WO2022228187A1 - Kit for detecting ureaplasma urealyticum, chlamydia trachomatis and neisseria gonorrhoeae and method therefor - Google Patents

Abstract

The present invention belongs to the field of molecular biology detection, and specifically relates to a kit for detecting Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae and and a method therefor. The present invention designs primer and probe sequences, obtains a new primer pair, and provides a kit comprising the primer pair. A method for using said reagents or a kit for PCR detection comprises the following steps: under the action of a primer pair, performing PCR amplification on a sample to be detected, and then performing detection. The present invention also optimizes detection conditions such as an annealing condition and the primer addition amount. The method of the present invention has excellent specificity, sensitivity and precision, and has good application prospects in the clinical detection of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae.

Description

A kind of kit and method for detection of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae technical field

The invention belongs to the field of molecular biology detection, and in particular relates to a kit and method for detection of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae.

Background technique

In recent years, the incidence of sexually transmitted diseases has increased year by year, and according to the World Health Organization, an estimated 500 million people worldwide suffer from syphilis, gonorrhea, chlamydia or trichomoniasis each year. Sexually transmitted diseases adversely affect the public health of individuals, communities and nations. Among the non-viral causes of STD infections, Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (NG) are common pathogens that cause STDs. These pathogenic bacteria cause a variety of clinical syndromes, such as urethritis, cervicitis, prostatitis, and vaginitis, which can lead to serious complications and long-term sequelae, including pelvic inflammatory disease, infertility, chronic pelvic pain, ectopic pregnancy, adult Neurological and cardiovascular disease, premature birth, neonatal death, severe disability or blindness in infants.

At present, the laboratory detection methods of Chlamydia trachomatis, Ureaplasma urealyticum and Neisseria gonorrhoeae mainly include isolation and culture methods, immunological methods, and molecular biological methods based on nucleic acid detection. The specificity and sensitivity of the culture method are high, but it has the disadvantages of long clinical detection time (at least 24h to 48h, or even longer), cumbersome process, the need to use special culture medium, and being easily affected by miscellaneous bacteria. Suitable for large-scale detection. Immunological methods are simple and quick, but have poor specificity and low sensitivity.

In view of the time-consuming and cumbersome operations of traditional isolation and culture, in recent years, the polymerase chain reaction (PCR) method has gradually shown its advantages in detection. Fluorescent PCR technology is a more sensitive, specific and accurate nucleic acid detection technology developed based on traditional PCR technology combined with spectral technology. , and the whole process avoids the problem of post-processing in traditional PCR and reduces pollution.

At present, some kits based on real-time fluorescent quantitative PCR technology for the detection of Chlamydia trachomatis, Ureaplasma urealyticum or Neisseria gonorrhoeae DNA have been put on the market, but there are generally problems of poor specificity and low sensitivity, and most of them are directed against Chlamydia trachomatis, Ureaplasma urealyticum or Neisseria gonorrhoeae. A single detection kit for Mycoplasma or Neisseria gonorrhoeae has a particularly poor detection effect on the mixture of multiple pathogenic microorganisms, and the processing process is cumbersome. However, since the mixed infection of STDs, that is, the proportion of patients infected with two or more STDs at the same time, is relatively high, establishing a method for simultaneous detection of the above pathogens can improve the efficiency of clinical diagnosis, which is of great significance.

Aiming at the mixed infection of a variety of venereal pathogens, it has been reported in the literature that based on PCR amplification, electrophoresis of different product lengths is performed to distinguish different venereal pathogens. However, since electrophoresis is required after PCR amplification, this process is not only time-consuming It also increases the risk of laboratory contamination from capping operations.

The Chinese invention patent application "CN202010329650.6 Primer set, product and application for triple detection of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum" discloses a triple detection primer set, which can be used for gonococcus, Chlamydia trachomatis and Ureaplasma urealyticum three Simultaneous detection of pathogens. However, the primers provided in this patent application have insufficient specificity for Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum, for example, the Chlamydia trachomatis primers can amplify the Chlamydia pneumoniae genome, resulting in insufficient detection accuracy.

SUMMARY OF THE INVENTION

In view of the defects of the prior art, the present invention provides a kit and method for the detection of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae, the purpose is to: improve the primers for the detection of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae to improve their specificity , thereby increasing the detection accuracy.

