CN106701915B - Preparation method and application of LH-PCR bacterial gene fingerprint of bacterial vaginosis - Google Patents

Abstract

The invention provides a method for preparing a bacterial gene fingerprint of bacterial vaginosis by applying LH-PCR technology, and application of the bacterial gene fingerprint in clinical accurate diagnosis of the bacterial vaginosis. The bacterial gene fingerprint spectrum can be used for clinical rapid diagnosis of bacterial vaginosis, avoids the defects of inaccurate diagnosis, complicated operation, long time consumption, large workload, high price of high-throughput sequencing and the like of the traditional bacterial isolation culture, and can accurately, rapidly and effectively detect the flora distribution condition in the vagina.

Description

Preparation method and application of LH-PCR bacterial gene fingerprint of bacterial vaginosis

Technical Field

The invention relates to a preparation method of a bacterial gene fingerprint, in particular to a method for preparing a bacterial gene fingerprint of bacterial vaginosis by applying LH-PCR technology.

Background

Bacterial Vaginosis (BV) is a vaginal infection caused by bacteria, is a common lower genital tract infectious disease of women in the childbearing age, and can cause various adverse pregnancy outcomes, such as spontaneous abortion, premature birth, amniotic fluid infection, postpartum endometritis, caesarean section incision infection, perinatal complications and the like. Recent studies have found that recurrent and persistent infection with bacterial vaginosis can also increase the risk of trichomonas vaginitis, candida albicans vaginitis, cervical cancer and Human Immunodeficiency Virus (HIV).

At present, the causes of bacterial vaginosis, particularly the difference of the vaginal flora constitution of patients suffering from bacterial vaginosis, are lack of intensive research. The traditional culture method cannot completely reflect the composition of vaginal flora, so that the research on pathogenic bacteria of bacterial vaginosis patients is limited, and sometimes, although the clinical evaluation of the patients is a cured state, the composition of the vaginal flora of the patients is not recovered to a normal state, and the pathogenic bacteria still exist, so that the bacterial vaginosis can relapse, the treatment of the disease is delayed, and the relapse rate of the disease is increased if the treatment is stopped only through the clinical evaluation.

With the rapid development of molecular biology technology in recent 20 years, the trend of using molecular biology methods to research the recurrence mechanism of bacterial vaginosis and the detailed diagnosis of different subtypes of bacterial vaginosis in clinic is a trend, and people are also in continuous depth on the research on the flora structure of bacterial vaginosis. Length polymorphism PCR (LH-PCR) uses fluorescently labeled probes to determine the relative amount of amplified sequences from different microorganisms, and the labeled fragments are separated by capillary electrophoresis and monitored by an automated fluorescent gene sequence analyzer. LH-PCR is a method for analyzing microbial structures based on the principle that certain specific genes exhibit polymorphisms in their inherent length due to insertion or splicing of genes or gene operons. Thus, such polymorphisms can be utilized to determine population information for microorganisms. The LH-PCR determined hypervariable region is mainly present in the small subunit of the ribosome (rrn).

LH-PCR is used as a molecular fingerprint spectrum technology based on PCR, and compared with a high-throughput sequencing technology, the LH-PCR has the advantages of sensitive technical operation, rapidness, simplicity, low components and the like. However, there is no report on the application of this technique to the medical field, particularly as a diagnostic technique for a disease.

Disclosure of Invention

The invention aims to provide a method for preparing a bacterial gene fingerprint of bacterial vaginosis by applying LH-PCR technology and application of accurately judging whether bacterial vaginosis pathogenic flora exists in a sample by applying the bacterial gene fingerprint. The bacterial gene fingerprint spectrum can be used for clinical rapid diagnosis of bacterial vaginosis, avoids the defects of inaccurate diagnosis, complicated operation, long time consumption, large workload, high price of high-throughput sequencing and the like of the traditional bacterial isolation culture, and can accurately, rapidly and effectively detect the flora distribution condition in the vagina.

In order to achieve the above object, the present invention provides a method for preparing LH-PCR bacterial gene fingerprint of bacterial vaginosis, wherein the bacteria are intravaginal bacteria, and the gene is 16S rRNA gene.

