CN112176079B

CN112176079B - Primer probe combination for detecting female vaginal microorganisms based on quadruple liquid drop digital PCR and application thereof

Info

Publication number: CN112176079B
Application number: CN202011164085.9A
Authority: CN
Inventors: 陈雯雯; 陈永欣; 王纪东; 郑佳莹; 黄炜塨; 刘晓蕾
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2023-06-23
Anticipated expiration: 2040-10-27
Also published as: CN112176079A

Abstract

The invention provides a primer probe combination for detecting female vaginal microorganisms based on quadruple liquid drop digital PCR and application thereof. The primer probe combination comprises: primer pairs and probes for amplifying and detecting the folE gene of Lactobacillus inertia, the gapR gene of Lactobacillus jensenii, the CbiQ1 gene of Lactobacillus crispatus and the murQ gene of Lactobacillus formans. The primer pair has better specificity, can avoid primer amplification of other strains during amplification, is used in combination with quadruple droplet digital PCR, classifies and quantitatively analyzes droplets amplified by the PCR, can determine the concentration of each lactobacillus in a sample, determines dominant microorganisms in female vagina, and realizes vaginal microorganism typing.

Description

Primer probe combination for detecting female vaginal microorganisms based on quadruple liquid drop digital PCR and application thereof

Technical Field

The invention relates to the technical field of microorganism detection, in particular to a primer probe combination for detecting female vaginal microorganisms based on quadruple liquid drop digital PCR and application thereof.

Background

There are a large number of microorganisms inside and outside the human body, which are widely involved in digestion, metabolism, immune defense and other processes. The vagina of a female is symbiotic with a plurality of microbial flora and has natural defense function on invasion of pathogens, and the vaginal microorganisms of healthy females are usually dominated by one of four types of lactobacillus including Lactobacillus Inertia (LI), lactobacillus Jensenii (LJ), lactobacillus Crispatus (LC) and lactobacillus formans (LG), and the lactobacillus can maintain the low pH environment of the vagina by producing lactic acid and hydrogen peroxide, and simultaneously produce bacteriocins and other antibacterial substances so as to prevent the reproduction of other pathogenic bacteria and maintain the normal vaginal ecosystem.

The vaginal microorganism plays an important role in maintaining the vaginal microecological health of women and preventing infection. When the micro-ecological disorder of the vagina, such as the decrease of the number of lactobacillus in the vagina and the large increase of gardnerella or other anaerobic bacteria, the flora in the vagina is disturbed, the natural defense function is destroyed, and the pathogen is easy to invade. Studies have shown that vaginal dysbacteriosis is closely related to female bacterial vaginitis, human Papillomavirus (HPV) or Human Immunodeficiency Virus (HIV) infection, and the like; women with vaginitis can affect sperm quality during pregnancy and even lead to infertility; even if pregnant, the pregnant woman is easy to cause intrauterine infection, birth canal infection and other links to infect the fetus, and serious consequences such as abortion, premature birth, congenital developmental deformity, mental retardation and the like are caused.

Currently, methods for vaginal flora analysis include microscopy, culture methods, high throughput sequencing, and the like. However, although the microscopic examination can directly check the cleanliness and the existence of trichomonas and fungal hyphae, the cost is low but the subjectivity is strong; the culture method has the advantages of complicated operation, long time consumption and poor repeatability, and is difficult to distinguish strains or strains with similar colony morphology; meanwhile, the high-throughput sequencing method comprises amplicon sequencing and metagenome sequencing, the types and the relative abundance of strains in a sample can be analyzed, but the absolute abundance information of the bacterial group is ignored in the sequencing, and a lot of strain information in the sequencing result is not the focus of research, so that the analysis of the result becomes complicated and difficult due to a large amount of sequencing result. However, the limitation of the sequencing depth, which cannot identify the strain with the relative abundance less than 0.1%, so that the strain of interest in the research cannot be identified by sequencing because of the low relative abundance; and the sequencing cost is high, the required period is long, so that the method is not suitable for routine detection.

