CN116574826A

CN116574826A - Vaginal flora molecular typing kit and application method thereof

Info

Publication number: CN116574826A
Application number: CN202310825643.9A
Authority: CN
Inventors: 刘忱; 刘亦金
Original assignee: Tianjin Jiuyiyuan Technology Development Co ltd
Current assignee: Tianjin Jiuyiyuan Technology Development Co ltd
Priority date: 2023-07-07
Filing date: 2023-07-07
Publication date: 2023-08-11

Abstract

The application provides a vaginal bacteria microecological molecular typing kit and a method for carrying out non-diagnostic vaginal bacteria microecological molecular typing by using the kit. In some embodiments, the molecular typing kit may comprise: amplification of primer sequences of Lactobacillus crispatus, lactobacillus jensenii, lactobacillus inertia, G.vaginalis, A.vaginalis, eggerthella, megasphaera, leptobacter leptospira/Sneathia, prevotela, all bacteria, SYBR Green I, accurate molecular typing of vaginal flora by fluorescent quantitative PCR detection, and enhancement of female reproductive health.

Description

Vaginal flora molecular typing kit and application method thereof

Technical Field

The application relates to the technical field of fluorescence quantitative PCR detection of vaginal microbial ecological molecule typing, in particular to a vaginal bacterial microbial ecological molecule typing kit and a method for non-diagnostic vaginal bacterial microbial ecological molecule typing by using the kit.

Background

Normal microorganisms are generally defined as populations of microorganisms that colonize the human body, at specific structural sites of the host, and which form as the host evolves over time, colonize the skin or mucosa of the host. The body surface, mouth, respiratory tract, gastrointestinal tract, urinary tract and genital tract are the main sites of microbial distribution in normal adult humans. Through long adaptation and biological evolution process with human body, the symbiotic state with human body is achieved, and a close connection is established between the two, so that the biological function of human body is perfected, and the biological function of human body is particularly improved. Localized microbiota plays an important role in immunomodulation.

Lactobacillus (Lactobacillus genus) is a resident bacterium that resides in the vagina of a female and plays an important role in maintaining the normal environment of the female reproductive organs. In the case of healthy women's vagina, lactic acid is constantly produced to maintain an acidic (pH 4.5 to 5.5) environment, thereby inhibiting overgrowth of other opportunistic bacteria and the infection caused thereby. The high-richness lactic acid stalk seedling is an identification of female genital tract health. Under physiological conditions, the vaginal flora, the host and the environment form a mutually restricted and mutually coordinated dynamic balance state, and the vaginal microecological environment in the childbearing period with the dominant lactobacillus is established together. Lactobacillus resists attack by foreign pathogens by a variety of mechanisms, including: decomposing glycogen located in the endothelial cells of the vagina to form lactic acid, maintaining a low pH (3.8-4.4) in the vagina; generating biosurfactant to resist invasion of external pathogenic bacteria and maintaining absolute predominance of lactic acid seedlings; producing antibiotic, hydrogen peroxide and other antibacterial and bactericidal substances; the lactobacillus adheres to the fine surface of the vaginal cell endothelium to form tight connection and form a mechanical barrier to prevent external invasive bacteria from entering blood circulation; lactic acid bacteria bind to the endothelial cell receptor of vagina, compete for mechanisms such as nutrition and space, promote and maintain female reproductive health, etc.

However, due to the influence of environmental changes, drug stimulation, changes of host self hormone level and immunity and other factors, the micro-ecological flora diversity in the vagina can change, the lactobacillus vaginalis is reduced, the pH value of the vagina is increased, various harmful bacteria are greatly proliferated, the micro-ecological balance in the vagina of the host is broken and changed, and the micro-ecological balance in the vagina is changed from a physiological combination state to a pathological combination state, so that the micro-ecological unbalance of female genital tract is caused, and the occurrence of diseases such as colpitis, cervical cancer and the like is further caused. Bacterial vaginitis (bacterial vaginosis, BV) affects 10% -37% of women as a common gynecopathy characterized by changes in the vaginal flora. The existence of BV greatly increases the risks of bad pregnancy such as premature birth, abortion, premature rupture of fetal membranes and the like, and the related flora of BV can obviously improve the probability of female suffering from pelvic inflammation. This phenomenon may be due to opportunistic bacteria generated by age, stress, hormonal imbalance, menstruation and by sexual intercourse in women. Vaginitis is a common disease experienced by many women, but if left alone for a long time, there is an increased risk of pelvic inflammation, and postoperative infection due to vaginitis can cause a number of complications.

