein, 45s at 60 ℃ is set as reading fluorescence, and a fluorescence channel is selected as TV-FAM and IC-VIC;

3) Analysis of results

After the PCR amplification is finished, analyzing the result by using analysis software of a fluorescence PCR instrument, and judging whether trichomonas vaginalis exists in the sample by detecting a fluorescence signal value of the FAM/VIC channel.

The invention designs the primer and the probe according to the trichomonas vaginalis iron redox protein (ferricoxin) gene diagnosis sequence and the human genome beta-action genome sequence, optimizes the PCR reaction condition to improve the detection sensitivity, and can observe the amplification result through the fluorescent signal value in real time. The fluorescent PCR detection kit has the advantages of strict design, simple operation, high sensitivity and strong specificity, can identify the infection of trichomonas vaginalis, has objective and accurate result judgment, and simultaneously is added with the human genome housekeeping gene beta-action gene as an internal standard to monitor the PCR detection process.

Drawings

Fig. 1: example 2-trichomonas vaginalis primer probe FAM fluorescence plot;

fig. 2: example 2-internal reference probe VIC fluorescence profile;

fig. 3: example 4-trichomonas vaginalis PCR reaction optimization results;

fig. 4: test example 2-kit specificity test results;

fig. 5: test example 3-sensitivity test results of kit;

fig. 6: test example 4-test kit reproducibility test results.

The specific embodiment is as follows:

example 1

Trichomonas vaginalis detection sequence: the gene sequence of the iron oxidation-reduction protein (ferrioxan) is shown as SEQ ID NO. 1;

internal standard detection sequence: the human genome beta-action gene sequence is shown as SEQ ID NO. 2.

Example 2

Trichomonas vaginalis and internal standard specific detection primer probes were designed according to the trichomonas vaginalis iron redox protein (ferrooxin) and human genome β -action gene sequences as follows:

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

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

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

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

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

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

The primer probe detects the trichomonas vaginalis sample, and the FAM and VIC channels corresponding to the trichomonas vaginalis and the internal standard have obvious amplification signals (see figure 1 and figure 2), so that the double detection purpose of the trichomonas vaginalis and the internal standard is realized.

Example 3

The composition structure of the trichomonas vaginalis fluorescence PCR detection kit is as follows:

1) Lysate: a mixed solution containing 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA and 30% absolute ethyl alcohol by volume percent concentration;

2) Magnetic beads: a solution containing 20mg/ml magnetic beads;

3) First washing liquid: a mixed solution containing 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA and 50% absolute ethanol by volume percent concentration;

4) And (2) a second washing solution: a solution of 70% absolute ethanol by volume;

5) Eluent: sterilizing and purifying water;

6) First PCR reaction solution: tris-HCl (pH 8.8) concentration of 40mM, mg of 2-10mM ²⁺ A concentration of dATP, dGTP, dCTP, dTTP, dUTP, and beta-Actin-VIC in concentrations of 500. Mu.M, 100. Mu.M, 1.5 pmol/. Mu.l each, and 0.75 pmol/. Mu.l each of the TV upstream primer (TV-F), the TV downstream primer (TV-R), the TV-FAM probe (TV-P-FAM), 1.5 pmol/. Mu.l each of the beta-Actin upstream primer (IC-F), the beta-Actin downstream primer (IC-R), and 0.5 pmol/. Mu.l each of the beta-Actin-VIC probe (IC-P-VIC);

7) Second PCR reaction solution: 2U/. Mu.l Taq enzyme, 0.5U/. Mu.l UNG enzyme.

Example 4

The PCR reaction optimization process of the kit comprises the following steps:

optimization of trichomonas vaginalis primer probe concentration: setting the final concentration gradient of trichomonas vaginalis primer in the first PCR reaction liquid to be 0.5 pmol/mu l, 1 pmol/mu l, 1.5 pmol/mu l and 2 pmol/mu l, and setting the corresponding final concentration gradient of the probe to be 0.25 pmol/mu l, 0.5 pmol/mu l, 0.75 pmol/mu l and 1 pmol/mu l; the PCR reaction liquid with the concentration gradients of the 4 different primer probes is used for synchronously carrying out fluorescent PCR amplification, when the final concentration of the primers is 1.5 pmol/mu l, 2 pmol/mu l and the final concentration of the probe is 0.75 pmol/mu l, the fluorescence signal value of trichomonas vaginalis is the highest, and the amplification curves are the most attractive, and the two are not obvious, so that the optimal primer probe concentration of trichomonas vaginalis is 1.5 pmol/mu l and the probe is 0.75 pmol/mu l;