A detection kit, comprising at least one of a primer pair for detecting Ureaplasma urealyticum, a primer pair for detecting Chlamydia trachomatis, and a primer pair for detecting Neisseria gonorrhoeae; the primer pair includes a forward primer and a reverse primer. to primer;

The primer pair for detecting Ureaplasma urealyticum is a nucleotide sequence as described in SEQ ID NO.1 and SEQ ID NO.4 or a nucleotide sequence having 95% homology with the aforementioned sequence;

The primer pair for detecting Chlamydia trachomatis is a nucleotide sequence as described in SEQ ID NO.2 and SEQ ID NO.5 or a nucleotide sequence having 95% homology with the aforementioned sequence;

The primer pair for detecting Neisseria gonorrhoeae is the nucleotide sequence described in SEQ ID NO.3 and SEQ ID NO.6 or the nucleotide sequence having 95% homology with the aforementioned sequence.

Preferably, the molar ratio of the amount of the forward primer and the reverse primer for detecting Ureaplasma urealyticum is 1-3:1-3;

And/or, the consumption molar ratio of the described forward primer and reverse primer for detecting Chlamydia trachomatis is 1-3:1-3;

And/or, the described consumption mol ratio of the forward primer for detecting gonococcus and the reverse primer is 1-3:1-3;

And/or, the consumption molar ratio of the forward primer for detecting Ureaplasma urealyticum, the forward primer for detecting Chlamydia trachomatis, and the forward primer for detecting Neisseria gonorrhoeae is 1-3:1-3:1 -3.

Preferably, the molar ratio of the amount of the forward primer for detecting Ureaplasma urealyticum and the reverse primer is 1:1;

And/or, the consumption mol ratio of the described forward primer and reverse primer for detecting Chlamydia trachomatis is 1:1;

And/or, the described consumption mol ratio of the forward primer for detecting gonococcus and the reverse primer is 1:1;

And/or, the molar ratio of the amount of the forward primer for detecting Ureaplasma urealyticum, the forward primer for detecting Chlamydia trachomatis, and the forward primer for detecting Neisseria gonorrhoeae is 1:1:1.

Preferably, the kit further comprises a probe set comprising at least one of a probe for detecting Ureaplasma urealyticum, a probe for detecting Chlamydia trachomatis, and a probe for detecting Neisseria gonorrhoeae;

The probe for detecting Ureaplasma urealyticum has the nucleotide sequence as described in SEQ ID NO.7, or a nucleotide sequence having at least 95% homology with the aforementioned nucleotide sequence;

The probe for detecting Chlamydia trachomatis has the nucleotide sequence as described in SEQ ID NO. 8, or a nucleotide sequence having at least 95% homology with the aforementioned nucleotide sequence;

The probe for detecting Neisseria gonorrhoeae has a nucleotide sequence as described in SEQ ID NO. 9, or a nucleotide sequence having at least 95% homology with the aforementioned nucleotide sequence.

Preferably, the probes in the probe set independently contain different fluorescent reporter groups and fluorescent quenching groups.

Preferably, the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3 or Dabcyl; the fluorescent reporter group is selected from FAM, HEX, ROX, CY5, VIC, TET or TAMRA.

Preferably, the molar ratio of the forward primer, reverse primer and probe for detecting Ureaplasma urealyticum is 1-3:1-3:1-4;

And/or, the consumption molar ratio of the forward primer, reverse primer and probe for detecting Chlamydia trachomatis is 1-3:1-3:1-4;

And/or, the consumption molar ratio of described forward primer, reverse primer and probe for detecting gonococcus is 1-3:1-3:1-4;

And/or, the molar ratio of the probe for detecting Ureaplasma urealyticum, the probe for detecting Chlamydia trachomatis, and the probe for detecting Neisseria gonorrhoeae is 1-3:1-3:1-4.

Preferably, the molar ratio of the forward primer, reverse primer and probe for detecting Ureaplasma urealyticum is 1:1:1;

And/or, the consumption molar ratio of described forward primer, reverse primer and probe for detecting Chlamydia trachomatis is 1:1:1;

And/or, the consumption molar ratio of described forward primer, reverse primer and probe for detecting gonococcus is 1:1:1;

And/or, the molar ratio of the probes for detecting Ureaplasma urealyticum, the probes for detecting Chlamydia trachomatis, and the probes for detecting Neisseria gonorrhoeae is 1:1:1.

The present invention provides a PCR detection method for Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae, comprising the following steps: using the above-mentioned kit to perform PCR amplification on a sample to be detected, and then detecting. The specific method of the detection can be selected from fluorescent probe detection or electrophoresis detection, preferably fluorescent probe detection.

