CN119082332A

CN119082332A - Application, primer probe combination and kit for target genes of Ureaplasma urealyticum and Ureaplasma microti

Info

Publication number: CN119082332A
Application number: CN202411422566.3A
Authority: CN
Inventors: 郑业焕; 孟纯阳; 胡明明; 李彬; 付光宇; 吴学炜
Original assignee: Autobio Diagnostics Co Ltd
Current assignee: Autobio Diagnostics Co Ltd
Priority date: 2024-10-12
Filing date: 2024-10-12
Publication date: 2024-12-06

Abstract

The invention belongs to the field of pathogen molecule detection, and particularly relates to application of ureaplasma urealyticum and tiny ureaplasma urealyticum target genes, primer probe combination and a kit. The kit provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that the detection and the distinction of gonococcus, tiny ureaplasma and ureaplasma urealyticum are simultaneously realized in a single-tube reaction system. The kit has the advantages of detection sensitivity up to 400copies/mL, good specificity, more accurate detection and capability of being stored at 2-8 ℃.

Description

Application of ureaplasma urealyticum and tiny ureaplasma urealyticum target genes, primer probe combination and kit

Technical Field

The invention relates to the technical field of pathogen molecule detection, in particular to application of ureaplasma urealyticum and tiny ureaplasma urealyticum target genes, primer probe combination and a kit.

Background

Neisseria gonorrhoeae is a causative agent of the classical sexually transmitted disease gonorrhea, which is widely prevalent worldwide, has a short incubation period of gonococci, can cause acute and chronic inflammatory reactions of the genital tract, and can cause the consequences of infertility, ectopic pregnancy, fetal dysplasia, etc. in severe cases. However, 5-20% of the adult men and 50% of the adult women are asymptomatic, and most of them are found to miss the optimal cure period when complications occur, so that gonorrhea is a serious sanitary problem at home and abroad.

Human ureaplasma is a prokaryotic microorganism belonging to the genus ureaplasma of the family mycoplasma, without cell walls, and the smallest microorganism found at present that can be propagated in inanimate medium. It is a conditionally pathogenic microorganism which mainly exists in the urogenital tract and respiratory tract and can invade the host mucosa when the organism is immunosuppressed. The transmission route is mainly sexually transmitted, and can be transmitted vertically through mother and infant or transmitted through body fluid at the time of birth of a fetus. It is currently known that human ureaplasma is largely divided into two biota (ureaplasma parvum and ureaplasma urealyticum), 14 serotypes. Micropuridines include serotypes 1,3,6 and 14, while the remaining 10 serotypes are ureaplasma urealyticum. The pathogenicity of human ureaplasma may be related to different biota, serotypes and host factors, but the mechanism is not yet clear, and a great deal of evidence indicates that the microplasma is easy to carry, and typing is helpful for the research of the pathogenesis.

Traditional neisseria gonorrhoeae and human ureaplasma detection methods include culture detection, antigen detection and nucleic acid amplification detection. The nucleic acid amplification detection has the characteristics of simplicity, rapidness and reliability, but most of the existing nucleic acid amplification detection kits have the defect of poor specificity, the target combination mode is few, the requirements on samples are strict, multiple groups of primers are required to be designed, the operation is complicated, and the detection reagent needs to be frozen for preservation. There is therefore a need in the art for a product that can simply, quickly and accurately distinguish between the pathogens mentioned above.

Disclosure of Invention

In view of this, the present invention provides applications, primer probe combinations and kits for understanding ureaplasma and tiny ureaplasma target genes. The kit provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that the detection and the distinction of gonococcus, tiny ureaplasma and ureaplasma urealyticum are simultaneously realized in a single-tube reaction system.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides application of a target gene in preparing a product for identifying ureaplasma urealyticum and ureaplasma parvum:

The target genes each have:

(I) A nucleotide sequence for identifying ureaplasma urealyticum as shown in SEQ ID NO.11, a nucleotide sequence for identifying ureaplasma parvum as shown in SEQ ID NO.12, and/or

(II) a nucleotide sequence which encodes the same protein as the nucleotide sequence shown in (I) but differs from the nucleotide sequence shown in (I) due to degeneracy of the genetic code, or

(III), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (I) or (II) and functionally identical or similar to the nucleotide sequence shown in (I) or (II), or

(IV) a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (I), (II) or (III).