Mg ²⁺ optimization of enzyme concentration: setting trichomonas vaginalis Mg ²⁺ The final concentration gradient in the first PCR reaction was 2mM, 4mM, 6mM, 10mM; using the above 4 different Mg ²⁺ Fluorescence PCR amplification is synchronously carried out on the PCR reaction liquid with concentration gradient, and when Mg ²⁺ At a concentration of 6mM, trichomonas vaginalis detection sensitivity is highest, and specificity is strongest;

optimization of Taq enzyme concentration: setting the final concentration gradient of the trichomonas vaginalis Taq enzyme in the second PCR reaction liquid to be 1U/mu l, 2U/mu l and 3U/mu l; the PCR reaction solutions with the 3 different Taq enzyme concentration gradients are used for synchronously carrying out fluorescence PCR amplification, when the Taq enzyme concentration is 2U/mu l and 3U/mu l, the detection fluorescence signal value of trichomonas vaginalis is highest, the amplification curve is beautiful, and the amplification curves are not obviously different, so that the optimal Taq enzyme concentration is 2U/mu l.

Optimization of PCR reaction conditions: the PCR reaction conditions are optimized as the first PCR reaction liquid: tris-HCl (pH 8.8) concentration of 40mM, mg of 6mM ²⁺ A concentration of dATP, dGTP, dCTP, dTTP, dUTP, and beta-Actin-VIC in concentrations of 500. Mu.M, 100. Mu.M, 1.5 pmol/. Mu.l each, and 0.75 pmol/. Mu.l each of the TV upstream primer (TV-F), the TV downstream primer (TV-R), the TV-FAM probe (TV-P-FAM), 1.5 pmol/. Mu.l each of the beta-Actin upstream primer (IC-F), the beta-Actin downstream primer (IC-R), and 0.5 pmol/. Mu.l each of the beta-Actin-VIC probe (IC-P-VIC); second PCR reaction solution: 2U/. Mu.l of Taq enzyme, 0.5U/. Mu.l of UNG enzyme (see FIG. 3).

Example 5

154 samples of female vaginal secretion swab were collected and tested by wet-chip microscopy and fluorescent PCR detection, respectively.

Wet film microscopic examination method

Vaginal secretions were smeared with a small amount of physiological saline and observed under high magnification.

(II) fluorescent PCR method

1) Extraction of sample DNA

(1) Taking a 1.5ml centrifuge tube, sequentially adding 600 μl of lysate, 20 μl of magnetic beads and 200 μl of sample (positive control and negative control are treated by the same method), covering a tube cover, reversing upside down, mixing completely, and standing at room temperature for 10min;

(2) Placing 1.5ml centrifuge tube on a magnetic rack, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(3) Adding 600 mu l of first washing liquid into a 1.5ml centrifuge tube, and fully and uniformly mixing the two washing liquids in an upside down manner; placing the magnetic beads on a magnetic frame, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(4) Adding 600 mu l of second washing liquid into a 1.5ml centrifuge tube, and fully and uniformly mixing the two washing liquids in an upside down manner; placing the magnetic beads on a magnetic frame, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(5) Standing at room temperature for 10min;

(6) Adding 40-100 μl of eluent into 1.5ml centrifuge tube, and mixing completely by upside down; placing the magnetic beads on a magnetic rack, standing for 30s, and transferring the eluent to a new 1.5ml centrifuge tube when the magnetic beads are completely adsorbed to the magnet position, thus obtaining the extracted DNA;

2) PCR amplification

(1) Preparing a PCR reaction tube with the number of samples to be detected being +2, and sequentially adding 8 mu l of a first PCR reaction liquid, 2 mu l of a second PCR reaction liquid and 10 mu l of extracted DNA samples;