Researchers have also developed fluorescent staining products for fungi, anaerobic bacteria and trichomonas in vaginal secretion samples, for example CN110940646a discloses a vaginal microorganism detection dual fluorescent staining solution comprising independent dual fluorescent staining solution a and dual fluorescent staining solution B; the double fluorescent staining solution A consists of a fluorescent staining agent, an auxiliary staining agent, a staining solution buffer reagent, a bacteriostat and water, and the double fluorescent staining solution B consists of a staining solution buffer reagent, an alkaline adjusting reagent, an anti-quenching agent, a bacteriostat and water. However, the method is complicated in preparation and staining, needs to smear and stain on a glass slide, and also needs to switch different microscope light sources in the observation process, so that the operation is complicated.

Therefore, it is important in the art to provide a detection method capable of rapidly distinguishing the dominant microbial flora of female vagina and judging whether the microbial flora of female vagina is disordered or not.

Disclosure of Invention

In view of the problems existing in the prior art, the invention aims to provide a primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR and application thereof. The primer probe combination can specifically amplify lactobacillus in female vagina microorganisms, so as to determine dominant microorganisms in female vagina and realize vagina microorganism typing.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR (ddPCR), comprising:

primer pairs and probes for amplifying and detecting the folE gene of Lactobacillus Inertia (LI), the gapR gene of Lactobacillus Jensenii (LJ), the CbiQ1 gene of Lactobacillus Crispatus (LC) and the murQ gene of Lactobacillus bifidus (LG).

In the invention, aiming at four common dominant colonies of female vaginal microorganisms, including lactobacillus inertia, lactobacillus jensenii, lactobacillus crispatus and lactobacillus formans, corresponding primer pairs for amplifying specific genes are designed, wherein the specific genes are only specific to one strain, and the other three strains and other vaginal microorganisms do not have the genes. The primer pair has better specificity, and can avoid the primer from amplifying other strains during amplification, so that the primer pair can well distinguish four lactobacillus strains, and then judges the concentration of the corresponding strain through fluorescence emitted by the corresponding fluorescent probe, and further judges which of the four strains is dominant by the vaginal microorganism, and if the fluorescence intensity of the four strains is lower, the vaginal microorganism flora is disordered.

As a preferable technical scheme of the invention, the nucleotide sequence of the primer pair for amplifying the folE gene is shown as SEQ ID NO. 1-2.

Preferably, the nucleotide sequences of the primer pair for amplifying the gapR gene are shown in SEQ ID NO. 3-4.

Preferably, the nucleotide sequences of the primer pair for amplifying the CbiQ1 gene are shown in SEQ ID NO. 5-6.

Preferably, the nucleotide sequences of the primer pair for amplifying the murQ gene are shown in SEQ ID NO. 7-8.

Preferably, the 5 'end of the probe is modified with a luminescent reporter fluorescent group, and the 3' end is modified with a fluorescence quenching group.

Preferably, the luminescent reporter fluorophore comprises either FAM, HEX, TET, JOE, CY or CY 5.

Preferably, the fluorescence quenching group includes any one of MGB, BHQ1 (Blackhole Quencher 1), BHQ2, or BHQ 3.

As a preferable technical scheme of the invention, the nucleotide sequence of the probe for detecting the folE gene is shown as SEQ ID NO. 9.

Preferably, the nucleotide sequence of the probe for detecting the gapR gene is shown in SEQ ID NO. 10.

Preferably, the nucleotide sequence of the probe for detecting the CbiQ1 gene is shown as SEQ ID NO. 11.

Preferably, the nucleotide sequence of the probe for detecting the murQ gene is shown as SEQ ID NO. 12.

Preferably, the working concentration of the probe for detecting the folE gene is 100 to 400nM, and may be, for example, 120nM, 150nM, 160nM, 180nM, 200nM, 220nM, 250nM, 300nM, 320nM, 350nM or 380nM, etc., preferably 200nM.

Preferably, the probe for detecting the gapR gene has an operating concentration of 200 to 500nM, for example, 220nM, 250nM, 280nM, 300nM, 320nM, 350nM, 380nM, 400nM, 420nM, 450nM or 480nM, and the like, preferably 300nM.