Furthermore, the preparation method of the LH-PCR bacterial gene fingerprint map of the bacterial vaginosis is to analyze the gene segment with the length of 310-380bp of the amplified segment.

Furthermore, the LH-PCR bacterial gene fingerprint map of the bacterial vaginosis is prepared by the method, wherein when the relative peak area of any one fragment in the fragment length of 340-374bp is more than 10%, the existence of pathogenic bacteria of the bacterial vaginosis is determined; when the relative peak area of all the fragments in the fragment length of 340-374bp is less than 10 percent, the normal flora is determined, and no pathogenic flora of the bacterial vaginosis exists.

Preferably, the LH-PCR bacterial gene fingerprint map preparation method of the bacterial vaginosis comprises the following steps: 5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-ATTACCGCGGCTGCTGG-3', wherein the 5' end of 5'-ATTACCGCGGCTGCTGG-3' is fluorescently labeled with FAM.

Further, the preparation method of the LH-PCR bacterial gene fingerprint map of the bacterial vaginosis comprises the following steps:

1. extracting bacterial genome DNA of vaginal secretion;

2. the 16S rRNA gene in the bacterial genome DNA was subjected to PCR amplification:

primer: 5'-AGAGTTTGATCCTGGCTCAG-3', and

5'-ATTACCGCGGCTGCTGG-3', wherein the 5' end is fluorescently labeled with FAM;

reaction system for PCR (50. mu.L): 5. mu.L of 10 Xbuffer, 1. mu.L of dNTPs, 1.5. mu.L of each primer, 1. mu.L of DNA polymerase, 2.5. mu.L of BSA, 1. mu.L of template DNA, ddH₂O is complemented to 50 mu L; setting negative control without adding template;

reaction procedure: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 45s, annealing at 55 deg.C for 1min, extension at 72 deg.C for 2min, 30 cycles, and storing at constant temperature of 4 deg.C;

3. capillary electrophoresis LH-PCR products:

enriching the PCR product by 1.0% agarose gel electrophoresis, and collecting 300-400bp fragments; then using Qiagen gel to recover and purify, mixing with Hi-Di formamide and GS500Liz internal dimension standard, denaturing the mixture in a PCR instrument at 95 ℃ for 5min, and then carrying out capillary electrophoresis detection, wherein the injection time is 10s, the injection and running voltage is 15.0kV, the running temperature is 60 ℃, and the running time is 70 min;

4. analysis of capillary electropherograms

The fingerprint electrophoresis pattern of the sample is collected by GeneScan 3.7, LH-PCR data is processed by BioNumerics6.0 software, and the fragment with the amplified fragment length of 310-380bp is analyzed.

Preferably, the LH-PCR bacterial gene fingerprint of bacterial vaginosis is prepared by a method wherein the bacterial species is selected from the group consisting of Mycobacterium, Ureapasma, Mycoplasma, Sneathia, Corynebacterium, Atopobium, Gardnerella, Mobilucus, Prevotella, Actinomyces, Staphylococcus, Anaerococcus, Peptoniphilus, Megasphaera, Lactobacillus.

Preferably, the preparation method of LH-PCR bacterial gene fingerprint of bacterial vaginosis is characterized in that the bacterial species and the corresponding amplified fragment length are as follows:

the invention also provides application of the bacterial gene fingerprint prepared by the preparation method in diagnosing bacterial vaginosis.

The invention also provides application of the bacterial gene fingerprint prepared by the preparation method in judging whether bacterial vaginosis pathogenic flora exists in a sample.

The invention also provides application of the bacterial gene fingerprint prepared by the preparation method in judging whether bacteria such as Mycobacterium, Urealasma, Mycoplasma, Sneathia, Corynebacterium, Atopobium, Gardnerella, Mobilucus, Prevotella, Actinomyces, Staphylococcus, Anaerococcus, Peptoniphilus, Megasphaera and Lactobacillus exist in a sample.