The invention also provides a primer probe combination, which comprises:

(I) An upstream primer for detecting gonococcus having a nucleotide sequence shown in SEQ ID NO.1, and

A downstream primer for detecting gonococcus with a nucleotide sequence shown as SEQ ID NO.2, and

Probe for detecting gonococcus having nucleotide sequence shown in SEQ ID NO.3, and/or

(II) an upstream primer for detecting ureaplasma minum having a nucleotide sequence shown in SEQ ID NO.4, and

Downstream primer for detecting ureaplasma minum having nucleotide sequence shown in SEQ ID NO.5, and

Probe for detecting ureaplasma parvum having nucleotide sequence shown in SEQ ID NO.6, and/or

(III) an upstream primer for detecting ureaplasma urealyticum having the nucleotide sequence shown in SEQ ID NO.4, and

Downstream primer for detecting ureaplasma urealyticum having nucleotide sequence shown as SEQ ID NO.5, and

Probe for detecting ureaplasma urealyticum having nucleotide sequence shown as SEQ ID NO.7, or

(IV) a nucleotide sequence encoding the same protein as the nucleotide sequence set forth in any one of (I) to (III) but differing from the nucleotide sequence set forth in any one of (I) to (III) due to degeneracy of the genetic code, or

(V), a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in any one of (I) to (IV), and functionally identical or similar to the nucleotide sequence shown in any one of (I) to (IV), or

(VI) a nucleotide sequence having at least 90% sequence homology to the nucleotide sequence of any one of (I) to (V).

In some embodiments of the invention, the fragment amplified by the primer pair for detecting ureaplasma urealyticum is shown as SEQ ID NO.11, and the fragment amplified by the primer pair for detecting ureaplasma parvum is shown as SEQ ID NO. 12.

In some embodiments of the invention, the primer probe combination further comprises a primer pair and a probe for amplifying the detection internal standard gene;

The internal standard gene comprises one or more of beta-globin, RNase P gene, action or GAPDH.

In some embodiments of the invention, the primer probe combination further comprises:

(I) An upstream primer for detecting an internal standard gene having a nucleotide sequence shown as SEQ ID NO.8, and

A downstream primer for detecting an internal standard gene having a nucleotide sequence shown as SEQ ID NO.9, and

A probe having a nucleotide sequence shown as SEQ ID NO.10 as an internal standard for detection, or

In some embodiments of the invention, the probe comprises a fluorophore comprising one or more of FAM, HEX, ROX, VIC, CY or cy 5.5.

In some embodiments of the invention, the probe shown as SEQ ID NO.3 is attached to a FAM fluorophore, the probe shown as SEQ ID NO.6 is attached to a ROX fluorophore, the probe shown as SEQ ID NO.7 is attached to a Cy5 fluorophore, and the probe shown as SEQ ID NO.10 is attached to a HEX fluorophore.

The invention also provides application of the primer probe combination in preparation of a reagent or a kit for detecting and/or identifying gonococcus, tiny ureaplasma and ureaplasma urealyticum.

The invention also provides application of the primer probe combination in preparation of a reagent or a kit for detecting genital tract inflammation.

The invention also provides a combined reagent, which comprises the primer probe combination.

The invention also provides a kit comprising any of the following and PCR reaction reagents:

(I) The primer probe combination, and/or

(II) the combined reagent.

In some embodiments of the invention, the PCR reagent comprises any one or more of dNTPs, DNA polymerase, UDG enzyme, PCR enhancer and magnesium ion.

In some embodiments of the invention, the PCR enhancer comprises DMSO.

In some embodiments of the invention, the magnesium ion comprises MgCl ₂.

In some embodiments of the invention, the reaction system of the kit comprises:

In some embodiments of the invention, the kit further comprises a negative quality control comprising an internal standard plasmid and a positive quality control comprising gonococcus, ureaplasma parvum, ureaplasma urealyticum, and an internal standard plasmid.