Preferably, the working concentration of the probe for detecting the CbiQ1 gene is 100 to 400nM, for example, 120nM, 150nM, 160nM, 180nM, 200nM, 220nM, 250nM, 300nM, 320nM, 350nM or 380nM, etc., preferably 200nM.

Preferably, the working concentration of the probe for detecting the murQ gene is 200 to 500nM, for example, 220nM, 250nM, 280nM, 300nM, 320nM, 350nM, 380nM, 400nM, 420nM, 450nM or 480nM, etc., preferably 300nM.

In the invention, the working concentration of the probe can influence the two-dimensional graph result of ddPCR, and the change of the probe concentration can lead the liquid drop to have different fluorescence intensities and be distributed at different positions. The working concentration in the invention can make the two-dimensional graph result of ddPCR clearer, and the droplet distribution is more uniform and will not influence each other.

The specific sequences are shown in Table 1 below:

TABLE 1

In a second aspect, the invention provides an application of the primer probe combination in preparing a female vaginal microorganism typing detection kit.

In a third aspect, the invention provides a kit for female vaginal microorganism typing detection, comprising the primer probe combination of the first aspect.

In a fourth aspect, the present invention also provides a method for using the kit for female vaginal genotyping detection according to the third aspect, comprising the steps of:

(1) Collecting a vaginal swab sample and extracting bacterial gDNA in the vaginal swab sample;

(2) Combining and mixing bacterial gDNA with a primer probe in the kit to prepare liquid drop digital PCR reaction liquid;

(3) Adding the liquid drop digital PCR reaction liquid into a sample hole of a liquid drop generator, and carrying out PCR amplification after obtaining micro drops;

(4) The droplets were counted and classified while fluorescent signals were detected, and analyzed to obtain concentrations of lactobacillus inertens, lactobacillus jensenii, lactobacillus crispatus and lactobacillus formans.

In the invention, a sample is amplified by utilizing a primer probe combination and a liquid drop digital PCR technology, the primer probe combination comprises primer pairs and probes for amplifying four specific genes, a reaction system is divided into micro drops by the liquid drop digital PCR technology, the PCR amplification can be independently carried out in the micro drops, the primer probe combination is mixed and then added into the reaction system, the primers can not be affected mutually, the specific amplification can still be better realized, and after fluorescence is detected by counting by using instruments such as micro drop counting and the like and fluorescent signal detection equipment, the liquid drops can be classified and quantitatively analyzed, so that the concentration of each lactobacillus in the sample can be determined. Based on the quantitative results of quadruple ddPCR for each bacterium, the highest concentration (significantly higher than the others) of lactobacillus was considered the dominant bacterium for the sample vagina. If the concentrations of the four lactobacilli are low, this means that the sample has a low number of lactobacilli, and the vaginal flora may be in a sub-healthy or disturbed state, requiring further examination.

The annealing temperature in the PCR amplification in the step (3) is 55-63 ℃, preferably 61 ℃.

Preferably, the reaction conditions for the PCR amplification in step (3) are: the pre-denaturation is carried out for 8 to 12 minutes at 94 to 98 ℃, and then the pre-denaturation is carried out for 35 to 40 cycles at 94 to 95 ℃ for 30 to 40 seconds and 55 to 63 ℃ for 60 to 90 seconds. Wherein the annealing and the extending steps are performed at the same temperature.

Preferably, the reaction conditions for the PCR amplification in step (3) are: the pre-denaturation was carried out at 95℃for 10min and then at 94℃for 30s and 61℃for 1min for 40 cycles.

Preferably, the step (3) further comprises a step of sealing the resulting droplets before the PCR amplification.