Any fingerprinting technique is intended to provide an overall picture of a population, rather than to distinguish between individual species. Therefore, it mainly reflects the evolution or difference of flora structure in the environment to analyze the correlation between disease state and flora structure. LH-PCR can easily track the change of flora structure by analyzing the peak shape change, can accurately analyze the flora structure of vaginal secretion, and can classify and identify main pathogenic bacteria. The method is relatively simple to operate, and the error generation probability is reduced by fewer operation steps, so that the reproducibility is very good. In addition, compared with LH-PCR (LH-PCR) and other fingerprint spectrum technologies such as T-RFLP (Terminal restriction fragment length polymorphism detection), PCR products do not need to be subjected to restriction enzyme cutting, gene analysis can be directly carried out, and the composition information of the microbial population can be obtained.

Drawings

FIG. 1: principal Component Analysis (PCA) of LH-PCR characteristic peaks for samples from group D0 (0), group SG-D7 (1), group FG-D7 (2), group SG-D30 (3), and group FG-D30 (4) 5.

FIG. 2: LH-PCR characteristic peaks for samples from group D0 (0), group SG-D7 (1), group FG-D7 (2), group SG-D30 (3), group FG-D30 (4) and 5 groups; the horizontal axis is the fragment length and the vertical axis is the relative peak area (in%) of each fragment length.

FIG. 3: example 2 LH-PCR profile of patient 1 before administration (D0); the horizontal axis is the fragment length and the vertical axis is the relative peak area (in%) of each fragment length.

FIG. 4: example 2 LH-PCR profile 7 days after patient 1 administration (D7); the horizontal axis is the fragment length and the vertical axis is the relative peak area (in%) of each fragment length.

FIG. 5: example 2 LH-PCR profile of patient 1 30 days after administration (D30); the horizontal axis is the fragment length and the vertical axis is the relative peak area (in%) of each fragment length.

FIG. 6: example 2 LH-PCR profile of patient 2 before dosing (D0); the horizontal axis is the fragment length and the vertical axis is the relative peak area (in%) of each fragment length.

FIG. 7: example 2 LH-PCR profile 7 days after administration of patient 2 (D7); the horizontal axis is the fragment length and the vertical axis is the relative peak area (in%) of each fragment length.

FIG. 8: example 2 LH-PCR profile of patient 2 30 days after administration (D30); the horizontal axis is the fragment length and the vertical axis is the relative peak area (in%) of each fragment length.

Detailed Description

The present invention is further described in detail by the following examples, and it will be appreciated by those skilled in the art that modifications may be made without departing from the scope and spirit of the invention.

Bacterial genomic DNA extraction kit (DP302) was purchased from: tiangen Biochemical technology Ltd.

All data were analyzed using SPSS15.0(Chicago, IL, USA) and if P <0.05, the data were statistically different.

Example 1: bacterial gene fingerprint spectrum of bacterial vaginosis prepared by LH-PCR technology

1. Study subjects and criteria:

1.1 study subjects: 63 women of childbearing age diagnosed with bacterial vaginosis were selected, treated with metronidazole gel on a standard 5 days basis and sampled before (0 days), 7 days after, and 30 days after drug administration.

1.2 grouping criteria: bacterial Vaginosis (BV) was diagnosed using Amsel clinical diagnostic criteria and Nugent laboratory diagnostic criteria. All secretion smears were read by 2 experienced microbiologists in the micro-ecological laboratory.

1.3 exclusion criteria:

women in gestation, lactation and menopause

② long-term taking hormone medicine for other diseases

③ those taking immunosuppressant

Fourthly, there are internal diseases such as heart, liver, kidney, internal secretion, etc. (mainly by inquiry)

(v) other vaginal infections such as trichomonas vaginalis and vulvovaginal candidiasis

(meth) acrylic acid metronidazole allergy

Seventhly, the compliance is poor

1.4 follow-up time: the patients with BV were followed up twice, 7 days after administration and 30 days after administration. The patient was recorded for symptoms, signs, pH and Nugent scores at each visit.

1.5 treatment methods and cure criteria: patients with BV in-group were dosed with metronidazole gel (37.5mg, qd, vaginal drug application) for 5 consecutive days. The treatment effect was evaluated on day 7 and day 30 after the administration, respectively. The standard of cure is as follows: simultaneously meets the micro-ecological evaluation healing standard and the clinical evaluation healing standard to be healing. Microecological evaluation recovery criteria: nugent scores 0-3 points. Clinical recovery criteria: the vaginal secretion has normal property, a Whiff test is negative, clue cells disappear, and the pH value of the vaginal secretion is less than 4.5.