The main microorganisms responsible for vaginitis include ganerna vaginalis and candida albicans, and Prevotella, anaerobic gram-positive cocci and Vibrio mobilis (Mobiluncus spp.), ureaplasma urealyticum and mycoplasma hominis. I.e. the kind of micro-organisms causing vaginitis is very large and may be caused by one or several micro-organisms.

Cervical cancer is one of the fourth most common cancers in women, with about 57 tens of thousands of new cases and 31 tens of thousands of deaths each year, and the incidence of cervical cancer in age-standardized worldwide is 13.1/10 ten thousand women. Cervical cancer has become the second most common cancer and the third leading cause of cancer death in women aged 15-44 in China. Bacteria symbiotic in the vagina of a host participate in the occurrence and development process of cervical diseases through affecting the infection and elimination of HPV which is a key causative factor of cervical cancer. Previous studies have shown that bacterial vaginitis promotes HPV infection by affecting the pH value of genital tract environment, immunological reaction, genital tract injury, etc., and inhibits the clearance of HPV; probiotic treatment may inhibit HPV infection by correcting vaginal bacterial micro-ecology.

The community structure of microorganisms in vagina is clarified, and a new effective means for targeted prevention and treatment of female reproductive system diseases is provided. However, the existing technical means for analyzing the ecology of the vaginal microorganisms are based on the conventional microscopic morphological examination and the conventional bacterial culture method, and it is difficult to effectively and accurately distinguish genetic diversity of microorganism populations; or by combining high-throughput sequencing with bioinformatics analysis, the method is difficult to popularize and apply in clinical detection.

Vaginal secretion detection is one of the conventional detection indexes of gynecology. Vaginal secretion detection, commonly known as "leucorrhea routine", is one of the most common applications in gynecological outpatient examinations, and is an important diagnostic basis for judging whether women suffer from vaginal inflammation. The traditional detection means is wet-chip microscopy, and the main detection items comprise epithelial cells, white blood cells, lactobacillus quantity, fungus, trichomonas and clue cells. The method is simple and direct, has high specificity, is also always favored as a gold standard, but cannot finely distinguish genetic diversity of microorganisms, and has high technical requirements on personnel due to manual operation and manual interpretation results, and can have differences of different personnel in result interpretation.

In recent years, the combined detection of hydrogen peroxide, leukocyte esterase, sialidase, acetylglucosaminidase activity and pH value in the leucorrhea by using a chemoenzymatic method to respectively correspond to the number and function of lactobacillus in the leucorrhea, and the infection of leucocytes, bacterial vaginosis BV, mold and trichomonas has gradually become hot spots in the detection of the leucorrhea, and the method is called as 'five-joint detection'. Judging the result of the five-joint inspection, wherein the hydrogen peroxide hole does not develop color to prompt the dysbacteriosis of the vagina, and judging the result+ and the corresponding microscopic inspection result are abnormal lactobacillus; leukocyte esterase positive corresponds to microscopic examination of leukocyte+; sialidase positive corresponds to microscopic clue cells; acetylglucosaminidase is positive and the pH is more than or equal to 4.8, and the corresponding microscopic examination shows that trichomonas+; positive acetylglucosaminidase and pH less than or equal to 4.6; then the corresponding mirror examination is to see fungi.

Gram staining has been widely used as a method for diagnosing vaginitis, but with the recently introduced non-culture-based gene detection method, vaginitis can be easily and rapidly diagnosed. For example, there is a method for detecting a microorganism causing vaginitis using a PCR (polymerase chain reaction) technique. The conventional PCR detection method adopts various methods of microorganisms related to sexually transmitted diseases as described above, which are not specific for detecting vaginitis, and are expensive, and the detection result can be obtained only about 3 to 5 days, so that it cannot be said that the method is a method for rapidly, simply and accurately detecting vaginitis only.

As an alternative to the conventional PCR test method, a method of diagnosing vaginitis by detecting the presence or absence of vaginitis-inducing microorganisms and vaginal flora using semi-quantitative multiplex PCR has been proposed, but the semi-quantitative multiplex PCR test method accurately analyzes the abundance of microorganisms. There is a problem in that the possibility of a result error is high. In other words, if vaginitis is diagnosed simply by the presence of vaginitis microorganisms, misdiagnosis may occur, and even vaginitis, vaginal flora may exist, which is not easy and has a wrong diagnosis problem.