Preferably, the PCR reaction system of the PCR amplification includes the following components:

PCR reaction mix 10-20μL;

DNF Buffer 2-5μL;

5.4 μL of the above primer pair solution;

Pathogen DNA 1-4 μL;

Water to make up the total volume of the PCR reaction system to 30 μL;

Wherein the solution of the primer pair includes the following components:

2×10 ^-12 -6×10 ^-12 mol of forward primer for detecting Ureaplasma urealyticum, preferably 6×10 ^-12 mol;

2×10 ^-12 -6×10 ^-12 mol of reverse primer for detecting Ureaplasma urealyticum, preferably 6×10 ^-12 mol;

The forward primer for detecting Chlamydia trachomatis is 2× ^{10-12-6×10-12 mol, preferably 6×10-12 mol ;}

The reverse primer for detecting Chlamydia trachomatis is 2× ^{10-12-6×10-12 mol, preferably 6×10-12 mol ;}

2× ^{10-12-6×10-12 mol of forward primer for detecting gonococcus, preferably 6×10-12 mol ;}

The reverse primer for detecting Neisseria gonorrhoeae is 2×10 ^-12 -6×10 ^-12 mol, preferably 6×10 ^-12 mol.

Preferably, the detection method is a fluorescence detection method;

The solution of the primer pair also includes the following components:

2× ^{10-12-6×10-12 mol of probe for detecting Ureaplasma urealyticum, preferably 6×10-12 mol ;}

2× ^{10-12-6×10-12 mol of probes for detecting Chlamydia trachomatis, preferably 6×10-12 mol ;}

The probe for detecting gonococcus is 2×10 ^-12 -6×10 ^-12 mol, preferably 6×10 ^-12 mol.

Preferably, the PCR amplification procedure is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 5s, annealing at 56°C for 15s, and extension at 72°C for 30s.

The "PCR reaction mix" used in the present invention refers to the PCR reaction solution in the prior art, which contains dNTP, polymerase required for the reaction, etc., and has been commercialized. For example, 2xTaqMan Fast qPCR Master Mix contains 25mM _MgCl2 , 25mM dNTPs, 100mM dUTP, Taq enzyme and UDG enzyme. The "DNF Buffer" used is a buffer solution, which has been commercialized. For example, in one embodiment the composition of DNF Buffer is 25 mM MgCl ₂ , 25 mM dNTPs, 100 mM dUTP, Taq enzyme and UDG enzyme.

The kit provided by the invention can achieve the purpose of simultaneously detecting three venereal disease pathogens of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae. And compared with the prior art, the redesigned amplification primers and probes of the present invention have better specificity, sensitivity and precision. Under preferred conditions, the kit and the detection method of the present invention are negative for other pathogens, and the detection limit for three venereal pathogens of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae is as low as 500 copies/ml, and the intra-batch precision is as low as 500 copies/ml. The coefficient of variation (CV,%) of Ct value is less than or equal to 5%, and the repeatability is good. This shows that the kit and the detection method provided by the present invention are very suitable for clinical detection.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 is the schematic flow chart of detection method;

Fig. 2 is the multiplex fluorescent PCR detection result of embodiment 1;

Fig. 3 is the result of the optimization of annealing temperature in Experimental Example 1;

Fig. 4 is the result of optimizing the amount of primers added in Experimental Example 2;

Fig. 5 is the specificity comparison result of the primer pair used to detect Ureaplasma urealyticum in the application and in the comparative document;

The specificity comparison result of the primer pair used in the detection of Chlamydia trachomatis in Fig. 6 in this application and in the comparative document;

The specificity comparison result of the primer pair used for detecting gonococcus in Fig. 7 in the present application and in the comparative document;

Fig. 8 is the specificity evaluation result of Experimental Example 4;

Fig. 9 is the detection limit evaluation result of Experimental Example 5;

Figure 10 shows the sensitivity and specificity evaluation results of Experimental Example 6;

FIG. 11 shows the precision evaluation results of Experimental Example 7. FIG.

Detailed ways

The reagents and materials used in the following examples and experimental examples include:

The amplification primers and detection probes included in the kit were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

The DNA templates (pathogen DNA) of three venereal disease pathogens of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae, extracted from patients;

2xTaqMan Fast qPCR Master Mix, purchased from Sangon Shanghai;

DNF buffer, purchased from Sangon Shanghai;

Water, Sterilized _ddH2O .