2. Sample preparation:

2.1. collecting samples: vaginal secretions were taken from 1/3 on the vaginal wall of the subject using two sterile cotton swabs, one of which was placed rapidly in a sterile centrifuge tube containing 1ml of PBS (pH:7.4) and placed in an ultra-low temperature freezer at-80 ℃ for genomic extraction. The other was spread evenly on a glass slide, stained with gram stain, observed under an oil mirror, and subjected to Nugent scoring.

2.2. Extraction of bacterial genomic DNA (using bacterial genomic extraction kit from tiangen Biochemical technology Co., Ltd and reagents therein):

1) the vaginal secretions were frozen and centrifuged at 10000rpm (about 11500 Xg) for 1min to remove the supernatant as completely as possible.

2) Add 180. mu.l buffer (20mM Tris, pH 8.0; 2 mna 2-EDTA; 1.2% Triton); lysozyme with the final concentration of 20mg/ml (the lysozyme is prepared by dissolving lysozyme dry powder in a buffer solution), and the lysozyme is treated for more than 30min at 37 ℃.

3) Add 20. mu.l proteinase K solution to the tube and mix well.

4) Add 220. mu.l buffer GB, shake for 15sec, stand at 70 ℃ for 10min, clear the solution, centrifuge briefly to remove beads on the inner wall of the tube cover.

5) Add 220. mu.l of absolute ethanol, mix well by shaking for 15sec, appear flocculent precipitate, centrifuge briefly to remove water droplets on the inner wall of the tube cover.

6) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (adsorption column is put into a collecting pipe), centrifuging at 12000rpm (about 13400 Xg) for 30sec, pouring off waste liquid, and putting adsorption column CB3 into the collecting pipe.

7) To adsorption column CB3, 500. mu.l of buffer GD (to which absolute ethanol was added before use) was added, and centrifuged at 12000rpm (about 13400 Xg) for 30sec, and the waste liquid was discarded, and adsorption column CB3 was put into the collection tube.

8) To adsorption column CB3, 600. mu.l of rinsing solution PW (previously examined whether or not absolute ethanol was added) was added, and centrifuged at 12000rpm (about 13400 Xg) for 30sec to remove the waste liquid, and adsorption column CB3 was put in the collection tube.

9) Operation 8 is repeated.

10) The adsorption column CB3 was returned to the collection tube, centrifuged at 12000rpm (about 13400 Xg) for 2min, and the waste liquid was discarded. The adsorption column CB3 is placed at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material (the purpose of this step is to remove the residual rinsing liquid in the adsorption column, and the residual ethanol in the rinsing liquid can affect the subsequent enzyme reaction (enzyme digestion, PCR, etc.) experiment).

11) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 μ l of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12000rpm (about 13400 Xg) for 2min, and collecting the solution into the centrifuge tube.

12) Mu.l RNase A (100mg/ml) solution was added thereto, shaken for 15sec, and left at room temperature for 5 min.

2.3. And (3) detecting the DNA concentration and purity:

and (3) detecting the concentration and purity of the total genome DNA by agarose gel electrophoresis and ultraviolet spectrophotometry respectively, and detecting that all sample DNA has an obvious absorption peak at OD260, the concentration of the DNA is more than 10 ng/mu l, the total amount is more than 0.1 mu g, and the ratio of OD260/OD280 is between 1.8 and 2.0, which indicates that the concentration and purity of the DNA extracted in the step 2.1 meet the experimental requirements of PCR and capillary electrophoresis.

3. Step of preparing bacterial gene fingerprint by LH-PCR technology

3.1. Bacterial PCR amplification

Selecting a primer

fD1(5'-AGAGTTTGATCCTGGCTCAG-3') and

5'FAM-PRUN518r(5'-ATTACCGCGGCTGCTGG-3')

bacterial 16S rRNA genes were amplified, with the 5 'end of 5' FAM-PRUN518r fluorescently labeled with FAM.