As a preferable technical scheme of the invention, the using method comprises the following steps:

(1) Collecting a vaginal swab sample, and extracting bacterial gDNA in the vaginal swab sample;

the sequence of the primer pair in the primer probe combination is shown as SEQ ID NO. 1-8, and the sequence of the probe in the primer probe combination is shown as SEQ ID NO. 9-12;

(3) Adding the liquid drop digital PCR reaction liquid into a sample hole of a liquid drop generator, adding droplet generation oil into an oil hole of the liquid drop generator, covering a gasket, preparing and obtaining droplets, transferring the droplets to a 96-hole PCR plate, and performing PCR amplification after sealing a film;

(4) After the PCR amplification is completed, the microdroplets are counted and classified, fluorescent signals are detected at the same time, and the concentrations of the lactobacillus inertia, the lactobacillus jensenii, the lactobacillus crispatus and the lactobacillus formans are obtained through analysis.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The invention provides a primer probe combination capable of amplifying and quantitatively analyzing inert lactobacillus, jean lactobacillus, lactobacillus crispatus and lactobacillus formans in a female vaginal swab sample by using quadruple droplet digital PCR, wherein the primer probe combination comprises four pairs of primer pairs and four corresponding probes, and the four pairs of primer pairs specifically amplify and detect folE genes of the inert lactobacillus, gapR genes of the jean lactobacillus, cbiQ1 genes of the lactobacillus crispatus and murQ genes of the lactobacillus formans respectively; the primer pair has better specificity, can avoid the primer from amplifying other strains during amplification, distinguishes four lactobacillus species through the amplification of specific genes, and judges the concentration of the corresponding strains through fluorescence emitted by the corresponding fluorescent probes;

(2) The kit for female vaginal microorganism typing detection by utilizing the primer probe combination provided by the invention is used in combination with liquid drop digital PCR, the primer pairs can not be mutually influenced, the specific amplification can still be well realized, and after counting by using instruments such as droplet counting and fluorescence signal detection equipment to detect fluorescence, the liquid drops can be classified and quantitatively analyzed; meanwhile, by adjusting the proportion of the probes and the reaction annealing temperature, clusters of various positive liquid drops are separated as far as possible, and the accuracy of quantitative results is improved.

Drawings

FIG. 1 is a distribution diagram of droplets detected after quadruple ddPCR in example 2.

FIG. 2 (A) is a graph showing fluorescence intensity obtained by fluorescent quantitative PCR of Lactobacillus bifidus and Lactobacillus jensenii in example 3.

FIG. 2 (B) is a graph showing fluorescence intensity obtained by subjecting Lactobacillus inertia and Lactobacillus crispatus to fluorescent quantitative PCR in example 3.

FIG. 3 is a distribution diagram of droplets detected after quadruple ddPCR in example 4.

FIG. 4 is a distribution diagram of droplets detected after quadruple ddPCR in example 5.

FIG. 5 is a distribution diagram of droplets detected after quadruple ddPCR in example 6.

Detailed Description

The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present invention and do not represent or limit the scope of the invention, which is defined by the claims.

In the examples below, the reagents used, unless otherwise specified, are available from manufacturers conventional in the art; the experimental methods used are all those known to the person skilled in the art.

Example 1

In this embodiment, a primer probe combination and a method for detecting vaginal microorganisms using the same are provided.

(1) Primer design

In order to distinguish the four lactobacillus species (LI, LJ, LC, LG) to be detected, in this embodiment, the specific genes of the four lactobacillus species are selected to design primers and probes, so that the specificity of the primers is ensured, and other strains are prevented from being amplified by the primers.

Wherein the specific genes are respectively the folE gene of Lactobacillus Inertia (LI), the gapR gene of Lactobacillus Jensenii (LJ), the CbiQ1 gene of Lactobacillus Crispatus (LC) and the murQ gene of Lactobacillus formans (LG). The sequences of the primers are shown below:

LI folE forward primer (SEQ ID NO: 1):

5′-GTGGCAGTAGGTGAAGATCC-3′；

LI folE reverse primer (SEQ ID NO: 2):

5′-TGGTCGATGATTGAGTGACG-3′；

LJ gapR forward primer (SEQ ID NO: 3):

5′-GCGAATTGGAGTTTGTTCCG-3′；

LJ gapR reverse primer (SEQ ID NO: 4):

5′-TTACCTCCGGTAGCTTTTGC-3′；

LC CbiQ1 forward primer (SEQ ID NO: 5):

5′-GGAATTTGTCAACGCGTATCT-3′；

LC CbiQ1 reverse primer (SEQ ID NO: 6):

5′-GATCGGGCAAAGTCTGTAGC-3′；

LG murQ forward primer (SEQ ID NO: 7):

5′-GGAAATTAAAAACTTAACTACGGAGC-3′；

LG murQ reverse primer (SEQ ID NO: 8):

5′-GTTTTGACCATGTCGATAGTTGAC-3′；

when in use, the above primers are mixed in equimolar amounts to prepare a primer mixture.