In some embodiments of the invention, the reaction procedure of the PCR comprises:

The present invention includes, but is not limited to, providing the following benefits:

the invention provides application of knowing ureaplasma urealyticum and tiny ureaplasma urealyticum target genes, primer probe combination and kit. The kit provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that the detection and the distinction of gonococcus, tiny ureaplasma and ureaplasma urealyticum are simultaneously realized in a single-tube reaction system. The kit has the advantages of detection sensitivity up to 400copies/mL, good specificity, more accurate detection and capability of being stored for 12 months at 2-8 ℃.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a diagram of detection results of the kit of preparation example 1 on gonococcus, tiny ureaplasma and ureaplasma urealyticum, wherein blue represents a gonococcus amplification curve, yellow represents a tiny ureaplasma urealyticum amplification curve, purple represents a ureaplasma urealyticum amplification curve, and green represents an internal standard amplification curve;

FIG. 2 shows a graph of the sensitivity results of the kit of the invention for detecting gonococci;

FIG. 3 shows a graph of the sensitivity results of the kit of the invention for detecting ureaplasma parvum;

FIG. 4 shows a graph of the sensitivity results of the kit of the invention for detecting ureaplasma urealyticum;

FIG. 5 shows a graph of the specificity test results of the kit of the present invention, wherein blue represents the gonococcus amplification curve, yellow represents the microplasma amplification curve, purple represents the ureaplasma urealyticum amplification curve, and green represents the internal standard amplification curve;

FIG. 6 shows a diagram of the results of single detection of ureaplasma urealyticum comparative primers;

FIG. 7 shows a graph of multiplex results for ureaplasma urealyticum comparative example primers, wherein blue represents gonococcus amplification curves, yellow represents microureaplasma amplification curves, purple represents ureaplasma urealyticum amplification curves, and green represents internal standard amplification curves.

Detailed Description

The invention discloses application of knowing ureaplasma urealyticum and tiny ureaplasma target genes, primer probe combination and kit, and the skilled person can properly improve the process parameters by referring to the content of the disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

The invention aims to provide a kit for detecting gonococcus, tiny ureaplasma and ureaplasma urealyticum, which can rapidly and accurately distinguish different pathogens, and has the advantages of high sensitivity, good specificity, capability of being stored at 2-8 ℃ and the like.

The invention provides a gonococcus, tiny ureaplasma and ureaplasma urealyticum detection kit, which comprises specific primers and probes. Wherein, the gonococcus primer probe is designed in the conserved region of the cryptic plasmid gene:

The nucleotide sequence of the primer upstream of gonococcus is (SEQ ID NO. 1): 5'-ACACCCAACC CGACCTCTTCTT-3';

The nucleotide sequence of the primer downstream of gonococcus is (SEQ ID NO. 2): 5'-GCGGCGGATT GACTTGGATGT-3';

the nucleotide sequence of the gonococcus probe is (SEQ ID NO. 3) 5'-CGGCGTTTGATGG CGGCAGCGTAAGAGC-3';

comparing the 14 serotypes of urease genes of human ureaplasma, the homology between the ureaplasma parvum (4 serotypes) is found to be 99.3 percent, the homology between the ureaplasma parvum (10 serotypes) is found to be 99.6 percent, the homology between the ureaplasma parvum and the ureaplasma parvum is found to be 89.7 percent, a primer probe is designed in a specific region to distinguish two biological groups, the ureaplasma parvum and the ureaplasma parvum share an upstream primer and a downstream primer, and different probes are distinguished:

the nucleotide sequence of the upstream primer of the microplasma is (SEQ ID NO. 4): 5'-CTAAATAT ACAATTAACCCAGCTATTGC-3';

the nucleotide sequence of the downstream primer of the microplasma is (SEQ ID NO. 5): 5'-GGTTTTGC ACCAAAGAATTTAGGTTCTC-3';

The nucleotide sequence of the microplasma probe is (SEQ ID NO. 6): 5'-GTGTTGATTCT TATATTGGTTCACTAGAAGTT-3';

the nucleotide sequence of the ureaplasma urealyticum upstream primer is (SEQ ID NO. 4): 5'-CTAAATAT ACAATTAACCCAGCTATTGC-3';