In addition, there was a study showing negative correlation using the inert lactobacillus l.inors, lactobacillus jensenii and atopomyces vaginalis a.vaginalis, but the lactobacillus-specific primers (Lactobacillus species-specific primers) used herein may frequently cross react with other microbial species, and the condition of the sample depends on primer dimer (dimer) and the like, and accurate quantitative determination has a limit.

Current examination means detect symptoms of disease but fail to clearly distinguish local microbial communities. Localized microbiota plays an important role in immunomodulation. In basic research work, researchers have developed research means for exploring the ecology of vaginal microorganisms by combining high-throughput sequencing with bioinformatics analysis, but these complex technical flows are difficult to popularize and apply in clinical diagnosis and treatment. To date, there is no clinical treatment that effectively enhances female reproductive health through microbial ecological regulation.

Therefore, in the technical field of the present application, there is a need to provide a method for accurately typing vaginal flora with low cost, simplicity, rapidness and accuracy, and a method for assisting in obtaining vaginal health conditions.

Disclosure of Invention

One object of the present application is to: provides a kit for detecting vaginal microorganism ecological molecule typing by fluorescence quantitative PCR. In some embodiments, it comprises a bacterial genome extraction kit, a fluorescent real-time quantitative PCR detection kit, specific primers for 9 bacteria that are dominant in the vagina (3 vaginal probiotics +6 vaginal pathogens), and primers for microbial 16srDNA that indicate the total amount of bacteria.

Another object of the application is: the detection method for the non-diagnostic purpose of the vaginal flora molecular typing kit is also provided, and the vaginal flora is subjected to accurate molecular typing by adopting a real-time fluorescence quantitative PCR method, and the detection method comprises the following steps of: (1) Bacterial genome extraction, preparing flora nucleic acid from vaginal secretion by adopting a bacterial genome extraction kit; (2) qPCR detection, wherein primers and experimental conditions in the kit are used for detection, and SYBR Green I real-time quantitative PCR detection kit is adopted for PCR amplification; (3) Generating a data report, standardizing a fluorescence quantitative PCR detection result by using a primer pair 10 to obtain the proportion of 3 vaginal probiotics and 6 vaginal pathogenic bacteria in total vaginal bacteria, and carrying out accurate molecular typing on vaginal flora.

Definition of the definition

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe polymers of any length consisting of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), such as greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than about 1000 bases, up to about 10,000 or more bases, and which can be enzymatically or synthetically produced (e.g., PNAs described in U.S. patent No. 5948902a and references cited therein) that can hybridize to naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, e.g., can participate in watson-crick base pairing interactions. Naturally occurring nucleotides include guanine, cytosine, adenine and thymine (G, C, A and T, respectively).

The term "oligonucleotide" refers to single stranded polymers of about 2 to 200 or more, up to about 500 nucleotides or more. Oligonucleotides may be synthetic or enzymatically produced, and in some embodiments, are less than 10 to 50 nucleotides in length. The oligonucleotide may comprise a ribonucleotide monomer (i.e. may be an oligoribonucleotide) or a deoxyribonucleotide monomer. For example, the oligoribonucleotides may be 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51-60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 200 nucleotides in length.

The terms "determining", "measuring", "evaluating", "analyzing" and "measuring" are used interchangeably herein to refer to any form of measurement and include determining the presence or absence of an element. These terms include quantitative and/or qualitative determinations. The evaluation may be relative or absolute. "evaluating presence" includes determining the number of something present and determining whether it is present.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In some embodiments of the application, a kit for fluorescent quantitative PCR detection of vaginal microbial ecological molecule typing is provided. In some embodiments, it comprises a bacterial genome extraction kit, a fluorescent real-time quantitative PCR detection kit, specific primers for 9 bacteria that are dominant in the vagina (3 vaginal probiotics +6 vaginal pathogens), and primers for microbial 16srDNA that indicate the total amount of bacteria.

In some embodiments of the application, the primers are as follows:

。

in some embodiments of the present application, a method for using a kit for detecting vaginal microbial ecological molecule typing by fluorescence quantitative PCR is provided, which adopts a real-time fluorescence quantitative PCR method to accurately type vaginal flora, and comprises the following steps:

(1) Bacterial genome extraction, preparing flora nucleic acid from vaginal secretion by adopting a bacterial genome extraction kit;

(2) qPCR assay, using primers in kit and experimental conditions, see Table below

The PCR amplification is carried out by adopting a SYBR Green I real-time quantitative PCR detection kit;

(3) Generating a data report, standardizing a fluorescence quantitative PCR detection result by using a primer pair 10 to obtain the proportion of 3 vaginal probiotics and 6 vaginal pathogenic bacteria in total vaginal bacteria, and carrying out accurate molecular typing on vaginal flora.