(2) Probe design

In order to save cost, FAM groups are modified on LI and LJ probes, HEX groups are modified on LC and LG probes, and then two targets can be detected on the same fluorescent channel. The sequence of the probe is as follows:

LI folE probe (SEQ ID NO: 9):

5′-FAM-TCGCAGTGGATTAGTAGAAACACC-MGB-3′；

LJ gapR probe (SEQ ID NO: 10):

5′-FAM-TCGTGGTGCGTTAGGTGAAAGCGTGGA-BHQ1-3′；

LC CbiQ1 probe (SEQ ID NO: 11):

5′-HEX-ACCTTGACGATTGCCTGCGTGACACGT-BHQ1-3′；

LG murQ probe (SEQ ID NO: 12):

5′-HEX-AACCCCGCGACTATGCACAT-MGB-3′；

(3) Extraction of gDNA from vaginal swab samples

1/3 of the secretion on the vaginal side wall and the secretion of the cervix were collected: before collecting cervical secretion, wiping mucus and attachments on the cervical surface by using a sterile cotton swab, extending a new sterile cotton swab into a cervical canal, and rotating clockwise for 3 weeks;

placing the swab in a 2mL centrifuge tube, adding 500 mu L of PBS and 50U of mutalysozyme, and incubating at 37 ℃ for 30min; bacterial gDNA was extracted using QIAamp DNA Mini Kit (Qiagen) and finally eluted with 100. Mu.L Buffer AE.

(4) Preparation of ddPCR reaction System, the specific reaction System is shown in Table 2 below:

TABLE 2

Component (A)	Volume of
		ddPCR Supermix for Probes(No dUTP)(Bio-Rad)	10μL
Primer mixture	1.8μL
		LI folE probe	0.4μL
LC CbiQ1 probe	0.4μL
		LJ gapR probe	0.6μL
LG murQ probe	0.6μL
		Sample to be measured	2μL
Sterile water	4.2μL
		Total volume of	20μL

After the preparation of the reaction system is completed, the concentration of each primer is 900nM;

LI folE probe was 200nM, LC CbiQ1 probe was 200nM, LJ gapR probe was 300nM, and LG murQ probe was 300nM.

(5) Droplet generation

After the prepared ddPCR amplification system is uniformly mixed, adding the mixture into a sample hole of a droplet generator QX200 Droplet Generator (matched with a Bio-rad ddPCR system), adding 70 mu L of droplet generation oil into an oil hole, covering a gasket, and putting the mixture into the droplet generator to prepare droplets;

(6) PCR amplification

Transferring 40 mu L of the microdroplet to a 96-well PCR plate, and performing amplification on a Bio-rad T100 PCR instrument after membrane sealing, wherein the PCR reaction conditions are as follows: pre-denaturation at 95℃for 10min,94℃for 30sec,61℃for 1min for 40 cycles;

(7) Interpretation of results

After the amplification is completed, transferring the PCR reaction plate to a Bio-rad ddPCR system matched with a QX200 Droplet Reader, and performing Droplet counting and fluorescent signal detection according to instrument and software operation instructions;

meanwhile, droplets were classified by QuantaSoft Analysis Pro version 1.0.596 software, and the concentration of LI, LJ, LC, LG was calculated.

Based on the quantitative results of quadruple ddPCR for each bacterium, the highest concentration of lactobacillus was considered the dominant bacterium for the sample vagina.

If the concentrations of the four lactobacilli are similar and lower than in the normal sample, this means that the sample has a lower number of lactobacilli and the vaginal flora may be in a sub-healthy or disturbed state, requiring further examination.