The nucleotide sequence of the ureaplasma urealyticum downstream primer is (SEQ ID NO. 5): 5'-GGTTTTGC ACCAAAGAATTTAGGTTCTC-3';

The nucleotide sequence of the ureaplasma urealyticum probe is (SEQ ID NO. 7) 5'-CATGGTATTGA CTCATACGTTGGATCAAT-3';

Internal standard is a human internal standard gene, for example, beta-globin, RNase P gene, action and GAPDH gene can be used. In one specific embodiment, the internal standard is the β -globin gene and the internal standard primer probe is as follows:

The nucleotide sequence of the internal standard upstream primer is (SEQ ID NO. 8): 5'-CTCAGTGTGGCA AAGGTGCC-3';

The nucleotide sequence of the internal standard downstream primer is (SEQ ID NO. 9): 5'-GAAGGCTCATGG CAAGAAAGTG-3';

the nucleotide sequence of the internal standard probe is (SEQ ID NO. 10): 5'-GGTTGTCCAGGTGA GCCAGGCCATCACT-3';

the reporter fluorophore is different for each probe, for example FAM, HEX, ROX, VIC, cy and cy5.5 can be used. In a specific embodiment, the reporter fluorophore of the gonococcal probe is FAM, the reporter fluorophore of the ureaplasma parvum is ROX, the reporter fluorophore of ureaplasma urealyticum is Cy5, and the reporter fluorophore of the internal standard is HEX.

Ureaplasma urealyticum target sequence 5'-3' (SEQ ID No. 11):

CTAAATATACAATTAACCCAGCTATTGCTCATGGTATTGACTCATACGTTGGATCAATCGAAGTAGGAAAATTAGCTGATATTGTTGCATGAGAACCTAAATTCTTTGGTGCAAAACC

the microureaplasma target sequence 5'-3' (SEQ ID No. 12):

CTAAATATACAATTAACCCAGCTATTGCACATGGTGTTGATTCTTATATTGGTTCACTAGAAGTTGGTAAATTAGCTGATATTGTTGCTTGAGAACCTAAATTCTTTGGTGCAAAACC

The kit comprises a PCR reaction liquid, wherein the PCR reaction liquid comprises a PCR reaction liquid 1 and a PCR reaction liquid 2, the PCR reaction liquid 1 comprises the primer probe combination, dNTPs, DNA polymerase, UDG enzyme and a PCR enhancer, and the PCR reaction liquid 2 comprises magnesium ions.

Specifically, the dNTPs are dATP, dGTP, dCTP, dUTP in combination, the dAT P concentration is 0.1-0.3 mM, the dGTP concentration is 0.1-0.3 mM, the dCTP concentration is 0.1-0.3 mM, and the dUTP concentration is 0.1-0.3 mM.

Specifically, the concentration of the DNA polymerase is 0.0125-0.0625U/. Mu.L, and in one embodiment the DNA polymerase may be Tth enzyme.

Specifically, the concentration of the UDG enzyme is 0.00375-0.0125U/. Mu.L.

Specifically, the PCR enhancer is DMSO, and the concentration is 0.0125% -0.0375% (DMSO to total volume ratio).

Specifically, the magnesium ion concentration is 1.5 mM-3 mM.

Specifically, the kit further comprises a positive quality control product and a negative quality control product. The positive quality control product is prepared from fragment plasmid of gonococcus cryptic plasmid gene, fragment plasmid of microplasma urease gene, and other components ureaplasma urealyticum urease gene fragment plasmid and beta-globin gene fragment plasmid, the negative quality control is beta-globin gene fragment plasmid.

If not specified, the application of ureaplasma urealyticum and tiny ureaplasma target genes, primer probe combination and raw materials and reagents used in the kit can be purchased from the market.