(2) Covering a PCR reaction tube cover, mixing the materials for a plurality of times in an upside down way, and placing the materials in a fluorescent PCR instrument after flashing and centrifuging for a plurality of seconds, wherein the PCR circulation conditions are as follows: cycle 1 (95 ℃ 10s, 5520s, 72 ℃ 20s,5 cycles), cycle 2 (95 ℃ 10s, 60 ℃ 45s,40 cycles) 2min at 37 ℃ 2min, 95 ℃ 5 min; wherein 45s at 60℃is set as the read fluorescence (fluorescence channel is selected as TV-FAM, IC-VIC)

Wet-chip microscopy and fluorescent PCR detection results:

the wet-chip microscopic examination method and the fluorescent PCR method are used for detecting 154 vaginal secretion swab samples, wherein 22 positive samples are detected by the wet-chip microscopic examination method, and 46 positive samples are detected by the fluorescent PCR method.

Test example 1 kit composition

1) Lysate: mixed solution of 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA and 30% absolute ethanol;

2) Magnetic beads: 20mg/ml of magnetic bead solution;

3) First washing liquid: 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA, 50% absolute ethanol;

4) And (2) a second washing solution: a solution of 70% absolute ethanol;

5) Eluent: sterilizing and purifying water;

6) First PCR reaction solution: tris-HCl (pH 8.8) concentration of 40mM, mg of 6mM ²⁺ A concentration of dATP, dGTP, dCTP, dTTP, dUTP, and beta-Actin-VIC in concentrations of 500. Mu.M, 100. Mu.M, 1.5 pmol/. Mu.l each, and 0.75 pmol/. Mu.l each of the TV upstream primer (TV-F), the TV downstream primer (TV-R), the TV-FAM probe (TV-P-FAM), 1.5 pmol/. Mu.l each of the beta-Actin upstream primer (IC-F), the beta-Actin downstream primer (IC-R), and 0.5 pmol/. Mu.l each of the beta-Actin-VIC probe (IC-P-VIC);

7) Second PCR reaction solution: 2U/. Mu.l Taq enzyme, 0.5U/. Mu.l UNG enzyme.

Test example 2 kit specificity test

Samples of trichomonas vaginalis, toxoplasma, chlamydia trachomatis, neisseria gonorrhoeae, ureaplasma urealyticum and human genome are taken, and amplified and detected by using the kit.

The results show (see fig. 4): only trichomonas vaginalis presents a positive result, other samples have no amplified signal, and the specificity detection standard is met.

Test example 3 sensitivity test of kit

Samples of trichomonas vaginalis subjected to digital PCR (polymerase chain reaction) are taken and respectively diluted to sensitivity gradient samples with the concentrations of 50000, 5000, 500 and 50copies/ml, and the samples are used for detection, wherein each concentration is repeated for 2 duplicate wells.

The detection result shows (see fig. 5): the detection of trichomonas vaginalis samples with the concentration of 50000, 5000 and 500copies/ml is positive, which indicates that the detection sensitivity of the kit to trichomonas vaginalis reaches 500copies/ml.

Test example 4 test kit repeatability test

The trichomonas vaginalis samples with digital PCR fixed values are respectively diluted to medium and low concentration samples with the concentration of 5000 and 1500copies/ml, and 5 multiple holes are detected by using the kit.

The detection result shows (see fig. 6): the trichomonas vaginalis samples with the concentration of 5000 and 1500 are positive in detection, and the variation coefficient CV values of 5 repeated detection results are respectively 1.07% and 1.15%, which shows that the kit has good repeatability in detecting trichomonas vaginalis.

Sequence listing

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<120> a specific trichomonas vaginalis fluorescent PCR detection kit

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ggctccggca tgtgcaaggc cggcttcgcg ggcgacgatg ccccccgggc cgtcttcccc 180

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Claims (3)

1. A specific trichomonas vaginalis fluorescent PCR detection kit, which is characterized by comprising the following reagents:

1) Lysate: a mixed solution containing 4M guanidine isothiocyanate, 20mM NaCl, 10mM EDTA and 30% absolute ethanol by volume percent concentration;

2) Magnetic beads: a solution containing 20mg/ml magnetic beads;

3) First washing liquid: a mixed solution containing 1.5M guanidine isothiocyanate, 10mM NaCl, 2mM EDTA and 50% absolute ethanol by volume percent concentration;