Example 2

In this example, the primer probe combinations and detection methods provided in example 1 were used to type plasmid mixtures of lactobacillus helveticus (LI), lactobacillus Jensenii (LJ), lactobacillus Crispatus (LC), lactobacillus formans (LG).

The method comprises the following specific steps:

(1) Designing primers aiming at LI folE, LJ gapR, LC CbiQ1 and LG murQ genes, amplifying gene fragments of LI folE, LJ gapR, LC CbiQ1 and LG murQ by using gDNA extracted from a LI, LJ, LC, LG strain separated clinically by the primers, and completing cloning and transformation by using pEASY-Blunt Cloning Kit;

(2) Plasmids were extracted from E.coli using TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 (TaKaRa);

(3) Plasmids of LI folE, LJ gapR, LC CbiQ1, LG murQ were mixed as test samples, wherein the concentration of each plasmid was 2X 10 ³ Copy/. Mu.L; the remaining detection steps were performed as described in reference example 1.

The ddPCR results are shown in FIG. 1, and the specific classification of droplets in FIG. 1 is shown in Table 3 below:

TABLE 3 Table 3

As can be seen from the colony types and ddPCR results in the binding table, four single positive droplets, double positive droplets, triple positive droplets and four positive droplets, namely negative droplets, LI positive (LI+), LJ positive (LJ+), LC positive (LC+), LG positive (LG+), were detected by performing ddPCR typing using the mixed plasmids (including LI folE, LJ gapR, LC CbiQ1, LG murQ); the obtained liquid drops can be well distinguished, the liquid drops are not mutually influenced, and adverse phenomena such as tailing and the like which influence the judgment of the result are not generated.

Example 3

The difference from example 2 is that amplification is not performed using ddPCR in this example, but is performed using only a general amplification method, namely: mixing the primer probe combination with a sample, directly preparing a PCR reaction system, and performing fluorescent quantitative PCR;

wherein, 20 mu L of reaction system is adopted for fluorescence quantitative PCR, and the reaction system comprises a reaction system a for detecting LG and LJ and a reaction system b for detecting LC and LI.

Reaction system a is shown in table 4 below:

TABLE 4 Table 4

Wherein, after the preparation of the reaction system is completed, the concentration of each primer is 300nM; LJ gapR probe was 200nM and LG murQ probe was 200nM.

Reaction system b is shown in table 5 below:

TABLE 5

Wherein, after the preparation of the reaction system is completed, the concentration of each primer is 300nM; the LI folE probe was 200nM and the LC CbiQ1 probe was 200nM.

The sample to be tested can be plasmid DNA or sample DNA, in this example, plasmid DNA is used as the sample to be tested, and the plasmid concentration corresponding to each strain is 2×10 ⁵ Copy/. Mu.L;

after incubation of the reaction system at 50℃for 2min, pre-incubation was performed at 95℃for 20s followed by a total of 40 thermal cycles of 3s at 95℃and 30s at 60 ℃.

Fluorescence quantitative PCR instrument was used, detection using FAM channel and HEX channel.

The obtained results are shown in FIG. 2 (A) and FIG. 2 (B), wherein FIG. 2 (A) is a fluorescence intensity curve obtained by detecting LG and LJ, and FIG. 2 (B) is a fluorescence intensity curve obtained by detecting LI and LC; while fluorescence quantitative PCR can only achieve double detection on the premise that only two kinds of fluorophores are used, ddPCR can achieve quadruple detection by using only two kinds of fluorophores and can provide absolute quantitative results.

Example 4

The difference from example 2 is that in this example, the annealing temperature was adjusted to 63℃and the remaining steps were kept the same as in example 2.

The results are shown in FIG. 3, in which the strains represented by the corresponding positions remain the same as in example 2.

Example 5

The difference from example 2 is that in this example, the annealing temperature was adjusted to 56.5℃and the remaining steps were kept the same as in example 2.

The results obtained are shown in FIG. 4, in which the strains represented by the corresponding positions remain the same as in example 2.