The kit adopts real-time fluorescence quantitative PCR to carry out molecular typing. Real-time quantitative PCR is an emerging nucleic acid quantitative technology developed on the basis of a conventional PCR technology, and has the following advantages compared with the conventional PCR:

a) Absolute quantification of the initial template is achieved;

b) The detection sensitivity is high, and the target gene with low copy can be detected;

c) Differences in minute copy numbers can be distinguished;

d) The samples with larger initial template content difference can be quantified simultaneously;

e) The detection design is flexible; saving time and labor.

The kit comprises standardized reagents and operation procedures, has stable and reliable technology, is easy to operate in clinical laboratories, and has the minimum detection limit of 10 compared with the prior art ² Copy/microliter, the sensitivity of the PCR is more than 100 times that of the common PCR, and the detection accuracy is 100%. The method has low requirements on reaction conditions and equipment, can directly extract vaginal secretion to prepare flora nucleic acid without further separating and culturing infectious germs, and therefore, the primer is usedThe combination molecularly imprinted vaginal microorganisms.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

The above-described aspects of the present application will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present application is limited to the following examples only. All techniques implemented based on the above description of the application are within the scope of the application.

Drawings

FIG. 1 shows the CT values of the L.crispatus assay in the examples of the present application.

FIG. 2 is an amplification curve of L.crispatus in the examples of the present application.

FIG. 3 is a graph showing the amplification melting curve of L.crispatus in the examples of the present application.

FIG. 4 is a graph showing melting peaks per unit time of amplification of L.crispatus in the examples of the present application.

FIG. 5 shows the detection of CT values by L.jensenii in the examples of the present application.

FIG. 6 is an amplification curve of L.jensenii in the examples of the present application.

FIG. 7 is a graph showing the melting curve of L.jensenii in the examples of the present application.

FIG. 8 is a graph showing melting peaks per unit time of amplification of L.jensenii in the examples of the present application.

FIG. 9 shows the detection CT value of L.jensenii in the example of the present application.

FIG. 10 is an amplification curve of L.Iners in the examples of the present application.

FIG. 11 is an amplification melting curve of L.Iners in the examples of the present application.

FIG. 12 is a graph showing melting peaks per unit time of amplification of L.Iners in examples of the present application.

FIG. 13 shows the CT values of the G.vaginalis assay in the examples of the present application.

FIG. 14 is an amplification curve of G.vaginalis in the examples of the present application.

FIG. 15 is a graph showing the amplification melting curve of G.vaginalis in the examples of the present application.

FIG. 16 is a graph showing melting peaks per unit time of amplification of G.vaginalis in the examples of the present application.

FIG. 17 is a graph showing the CT values detected by A.vaginae in the examples of the present application.

FIG. 18 is an amplification curve of A. Vaginae in the examples of the present application.

FIG. 19 is a graph showing the amplification melting curve of A. Vaginae in the examples of the present application.

FIG. 20 is a graph showing melting peaks per unit time for amplification of A.vaginae in the examples of the present application.

Fig. 21 is an Eggerthella detection CT value in an embodiment of the application.

FIG. 22 is an amplification curve of Eggerthella in an example of the present application.

FIG. 23 is a graph showing the amplification melting curve of Eggerthella in an example of the present application.

FIG. 24 is a graph showing melting peaks per unit time for Eggerthella amplification in an example of the present application.

FIG. 25 shows the measurement of CT values by Megasphaera in the examples of the present application.

FIG. 26 is an amplification curve of Megasphaera in the examples of the present application.

FIG. 27 is a graph showing the melting curve of Megasphaera amplification in the examples of the present application.

FIG. 28 is a graph showing melting peaks per unit time of Megasphaera amplification in the examples of the present application.

FIG. 29 shows the detection CT values of Leptotrichia/Sneathia in the examples of the present application.

FIG. 30 is an amplification curve of Leptotrichia/Sneathia in the examples of the present application.

FIG. 31 is a graph showing the amplification melting curve of Leptotrichia/Sneathia in the examples of the present application.

FIG. 32 is a graph showing melting peaks per unit time of amplification of Leptotrichia/Sneathia in the examples of the present application.

FIG. 33 shows the CT number of the Prevotella test in the example of the present application.

FIG. 34 is an amplification curve of Prevotella in the example of the present application.