Example 1 Simultaneous detection of three STD pathogens: Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)

The detection process of this embodiment is shown in Figure 1, and the details are as follows:

1. Design of amplification primers and detection probes

The primer pairs and probes of this example are shown in Table 1:

Table 1 Primer pairs and probe sequences

2. Test sample collection (nucleic acid extraction)

Collect genital tract secretion specimens for detection of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae according to the methods of the prior art, eluate the collected specimens with sterile saline, and extract bacterial nucleic acids with reference to the instructions for bacterial genome extraction. Nucleic acid extraction is used for subsequent detection.

3. Preparation of reaction solution

Prepare PCR reaction solution according to the following table:

Table 2 PCR reaction solution

Element	Volume (μL)
2×TaqMan Fast qPCR Mix	15
Forward primer (10μmol/L)	Three kinds of 0.6 each
Reverse primer (10μmol/L)	Three kinds of 0.6 each

Probe (10μmol/L)	Three kinds of 0.6 each
DNF Buffer	3
water	3
Pathogen DNA	3

Prepare the negative control substance reaction solution according to the following table:

Table 3 negative control substance reaction solution

Element	Volume (μL)
2×TaqMan Fast qPCR Mix	15
Forward primer (10μmol/L)	Three kinds of 0.6 each
Reverse primer (10μmol/L)	Three kinds of 0.6 each
Probe (10μmol/L)	Three kinds of 0.6 each
DNF Buffer	3
water	6

4. Multiplex fluorescent PCR detection

After mixing the detection sample with the reaction solution, PCR amplification was carried out. The PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 5s, annealing at 56°C for 15s, and extension at 72°C for 30s. Fluorescence signals were collected simultaneously under the HEX, ROX and CY5 channels for 50 cycles. According to the fluorescence intensity detected by the three channels, it is judged whether the detected sample is negative or positive for the three STD pathogens.

For the positive detection samples containing three STD pathogens, the results of multiplex fluorescence PCR are shown in Figure 2. The detection probe of UU is modified with -FAM fluorescent reporter group, and the amplification curve is green; the detection probe of CT is -ROX fluorescence The reporter group was modified, and the amplification curve was orange; the detection probe of NG was modified with -Cy5 fluorescent reporter group, and the amplification curve was purple. The amplification results showed that there was no mutual interference during the amplification of the nucleic acids of the three pathogenic bacteria. Or inhibition, all have good amplification, indicating that the specificity of the primers and probes of the present invention is very good.

Experimental Example 1 Annealing Temperature Optimization

Annealing temperature is an important factor affecting the specificity of PCR reaction. If the annealing temperature is too low, it may cause non-specific amplification and reduce the specific amplification efficiency. If the annealing temperature is too high, it will affect the binding of the primer and the template and reduce the PCR amplification efficiency. Since there are more than one pair of primers in multiplex fluorescent PCR, the correct control of the annealing temperature is even more important.

In this experimental example, the same positive samples were detected according to the method of Example 1, the difference was that the annealing temperature of the multiplex fluorescence PCR was set to 55.0, 56.0, 58.0, 60.9, 64.5, 67.5, 69.2, 70.0 °C in sequence, and the results are shown in Figure 3. Show. The optimum annealing temperature was selected as 56.0°C.

Experimental Example 2 Optimization of the addition amount of amplification primers

In this experimental example, the same positive samples were detected according to the method in Example 1. The principle of adding equal amounts of each primer pair was the principle. The results are shown in Figure 4. The optimal amount to be added was 0.6 μL of each primer.

Experimental Example 3 Specificity test of primers and probes

This experimental example examines the specificity of the primers disclosed in Example 1 and the Chinese invention patent application "CN202010329650.6 Triple Detection Primer Set, Product and Application of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum". Compare with the database to understand its amplification product and Tm value.

Figure 5 shows the specific comparison results of the primer pairs used in the detection of Ureaplasma urealyticum in Example 1 and the comparative documents. It can be seen from the results that the amplification products obtained by the primers used in this application are all Ureaplasma urealyticum genes. , there are no other non-purpose species. The primers used in the comparison document can not only amplify part of Ureaplasma urealyticum but also can amplify other products, such as Ureaplasma parvum.

Figure 6 shows the specific comparison results of the primer pairs used in the detection of Chlamydia trachomatis in Example 1 and the comparative documents. It can be seen from the results that the amplification products obtained by the primers used in this application are all Chlamydia trachomatis genes, not Other non-purpose species exist. The primers used in the comparison document can not only amplify part of Chlamydia trachomatis but also other products, such as Chlamydia pneumoniae.