The invention is further illustrated by the following examples:

Preparation example 1 gonococcus, microureaplasma and ureaplasma urealyticum detection kit

The embodiment provides a gonococcus, tiny ureaplasma and ureaplasma urealyticum detection kit, which comprises the following components in table 1:

TABLE 1 major Components of the kit

Preparation example 2 gonococcus, microureaplasma and ureaplasma urealyticum detection kit

The embodiment provides a gonococcus, tiny ureaplasma and ureaplasma urealyticum detection kit, which comprises the following components in table 2:

TABLE 2 major Components of the kit

Preparation example 3 gonococcus, microureaplasma and ureaplasma urealyticum detection kit

The embodiment provides a gonococcus, tiny ureaplasma and ureaplasma urealyticum detection kit, which comprises the following components in part by weight, see table 3:

TABLE 3 major Components of the kit

Comparative example gonococcus, microureaplasma and ureaplasma urealyticum detection kit

Based on the kit of example 1, the specific primers and probes therein were replaced with primer probe sequences shown in Table 4.

TABLE 4 comparative primer probe sequences

Name of the name	Numbering device	5'-3'
			Upstream primer	SEQ ID NO.13	AATGAAACTCAGCAACTCCAAATCT
Downstream primer	SEQ ID NO.14	CCGTTTGCAGTTTTCTTTAATTTCT
			Probe with a probe tip	SEQ ID NO.15	GCTGCCGTTGACTACAACGACTTAGAAAAC

Example qualitative detection of gonococci, microureaplasma and De-ureaplasma

1. Reagent preparation

And according to the quantity of samples to be detected, negative quality control and positive quality control, taking corresponding amounts of the PCR reaction liquid 1 and the PCR reaction liquid 2 according to the proportion of the kit (20 mu L/human PCR reaction liquid 1+20 mu L/human PCR reaction liquid 2) in preparation examples 1-3, fully and uniformly mixing to obtain a PCR-MIX mixed liquid, and carrying out instantaneous centrifugation for later use.

2. Sample processing

(1) And placing the swab to be tested into 1mL of physiological saline for rinsing, and reserving the rinsed liquid or directly detecting a urine sample.

(2) Taking 600 mu L of rinsed liquid sample or urine sample, and extracting nucleic acid of gonococcus, micropurium and ureaplasma with a magnetic bead method nucleic acid extraction reagent (recorded number: yu Zheng Xiebei 20180037) of Zhengzhou Anji bioengineering Co., ltd.) in the sample to be detected as extracted DNA of the sample to be detected for later use;

(3) Meanwhile, 600 mu L of negative/positive quality control substances are taken and matched with a magnetic bead method nucleic acid extraction reagent of Zhengzhou Anji bioengineering Co., ltd to extract DNA nucleic acid of the negative/positive quality control substances for later use;

(4) Taking a plurality of PCR reaction tubes, adding 40 mu L of a PCR-MIX mixed solution, respectively adding 40 mu L of extracted sample DNA to be detected and 40 mu L of each negative/positive quality control product nucleic acid product, covering a tube cover (after removing bubbles), and performing instantaneous centrifugation for 10 seconds.

3. Fluorescent PCR reaction

(1) Placing the PCR reaction tube into a sample tank of an amplification instrument, and setting the names of samples to be detected according to the corresponding sequence;

(2) Selecting a fluorescence detection channel, namely selecting FAM/ROX/Cy5 channel detection targets;

(3) The PCR reaction procedure was 50℃for 2min, 95℃for 2min, (95℃for 10s,60℃for 22s,45 cycles) and 40℃for 10s.

(4) Analysis of results

After the PCR reaction is finished, the detection result is judged according to the Ct value. The intersection point of the amplification curve and the threshold line is called a Ct value (namely cycle threshold, which is a cycle number experienced when a fluorescent signal in a PCR reaction tube reaches a set threshold), and the instrument software can judge the detection result according to the Ct value of each sample. For the sample with Ct value less than or equal to 38, the positive is reported, for the sample with Ct value greater than 38 and internal standard detection is positive (Ct value less than or equal to 35), the negative is reported, for the sample with Ct value less than or equal to 35, the negative is reported, if the internal standard Ct value greater than or equal to 35 or no display, the detection result of the sample is invalid, the reason is searched and eliminated, and the sample is repeatedly detected.

The PCR detection results of gonococcus, tiny ureaplasma and ureaplasma urealyticum are shown in Table 5 and FIG. 1, and the kit can well detect three pathogens.