FIG. 35 is a graph showing the amplification melting point of Prevoltella in the examples of the present application.

FIG. 36 is a graph showing melting peaks per unit time of amplification by Prevoltella in the examples of the present application.

FIG. 37 shows the CT number of the Prevotella test in the example of the present application.

FIG. 38 is an amplification curve of all bacteria in the examples of the present application.

FIG. 39 is a graph showing the melting curve of all bacteria in the examples of the present application.

FIG. 40 is a graph showing melting peaks per unit time for the amplification of all bacteria in the examples of the present application.

FIG. 41 is a map of specific detection of 10 bacterial groups representing amplification curves of 10 bacterial groups by the real-time fluorescent quantitative PCR method in the example of the present application.

Detailed Description

In order to specifically illustrate the general design concept of the present application, specific experimental parameters are shown below as examples, but should not be construed as a reason for limiting the scope of the present application.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

FIGS. 1 to 4 are diagrams showing specific detection L.crispatus patterns of a real-time fluorescent quantitative PCR method according to an embodiment of the present application.

FIGS. 5 to 8 are diagrams showing specific detection L.jensenii by the real-time fluorescent quantitative PCR method according to the present application.

FIGS. 9 to 12 are specific detection L.Iners spectra of the real-time fluorescent quantitative PCR method in the examples of the present application.

FIGS. 13 to 16 are graphs of the specific detection G.vaginalis patterns of the real-time fluorescent quantitative PCR method according to the embodiment of the present application.

FIGS. 17 to 20 are specific detection A. Vaginae patterns of the real-time fluorescent quantitative PCR method in the examples of the present application.

FIGS. 21 to 24 are specific detection Eggerthella patterns of a real-time fluorescent quantitative PCR method according to an embodiment of the present application.

FIGS. 25 to 28 are diagrams showing specific detection Megasphaera patterns of a real-time fluorescent quantitative PCR method according to an embodiment of the present application.

FIGS. 29 to 32 are graphs of specific detection Leptotrichia/Sneathia patterns of a real-time fluorescent quantitative PCR method in an embodiment of the present application.

FIGS. 33 to 36 show the specific detection Prevotella pattern of the real-time fluorescent quantitative PCR method according to the embodiment of the present application.

FIGS. 37 to 40 are diagrams of specific detection all bacteria of the real-time fluorescent quantitative PCR method according to the embodiment of the present application.

Example 1

The kit comprises a bacterial genome extraction kit, a fluorescent real-time quantitative PCR detection kit, 9 specific primers for bacteria (3 vaginal probiotics and 6 vaginal pathogenic bacteria) dominant in vagina and a primer for 16srDNA of the microorganism indicating the total bacterial amount, and has high specificity and sensitivity, and a target gene is selected according to the existing literature and GenBank gene sequence.

Referring to sequences in GenBank, a primer set capable of specifically recognizing and amplifying reaction products is designed by sequence homology analysis, and the primer entrusts the synthesis of large genes, wherein the primer set is shown in table 1:

the method adopts a real-time fluorescence quantitative PCR method to accurately carry out molecular typing on vaginal flora, and comprises the following steps:

(1) Bacterial genome extraction, bacterial genome extraction kit was used to prepare flora nucleic acid from vaginal secretions, 3 parallel experiments were performed per bacterial sample: 10 ² Copy/microliter, 10 ³ Copy/microliter, 10 ⁴ Copy/microliter;

(2) qPCR detection was performed using the primers in the kit and experimental conditions, first real-time fluorescent quantitative PCR was performed using the primer pairs for each bacterium in Table 1, and the amplification conditions were searched, and then multiplex real-time fluorescent quantitative PCR was performed using all the primers in Table 1. The apparatus used was a PikoReal 96 real-time PCR apparatus (Thermo Scientific, massachusetts, usa) for real-time PCR. Real-time PCR conditions for specific detection of vaginal flora without cross-reaction with other microbial strains were established and the conditions for optimal results among the established real-time quantitative fluorescent PCR amplification conditions were applied to this example.

1ng of genomic DNA (template) of a standard strain extracted using a DNA extraction kit, and 1.5. Mu.l of 100pmol of a primer (primer set in Table 1 of the present application) were added to the PCR reaction solution. In addition, 10. Mu.l of 2 Xreal-time qPCR master mix (real-time qPCR master mixture) was added to the PCR reaction solution, and then three-stage distilled water was added until the PCR reaction solution reached 20. Mu.l.