Reaction system for PCR (50. mu.L): 5. mu.L of 10 Xbuffer, 1. mu.L of dNTPs, 1.5. mu.L of each primer, 1. mu.L of DNA polymerase, 2.5. mu.L of BSA, 1. mu.L of template DNA, ddH₂O make up to 50. mu.L.

While a negative control without template addition was set.

The reaction procedure is as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 45s, annealing at 55 deg.C for 1min, extension at 72 deg.C for 2min, 30 cycles, and storing at constant temperature of 4 deg.C.

3.2. Capillary electrophoresis LH-PCR product

The PCR product of step 3.1 was enriched by 1.0% agarose gel electrophoresis and the 300-400bp fragment was collected. Then, the gel was excised using Qiagen gel purification kit, recovered and purified, and mixed with Hi-Di formamide and GS500Liz internal size standard, and the mixture was first put into a PCR instrument (Peltier Thermal Cycler DNAEngine), denatured at 95 ℃ for 5min, and immediately put into an ice box. And placing into ABI PRISM 310 genetic analyzer (Applied Biosystems, USA) for capillary electrophoresis detection, with injection time of 10s, injection and operation voltage of 15.0kV, operation temperature of 60 deg.C, and operation time of 70 min.

3.3. Analysis of capillary electropherograms

The fingerprint chromatogram of each sample was collected using GeneScan 3.7. The LH-PCR data was further processed using BioNumerics6.0 software. The electropherograms were converted to the BioNumerics curve format and the fragments with an amplified fragment length of 310-380bp were analyzed using normalized internal size criteria.

4. Preparation of bacterial gene fingerprint

4.1. Grouping of sample data:

63 study subjects were dosed continuously for 5 days according to step 1.5, followed up on day 7 after dosing, and clinical assessments were all cured; the follow-up on the 30 th day after the administration of the medicine shows that 42 patients are cured without relapse, 21 patients are relapsed, and the cure rate is 66.67%.

For the 63 subjects, 189 samples were collected on day 0, day 7 and day 30, respectively. Based on the above treatment results, the sample data were divided into 5 groups:

group 0: group D0 (samples taken from patients before administration, for 63 cases),

Group 1: SG-D7 group (42 samples taken at 7 days in patients who did not relapse 30 days after drug administration),

And 2, group: FG-D7 group (21 samples taken at 7 days in patients who relapsed 30 days after drug administration),

And 3, group: SG-D30 group (42 samples taken at 30 days from 30 days of non-relapsed patients after drug administration),

4 groups are as follows: FG-D30 group (samples taken at 30 days of 30-day relapse patients after drug administration, 21 total).

4.2. LH-PCR characteristic peak

By analyzing LH-PCR amplified fragments of 189 vaginal secretion samples (63 patients, each patient sampling 3 times), the LH-PCR scanning peaks were mainly concentrated at 340-375bp, but the characteristic peaks of LH-PCR were different from one sample data set to another.

FIG. 1 shows the main Component Analysis (PCA) of LH-PCR characteristic peaks of 5 groups of samples of group D0, group SG-D7, group FG-D7, group SG-D30 and group FG-D30, as shown in FIG. 1:

for the sample of D30, the vaginal microflora of the SG-D30 cluster (filled circles) and the FG-D30 cluster (open circles) clearly divided into two clusters, indicating that the colonies of the cured group (non-relapsed group) and the relapsed group are clearly different;

the FG-D30 cluster (open circles) overlapped the microbial colony distribution of the D0 cluster (open squares), indicating that the colonies in the relapsed and untreated groups were similar;

the sample distributions of the SG-D7 group (filled triangles) and the FG-D7 group (open triangles) were partially overlapping but not identical, indicating that even though the clinical evaluation was curative at day 7, the subsequent recurrent and non-recurrent sample colonies differed in intermediate states.

Therefore, based on the LH-PCR characteristic peak formed by the invention, the difference of vaginal flora at different time points can be obviously distinguished by analyzing the main components of the microbial flora in the vagina of BV and healthy women, and diseases can be obviously distinguished from the vaginal flora in a healthy state.