TABLE 5 qualitative detection of gonococci, microureaplasma and De-ureaplasma

Effect example 1 sensitivity detection

The reagent kit in preparation example 1 is adopted to carry out sensitivity detection on each target according to the method of the embodiment, and the concentration of the sample to be detected is 10800, 3600, 1200 and 400copies/mL respectively so as to simulate clinical samples and carry out multiplex PCR detection. The results are shown in figures 2-4, which show that each channel of the sample as low as 400copies/mL can still be accurately detected, and the detection rate is 100%, which shows that the sensitivity of the kit is 400copies/mL.

Effect example 2 specificity detection

The kit in preparation example 1 is adopted, the sample to be tested in the example is replaced by common pathogens of genital tract and other pathogens with similar infection symptoms, such as chlamydia trachomatis (1E+7copies/mL), herpes simplex virus type 1 (1E+7copies/mL), streptococcus B (1E+6CFU/mL), neisseria meningitidis (1E+6CFU/mL), mycoplasma genitalium (1E+7copies/mL), mycoplasma hominis (1E+7copies/mL), human papillomavirus 16 type (1E+7copies/mL), human cytomegalovirus (1E+7copies/mL), mycoplasma pneumoniae (1E+7copies/mL), lactobacillus paracasei (1E+6CFU/mL), gardnerella vaginalis (1E+6CFU/mL) and candida albicans (1E+6CFU/mL), and the detection results are shown in fig. 5, wherein a blue line represents a coccus test curve represents a negative curve, a human papillomavirus 16 type (1E+7copies/mL), a human cytomegalovirus (1E+7copies/mL), a lactobacillus paracasei (1E+6CFU/mL), a vaginal gardner (1E+6CFU/mL), and the detection results represent a blue-line represents a yellow urea amplification curve. The invention has no cross reaction with other pathogens similar to common pathogens of genital tract and infection symptoms (such as chlamydia trachomatis, herpes simplex virus, group B streptococcus, neisseria meningitidis, mycoplasma genitalium, mycoplasma hominis, papillomavirus type 16, human cytomegalovirus, mycoplasma pneumoniae, lactobacillus paracasei, gardnerella vaginalis and candida albicans).

Effect example 3 stability detection

The reagent can be stored at 2-8 ℃, the actual stability of the reagent is evaluated by a 37 ℃ heat stability acceleration test, the reagent is accelerated for 0, 7, 14 and 21 days at 37 ℃, the kit in preparation example 1 is adopted, three samples (4E+4/mL, 4000/mL and 400/mL, respectively, the concentration of the ureaplasma parvum and the concentration of the ureaplasma urealyticum are 5E+4CCU/mL, 5000CCU/mL and 500CCU/mL respectively) are detected according to the method of the embodiment, the Ct value difference is not higher than 1.33 Ct compared with 0 day, the difference is small, and the stability is shown in the following table:

Table 6 stability test

Effect example 4 selection of primer probe sequences

In a multiple pathogen detection system, the primer probe sequences in the same reaction are more, a dimer is formed between the primer and (or) the probe, and the primer probes are mutually interfered, and all factors have influence on the sensitivity. The inventors also designed some other primer probe combinations (sequences as in Table 4) and used the kit of comparative examples to detect gonococci, ureaplasma parvum and ureaplasma urealyticum as described in the examples. Specific detection results are shown in fig. 6-7, and the comparative primer probe for ureaplasma urealyticum can be seen from the figures, the detection result of a single detection system is better, but in a multiple system, the detection is influenced, the Ct value is worse, and the superiority of the combination of the invention is further illustrated.

Effect example 5 comparison of different detection methods

Experimental grouping:

Test group test samples of different concentrations (clinical samples of known concentrations were diluted 4-fold to 6400, 1600, 400, 100 copies/mL) were taken and tested by the method of example using the kit of preparation 1.