As is clear from a comparison of example 2 with examples 4 and 5, the amplification effect is better at lower temperatures, the fluorescence value is higher, the droplet clusters are more separated, but the tailing is severe. At higher temperatures, the specificity is stronger, the non-specific amplification is reduced, the tailing is reduced, but the amplification effect is poor.

Thus, taking into account the combination of specificity, amplification efficiency and better differentiation between different droplet clusters, 61℃was chosen as annealing temperature in the present invention.

Example 6

The difference from example 2 is that in this example, the concentrations of LI folE probe, LC CbiQ1 probe, LJ gapR probe and LG murQ probe are all 200nM, and the rest of the procedure remains the same as in example 2.

The results obtained are shown in FIG. 5, in which the strains represented by the corresponding positions remain the same as in example 2.

As can be seen from the figure, although the droplet clusters of LI and LJ can be separated, the droplet clusters of LC and LG cannot be separated, the detection result is unclear as compared with example 2, and four kinds of lactobacillus cannot be distinguished well and the concentration thereof can be obtained.

In conclusion, the primer probe combination for the female vaginal swab sample by utilizing the quadruple liquid drop digital PCR can specifically amplify and detect the folE gene of the inert lactobacillus, the gapR gene of the jean lactobacillus, the CbiQ1 gene of the curly lactobacillus and the murQ gene of the format lactobacillus, can avoid the primer amplification of other strains during amplification, simultaneously, the primer pairs can not be mutually influenced by the quadruple liquid drop digital PCR technology, can still better realize the specific amplification, and can classify and quantitatively analyze liquid drops after counting by instruments such as a liquid drop count instrument and fluorescent signal detection equipment to detect fluorescence.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

SEQUENCE LISTING

<110> Shenzhen university

<120> a primer probe combination for detecting female vaginal microorganisms based on quadruple droplet digital PCR

<130> 20201019

<160> 12

<170> PatentIn version 3.3

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Claims

1. The primer probe combination for detecting female vaginal microorganisms based on quadruple liquid drop digital PCR is characterized by comprising primer pairs and probes for amplifying and detecting folE gene of inert lactobacillus, gapR gene of jean lactobacillus, cbiQ1 gene of frizzled lactobacillus and murQ gene of format lactobacillus;

the nucleotide sequence of the primer pair for amplifying the folE gene is shown as SEQ ID NO. 1-2;

the nucleotide sequence of the primer pair for amplifying the gapR gene is shown in SEQ ID NO. 3-4;

the nucleotide sequence of the primer pair for amplifying the CbiQ1 gene is shown as SEQ ID NO. 5-6;

the nucleotide sequence of the primer pair for amplifying the murQ gene is shown in SEQ ID NO. 7-8;

the nucleotide sequence of the probe for detecting the folE gene is shown as SEQ ID NO. 9;

the nucleotide sequence of the probe for detecting the gapR gene is shown as SEQ ID NO. 10;

the nucleotide sequence of the probe for detecting the CbiQ1 gene is shown as SEQ ID NO. 11;

the nucleotide sequence of the probe for detecting the murQ gene is shown as SEQ ID NO. 12.

2. The primer probe combination of claim 1, wherein the probe is modified at the 5 'end with a luminescent reporter fluorophore and at the 3' end with a fluorescence quenching group.

3. The primer probe combination of claim 2, wherein the luminescent reporter fluorophore comprises any one of FAM, HEX, TET, JOE, CY or CY 5.

4. The primer probe combination of claim 2, wherein the fluorescence quenching group comprises any one of MGB, BHQ1, BHQ2, or BHQ 3.

5. The primer probe combination according to any one of claims 1 to 4, wherein the working concentration of the probe for detecting the folE gene is 100 to 400nM;

the working concentration of the probe for detecting the gapR gene is 200-500 nM;

the working concentration of the probe for detecting the CbiQ1 gene is 100-400 nM;

the working concentration of the probe for detecting the murQ gene is 200-500 nM.

6. Use of a primer probe combination according to any one of claims 1 to 5 for the preparation of a kit for female vaginal genotyping detection.

7. A kit for female vaginal genotyping, comprising the primer probe combination of any one of claims 1-5.