4) And (2) a second washing solution: an absolute ethanol solution with the volume percentage concentration of 70%;

5) Eluent: sterilizing and purifying water;

6) First PCR reaction solution: 40mM Tris-HCl solution at pH 8.8, 2-10mM Mg ²⁺ Solutions, 500. Mu.M dATP, dGTP, dCTP, 100. Mu.M dTTP, 400. Mu.M dUTP, 1.5 pmol/. Mu.l TV-F upstream primer, 0.75 pmol/. Mu.l TV-P-FAM probe, 1.5 pmol/. Mu.l beta-action upstream primer IC-F, 1.5 pmol/. Beta-action downstream primer IC-R, and 0.5 pmol/. Mu.l beta-action-VIC probe IC-P-VIC, respectively;

7) Trichomonas vaginalis specific primer probe:

TV-F：5’-cgaatgctctctcaagtttgc-3’；

TV-R：5’-ccttctcatcatcctcagca-3’；

TV-P-FAM：5’-FAM-ctttggaacaatcacagccgtcaag-BQ1-3’；

8) Human genome beta-action internal standard specific primer probe:

IC-F：5’-tccctggagaagagctacga-3’；

IC-R：5’-agcactgtgttggcgtacag-3’；

IC-P-VIC：5’-VIC-cctgtggcatccacgaaactacctt-BQ1-3’；

9) Second PCR reaction solution: 2U/. Mu.l Taq enzyme, 0.5U/. Mu.l UNG enzyme.

2. A specific trichomonas vaginalis fluorescent PCR assay kit according to claim 1, further comprising:

1) Positive control: positive controls including trichomonas vaginalis genomic DNA and human genomic DNA were used to monitor the PCR process;

2) Negative control: the negative control was human genomic DNA and used to monitor the PCR process.

3. A method for detecting trichomonas vaginalis using the kit of claim 1, comprising the steps of:

1) Extraction of sample DNA

(1) Taking 1.5ml centrifuge tube, sequentially adding 600 μl of lysate, 20 μl of magnetic beads and 200 μl of sample, treating positive control and negative control by the same method, covering tube cover, reversing upside down, mixing completely,

(2) Standing at room temperature for 10min;

(3) Placing 1.5ml centrifuge tube on a magnetic rack, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(4) Adding 600 mu l of first washing liquid into a 1.5ml centrifuge tube, and fully and uniformly mixing the two washing liquids in an upside down manner; placing the magnetic beads on a magnetic frame, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(5) Adding 600 mu l of second washing liquid into a 1.5ml centrifuge tube, and fully and uniformly mixing the two washing liquids in an upside down manner; placing the magnetic beads on a magnetic frame, standing for 15s, and discarding the supernatant when the magnetic beads are completely adsorbed to the magnet position;

(6) Standing at room temperature for 10min;

(7) Adding 40-100 μl of eluent into 1.5ml centrifuge tube, and mixing completely by upside down; placing the magnetic beads on a magnetic rack, standing for 30s, and transferring the eluent to a new 1.5ml centrifuge tube when the magnetic beads are completely adsorbed to the magnet position, thus obtaining the extracted DNA;

2) PCR amplification

(1) Preparing a PCR reaction tube with the number of samples to be detected being +2, and sequentially adding 8 mu l of a first PCR reaction liquid, 2 mu l of a second PCR reaction liquid and 10 mu l of extracted DNA samples;

(2) Covering a PCR reaction tube cover, mixing the materials for a plurality of times in an upside down way, and placing the materials in a fluorescent PCR instrument after flashing and centrifuging for a plurality of seconds, wherein the PCR circulation conditions are as follows: 2min at 37 ℃ and 5min at 95 ℃,

cycle 1:

95℃for 10s, 5520s, 72℃for 20s,5 cycles;

cycle 2:95 ℃ for 10s, 60 ℃ for 45s and 40 cycles;

wherein, 45s at 60 ℃ is set as reading fluorescence, and a fluorescence channel is selected as TV-FAM and IC-VIC;

3) Analysis of results

After the PCR amplification is finished, analyzing the result by using analysis software of a fluorescence PCR instrument, and judging whether trichomonas vaginalis exists in the sample by detecting a fluorescence signal value of the FAM/VIC channel.