Figure 7 shows the specific comparison results of the primer pairs used in the detection of Neisseria gonorrhoeae in Example 1 and the comparative documents. It can be seen from the results that the amplification products obtained by the primers used in this application are all Neisseria gonorrhoeae genes. Other non-purpose species exist. The primers used in the comparison document can not only amplify part of gonococcus, but also can amplify other products, such as Neisseria meningitidis and Neisseria meningitidis.

The above results show that the specificity of the three primer pairs used in this application is higher than that of the reference document. In addition, the Tm value of the primers used in Example 1 is close to 60°C, which is higher than the Tm value of the primers used in the comparison document, which also shows that the specificity of the primers in Example 1 is higher than that in the comparison document.

Experimental Example 4 Evaluation of Response Specificity

In order to determine the specificity of the established multiplex fluorescence PCR method, the method was detected according to the method of Example 1. The results showed that: the detection of the three mixed pathogenic bacteria genomes of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae as templates all showed the target amplification curve, Other non-target pathogens (herpes simplex virus type 2, human papilloma virus type 6, human papilloma virus type 16) were all negative, indicating that the method has good specificity (Figure 8).

Experimental Example 5 Evaluation of detection limit

Dilute the positive quality control substances of the three pathogenic bacteria Ureaplasma trachomatis, Chlamydia trachomatis and Neisseria gonorrhoeae to the concentration of 50copies/ml, 500copies/ml and 5000copies/ml, and then perform detection according to the conditions of Example 1. The results are shown in Figure 9. The nucleic acid concentrations of the three pathogenic bacteria UU, CT and NG were in the range of 5×10 ² to ⁵ ×10 ⁴ copies/mL. /ml, which shows that the multiplex fluorescence PCR method is very sensitive and meets the clinical detection standard.

Experimental Example 6 Evaluation of Diagnostic Sensitivity and Specificity

The diagnostic sensitivity and specificity were evaluated in this experimental case, 30 negative clinical specimens and 30 positive specimens were collected. Among the positive specimens, 23 were single infection specimens, and 7 were mixed infection specimens. The above samples were detected by the method of Example 1 and the PCR kits currently used in clinics. Among them, the PCR kits used for comparison are Ureaplasma urealyticum (UU) nucleic acid determination kit (fluorescent PCR method) (Zhijiang Bio), Chlamydia trachomatis (CT) nucleic acid determination kit (fluorescent PCR method) (Zhijiang Bio) ), Neisseria gonorrhoeae (NG) nucleic acid assay kit (fluorescent PCR method) (Zhijiang Bio).

The test results show that the method of Example 1 is close to the test results of the PCR kits currently used in clinics (Figure 10). Taking the clinical test results as the gold standard, the diagnostic sensitivity and specificity are preliminarily evaluated, and the diagnostic sensitivity is Sen=94.87% , the diagnostic specificity Sep=96.67%.

Experimental Example 7 Diagnostic Precision Evaluation

In this experiment, in order to prove the stability of the method, the intra-assay precision was evaluated. A group of positive samples was repeatedly tested 5 times, and the Ct value of each result was obtained (Fig. 11). Coefficient of variation of pathogen detection results, CV _UU = 4.21%, CV _CT = 3.20%, CV _NG = 0.76%

The coefficient of variation CV is less than 5%, it can be seen that the method of the present invention is not only sensitive, fast, but also has strong repeatability, and is suitable for the current clinical detection situation.

To sum up, the present invention provides a method for simultaneous detection of three venereal pathogens of Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae based on the multiplex fluorescent PCR technology. The method of the invention designs primers and probe sequences, and optimizes detection conditions such as annealing conditions and primer addition amount, thereby improving the specificity, sensitivity and precision of the detection method. It has a good prospect in clinical application.