The control group is to take samples to be detected with different concentrations (clinical samples with known concentrations are diluted to 6400, 1600, 400 and 100copies/mL in a 4-time gradient mode), and compare the samples with the detection method in the published patent CN200910164977 (positive judgment value 40), the sequence of a primer probe is shown in a table 7, the reaction system is shown in a table 8, and the PCR amplification program is 50 ℃ for 2min, 95 ℃ for 10s,60 ℃ for 50s and 40 cycles.

As a result, as shown in Table 9, it was found that the detection of 400copies/mL of the gonococcus of the present invention was still stable (experimental group), but the control group had failed to detect, so that the detection ability of the present invention was superior to that of the control reagent.

TABLE 7 control primer probe sequences

Name of the name	Numbering device	5'-3'
			Upstream primer	SEQ ID NO.16	CTTCTCGGGTGGCGAGTGG
Downstream primer	SEQ ID NO.17	CCAGTAATTCCGATTAACGCTCG
			Probe with a probe tip	SEQ ID NO.18	CGGGTCTGAGAGGATGATCCGCC

Table 8 control reaction System

Table 9 comparison of different detection methods

Note that negative (-), positive (+).

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. Application of target genes in the preparation of products for identifying Ureaplasma urealyticum and Ureaplasma microti:

The target genes respectively have:

(I), a nucleotide sequence for identifying Ureaplasma urealyticum as shown in SEQ ID NO.11, a nucleotide sequence for identifying Ureaplasma microti as shown in SEQ ID NO.12; and/or

(II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

(III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

2. A primer-probe combination, characterized in that the primer-probe combination comprises:

(I), an upstream primer for detecting gonococci having a nucleotide sequence as shown in SEQ ID NO.1; and

A downstream primer for detecting gonococci having a nucleotide sequence as shown in SEQ ID NO.2; and

A probe for detecting gonococci having a nucleotide sequence as shown in SEQ ID NO.3; and/or

(II), an upstream primer for detecting Ureaplasma microti having a nucleotide sequence as shown in SEQ ID NO.4; and

A downstream primer for detecting Ureaplasma microti having a nucleotide sequence as shown in SEQ ID NO.5; and

A probe for detecting Ureaplasma microti having a nucleotide sequence as shown in SEQ ID NO.6; and/or

(III), an upstream primer for detecting Ureaplasma urealyticum having a nucleotide sequence as shown in SEQ ID NO.4; and

A downstream primer for detecting Ureaplasma urealyticum having a nucleotide sequence as shown in SEQ ID NO.5; and

A probe for detecting Ureaplasma urealyticum having a nucleotide sequence as shown in SEQ ID NO.7; or

(IV) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in any one of (I) to (III) but different from the nucleotide sequence shown in any one of (I) to (III) due to the degeneracy of the genetic code; or

(V) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in any one of (I) to (IV), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in any one of (I) to (IV); or

(VI) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of any one of (I) to (V).

3. The primer-probe combination according to claim 2, further comprising a primer pair and a probe for amplifying and detecting an internal standard gene;

The internal standard gene includes one or more of β-globin, RNase P gene, Actin or GAPDH.

4. The primer-probe combination according to claim 2 or 3, wherein the probe comprises a fluorescent group; the fluorescent group comprises one or more of FAM, HEX, ROX, VIC, CY5 or Cy5.5.

5. Use of the primer-probe combination according to any one of claims 2 to 4 in the preparation of a reagent or kit for detecting and/or identifying Neisseria gonorrhoeae, Ureaplasma parvum or Ureaplasma urealyticum.

6. Use of the primer-probe combination according to any one of claims 2 to 4 in the preparation of a reagent or a kit for detecting genital tract inflammation.

7. A combination reagent, characterized in that it comprises the primer-probe combination according to any one of claims 2 to 4.

8. A kit, characterized in that it comprises any of the following items and PCR reaction reagents:

(I) a primer-probe combination as claimed in any one of claims 2 to 4; and/or

(II) The combined reagent according to claim 7.

9. The kit according to claim 8, wherein the PCR reaction reagents include dNTPs, DNA polymerase, PCR enhancer, and enzyme activator.

10. The kit according to claim 9, wherein the PCR enhancer comprises one or more of DMSO, ammonium sulfate, and betaine;

The enzyme activator includes magnesium ions, manganese ions or a combination thereof.