Then, denaturation was performed using a Picoreal 96 real-time PCR apparatus at 95℃for 7 minutes, and the conditions of 95℃for 5 seconds and 60℃for 30 seconds were repeated through 40 cycles.

On the other hand, in order to confirm the result of the real-time PCR amplification reaction and quantitatively analyze it, a fluorescent material SYBR Green I was used for labeling. Meanwhile, the fluorescent material for labeling is not limited to the above example, and various fluorescent materials known in the art may be used.

Quantification in Real-timeepcr is a method of monitoring the progress of each cycle during PCR, measuring the amplified products in the exponential phase in Real time. At this point, an important concept is that Ct (threshold cycle) is a number. The value is that the fluorescence intensity generated by the amplification of the primer group obviously increases to the cycle times exceeding the baseline level, namely, the fluorescence intensity standard curve is obtained by comparing the fluorescence intensity with the microbial 16srDNA indicating the total bacterial amount, and then the fluorescence quantitative PCR detection result is standardized by the primer pair 10 to obtain the proportion of 3 vaginal probiotics and 6 vaginal pathogenic bacteria in the total vaginal bacteria, the accurate molecular typing is carried out on the vaginal flora, and the proportion of 3 vaginal probiotics and 6 vaginal pathogenic bacteria in the total vaginal bacteria is quantitatively analyzed.

As can be seen from FIGS. 1-41, 3 vaginal probiotics and 6 vaginal pathogenic bacteria as well as control total bacteria can be successfully amplified by using the primer fluorescence quantitative PCR of the application. The PCR amplification conditions after fumbling were: denaturation at 95℃for 7 min and repetition of 95℃for 5 sec and 60℃for 30 sec was performed by 40 cycles. The minimum detection limit reaches 102 copies/microliter.

Example 2

Quantitative PCR (qPCR) for samples to detect the presence of 6 vaginal pathogens, primer designs were developed for each organism based on computer analysis of published 16S rRNA gene sequences, as shown in Table 1. Primers were screened for multiplex qPCR compatibility and lack of cross-reactivity. The PCR products were specifically amplified to monitor the concentration-dependent decrease in fluorescence and confirm the amplified products by measuring the peak melting temperature (Tm) after amplification of the desired products was completed.

The method given in example 1 was used to perform accurate molecular typing, qPCR detection, multiplex fluorescent quantitative PCR detection using the primers and experimental conditions in the kit, real-time fluorescent quantitative PCR using the primer pairs for the bacteria in Table 1, and normalization of the fluorescent quantitative PCR detection results using primer pair 10, to obtain the proportion of 3 vaginal probiotics and 6 vaginal pathogenic bacteria in total bacteria in the vagina.

Materials were studied using real-time PCR. As can be seen from FIG. 41, the results were obtained that 3 vaginal probiotics and 6 vaginal pathogens as well as total bacteria were amplified effectively at the same time, 100% total bacteria/sample. Crispaties 15%/sample, L.jensenii 20%/sample, L.Iners 30%/sample, G.vaginalis 5%/sample, A.vaginalis 10%/sample, eggerthella 5%/sample, megasphaera 5%/sample, leptotrichia/Sneathia 5%/sample, prevoltella 5%/sample. From this, it can be initially judged that the sample is dominated by vaginal probiotics.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A vaginal flora molecular typing kit, comprising:

the kit comprises a bacterial genome extraction kit, a fluorescent real-time quantitative PCR detection kit, specific primers of 9 bacteria dominant in vagina and primers of microbial 16srDNA (deoxyribonucleic acid) indicating total bacterial amount, wherein the 9 bacteria dominant in vagina comprise 3 vaginal probiotics and 6 vaginal pathogenic bacteria, and the primers are as follows:

。

2. the vaginal flora molecular typing kit according to claim 1, further comprising a DNA polymerase, dntps, PCR buffer and a labeling material SYBR Green I for PCR amplification products.

3. A method of use for a kit for molecular typing of vaginal flora according to claims 1-2, characterized in that the accurate molecular typing of vaginal flora is performed by a real-time fluorescent quantitative PCR method comprising the steps of:

The PCR amplification is carried out by a marking material SYBR Green I real-time quantitative PCR detection kit;

4. The method of claim 3, wherein the 3 vaginal probiotics are l.crispatus, l.jensenii, l.inas.

5. A method of use according to claim 3, wherein the 6 vaginal probiotics are g.vaginalis, a.vaginalis, eggerthella, megasphaera, leptotrichia/Sneathia, prevotella.