Claims (10)

1. A detection kit is characterized in that: comprising at least one of a primer pair for detecting Ureaplasma urealyticum, a primer pair for detecting Chlamydia trachomatis, and a primer pair for detecting Neisseria gonorrhoeae; the primer pair includes a positive Forward and reverse primers;

   The primer pair for detecting Ureaplasma urealyticum is a nucleotide sequence as described in SEQ ID NO.1 and SEQ ID NO.4 or a nucleotide sequence having 95% homology with the aforementioned sequence;

   The primer pair for detecting Chlamydia trachomatis is a nucleotide sequence as described in SEQ ID NO.2 and SEQ ID NO.5 or a nucleotide sequence having 95% homology with the aforementioned sequence;

   The primer pair for detecting Neisseria gonorrhoeae is the nucleotide sequence described in SEQ ID NO.3 and SEQ ID NO.6 or the nucleotide sequence having 95% homology with the aforementioned sequence.
2. The test kit according to claim 1, wherein the amount of the forward primer and the reverse primer for detecting Ureaplasma urealyticum is in a molar ratio of 1-3:1-3;

   And/or, the consumption molar ratio of the described forward primer and reverse primer for detecting Chlamydia trachomatis is 1-3:1-3;

   And/or, the described consumption mol ratio of the forward primer for detecting gonococcus and the reverse primer is 1-3:1-3;

   And/or, the consumption molar ratio of the forward primer for detecting Ureaplasma urealyticum, the forward primer for detecting Chlamydia trachomatis, and the forward primer for detecting Neisseria gonorrhoeae is 1-3:1-3:1 -3.
3. The test kit according to claim 2, wherein: the molar ratio of the amount of the forward primer for detecting Ureaplasma urealyticum and the reverse primer is 1:1;

   And/or, the consumption mol ratio of the described forward primer and reverse primer for detecting Chlamydia trachomatis is 1:1;

   And/or, the described consumption mol ratio of the forward primer for detecting gonococcus and the reverse primer is 1:1;

   And/or, the molar ratio of the amount of the forward primer for detecting Ureaplasma urealyticum, the forward primer for detecting Chlamydia trachomatis, and the forward primer for detecting Neisseria gonorrhoeae is 1:1:1.
4. The kit according to claim 1, characterized in that: the kit further comprises a probe set, and the probe set comprises a probe for detecting Ureaplasma urealyticum, a probe for detecting Chlamydia trachomatis, a probe for detecting at least one of the probes for Neisseria gonorrhoeae;

   The probe for detecting Ureaplasma urealyticum has the nucleotide sequence as described in SEQ ID NO.7, or a nucleotide sequence having at least 95% homology with the aforementioned nucleotide sequence;

   The probe for detecting Chlamydia trachomatis has the nucleotide sequence as described in SEQ ID NO. 8, or a nucleotide sequence having at least 95% homology with the aforementioned nucleotide sequence;

   The probe for detecting Neisseria gonorrhoeae has a nucleotide sequence as described in SEQ ID NO. 9, or a nucleotide sequence having at least 95% homology with the aforementioned nucleotide sequence.
5. The kit according to claim 4, wherein the probes in the probe set independently contain different fluorescent reporter groups and fluorescent quenching groups.
6. The kit according to claim 5, wherein the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3 or Dabcyl; the fluorescent reporter group is selected from FAM, HEX, ROX, CY5, VIC, TET or TAMRA.
7. The kit according to any one of claims 4-6, wherein the amount of the forward primer, reverse primer and probe for detecting Ureaplasma urealyticum is in a molar ratio of 1-3:1-3 :1-4;

   And/or, the consumption molar ratio of the forward primer, reverse primer and probe for detecting Chlamydia trachomatis is 1-3:1-3:1-4;

   And/or, the consumption molar ratio of described forward primer, reverse primer and probe for detecting gonococcus is 1-3:1-3:1-4;

   And/or, the molar ratio of the probe for detecting Ureaplasma urealyticum, the probe for detecting Chlamydia trachomatis, and the probe for detecting Neisseria gonorrhoeae is 1-3:1-3:1-4.
8. The kit according to claim 7, characterized in that: the molar ratio of the amounts of the forward primer, reverse primer and probe for detecting Ureaplasma urealyticum is 1:1:1;

   And/or, the consumption molar ratio of described forward primer, reverse primer and probe for detecting Chlamydia trachomatis is 1:1:1;

   And/or, the consumption molar ratio of described forward primer, reverse primer and probe for detecting gonococcus is 1:1:1;

   And/or, the molar ratio of the probes for detecting Ureaplasma urealyticum, the probes for detecting Chlamydia trachomatis, and the probes for detecting Neisseria gonorrhoeae is 1:1:1.
9. A PCR detection method for Ureaplasma urealyticum, Chlamydia trachomatis and Neisseria gonorrhoeae, characterized in that it comprises the steps of: using the kit according to any one of claims 1-8 to perform PCR amplification on a sample to be detected, and then detect.
10. The method according to claim 9, wherein the PCR amplification procedure is as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 5s, annealing at 56°C for 15s, and extension at 72°C for 30s.

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