The LH-PCR signature was further tested by Kruskal Wallis Test, and the results are shown in FIG. 2 and Table 1. FIG. 2 shows LH-PCR characteristic peaks for 5 sets of samples of set D0, set SG-D7, set FG-D7, set SG-D30 and set FG-D30. In each group of peak spectra, the vertical axis is the relative peak area of each fragment, the horizontal axis is 14 fragments with significant difference in length after Kruskal Wallis Test, and the fragments are 340bp, 341bp, 343bp, 344bp, 345bp, 347bp, 349bp, 353bp, 354bp, 356bp, 361bp, 363bp, 368bp and 375bp sequentially from left to right. Table 1 shows the interquartile range of relative peak areas for these 14 fragment lengths.

TABLE 1 interquartile range of relative peak areas for fragment lengths with significant differences

From the results shown in FIG. 2 and Table 1, it can be calculated that when the relative peak area (interquartile range) of any one of the fragments with the fragment length of 340-374bp is greater than 10%, the pathogenic bacteria of bacterial vaginosis are determined to be present; when the relative peak areas (quartile spacing) of all the fragments in the fragment length of 340-374bp are less than 10 percent, the normal flora is determined, and no pathogenic flora of the bacterial vaginosis exists.

In order to determine the bacterial species corresponding to the gene fragment lengths that produce significant differences, applicants compared the analysis results of LH-PCR with the results of high throughput sequencing.

Wherein high throughput sequencing is performed by using a standard 454/Roche GS-FLX scheme to complete sequencing work on the V1-V3 region of the 16S rRNA gene. High throughput sequencing yielded 1,622,359 high quality reads with an average of 8071 sequences per sample (3424-18517). A total of 8967 OTUs were detected for all samples, and the number of OTUs per sample ranged from 54-706. After screening of the original sequences, comparison of bacterial species was done by aligning the RDP database. Finally, the bacterial species corresponding to the different fragment lengths were confirmed to be shown in table 2:

TABLE 2 bacterial species and corresponding fragment lengths

Example 2: diagnosis of bacterial vaginosis by applying LH-PCR map of the invention

Patient 1:

the LH-PCR map (figure 3) before administration (day 0) shows that the relative peak areas of 343bp, 353bp and 366bp are more than 10%, so that the diagnosis is that the bacterial vaginosis is caused. And meanwhile, microecological evaluation and clinical standard evaluation are carried out for verification, the Nugent score is 9, the vaginal secretion is thin and has fishy smell, the Whiff test is positive, clue cells are positive, the pH value of the vaginal secretion is 5.1, and the result of clinical diagnosis of the vaginal secretion is consistent with the result of diagnosis through the LH-PCR gene fingerprint.

The patient is treated by metronidazole gel for 5 days according to the standard, and is analyzed 7 days after the drug is taken, at the moment, the micro-ecological evaluation and the clinical standard evaluation of the patient show that BV is cured, the LH-PCR map (figure 4) analysis shows that the relative peak area of all the fragments in the fragment length of 340-374bp is less than 10 percent, according to the map analysis of the embodiment 1, the patient is cured at the moment, the flora is recovered to a normal state, no pathogenic bacteria exist, and the recurrence risk is predicted.

At 30 days after the administration, LH-PCR analysis was performed at the 3 rd time, and the map (FIG. 5) showed that the relative peak area of all the fragments in the fragment length of 340-374bp was < 10%, which was diagnosed as the bacterial vaginosis did not recur and was cured. Meanwhile, the microecological evaluation and the clinical standard evaluation of the sample at 30 days are also verified, the Nugent score is 1, the vaginal secretion has normal physical properties, the Whiff test is negative, the clue cell is negative, the pH value of the vaginal secretion is 4.1, the clinical diagnosis is that the vaginal secretion is also cured and does not relapse, the result is consistent with the result diagnosed by the LH-PCR gene fingerprint, and the result is correct to the analysis and prediction of the LH-PCR in 7 days after the medicine is taken.

Patient 2:

the LH-PCR map (figure 6) before administration (day 0) shows that the relative peak areas of 343bp and 353bp are more than 10%, and the diagnosis is that the bacterial vaginosis is caused. And meanwhile, microecological evaluation and clinical standard evaluation are carried out for verification, the Nugent score is 9, the vaginal secretion has thin physical property and fishy smell, the Whiff test is positive, the clue cell is positive, the pH value of the vaginal secretion is 4.8, and the clinical diagnosis shows that the vaginal secretion has bacterial vaginosis, which is consistent with the diagnosis result through the LH-PCR gene fingerprint spectrum.

The patient is treated by metronidazole gel for 5 days according to the standard, and is analyzed 7 days after the drug is taken, at the moment, the micro-ecological evaluation and the clinical standard evaluation of the patient show that BV is cured, but the analysis of an LH-PCR map (figure 7) shows that the relative peak areas of two parts of 364bp and 368bp are more than 10 percent, according to the map analysis of the embodiment 1, the flora is not recovered to be normal, the flora also belongs to an abnormal flora state, pathogenic bacteria still exist, and the possibility of subsequent relapse risk of the pathogenic bacteria is predicted.

After 30 days after the administration, LH-PCR analysis is carried out on the 3 rd time, and the map (figure 8) shows that the relative peak areas of 353bp and 366bp are more than 10 percent, and the diagnosis is that the bacterial vaginosis still exists. Meanwhile, the microecological evaluation and the clinical standard evaluation of the sample at 30 days are also verified, the Nugent score is 8, the vaginal secretion has thin physical state and fishy smell, the Whiff test is positive, the clue cell is positive, the pH value of the vaginal secretion is 5.2, the vaginal secretion is also clinically diagnosed to be suffering from bacterial vaginosis, the result is consistent with the result diagnosed by the LH-PCR gene fingerprint spectrum of the invention, and the result is correct to the LH-PCR analysis and prediction of 7 days after the drug is taken.

Claims (4)

1. A method for preparing LH-PCR bacterial gene fingerprint map, wherein the bacteria is intravaginal bacteria, and the gene is 16S rRNA gene;

the preparation method is that the LH-PCR characteristic peak of the gene fragment with the amplified fragment length of 310-380bp is subjected to KruskalWallis Test;

when the relative peak area of any fragment in the fragment length of 340-374bp is more than 10 percent, determining that the pathogenic bacteria of the bacterial vaginosis exist; when the relative peak area of all the fragments in the fragment length of 340-374bp is less than 10 percent, determining that no pathogenic flora of the bacterial vaginosis exists;

the primer of the LH-PCR is as follows: 5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-ATTACCGCGGCTGCTGG-3', wherein the 5' end of 5'-ATTACCGCGGCTGCTGG-3' is fluorescently labeled with FAM;

the species of bacteria is selected from the group consisting of Mycobacterium, Ureapasma, Mycoplasma, Sneathia, Corynebacterium, Atopobium, Gardnerella, Mobilucus, Prevotella, Actinomyces, Staphylococcus, Anaerococcus, Peptoniphilus, Megasphaera, Lactobacillus.

2. The method of claim 1, comprising the steps of:

1) extracting bacterial genome DNA of vaginal secretion;

2) the 16S rRNA gene in the bacterial genome DNA was subjected to PCR amplification:

3) primer: 5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-ATTACCGCGGCTGCTGG-3', wherein the 5' end of 5'-ATTACCGCGGCTGCTGG-3' is fluorescently labeled with FAM;

4) reaction system of PCR: 5 μ L of 10 Xbuffer, 1 μ L of dNTPs, 1.5 μ L of each primer, 1 μ L of DNA polymerase, 2.5 μ L of BSA, 1 μ L of template DNA, and the complement of ddH2O to 50 μ L; setting negative control without adding template;

5) reaction procedure: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 45s, annealing at 55 deg.C for 1min, extension at 72 deg.C for 2min, 30 cycles, and storing at constant temperature of 4 deg.C;

6) capillary electrophoresis LH-PCR products:

7) enriching the PCR product by 1.0% agarose gel electrophoresis, and collecting 300-400bp fragments; then using Qiagen gel to recover and purify, mixing with Hi-Di formamide and GS500Liz internal dimension standard, denaturing the mixture in a PCR instrument at 95 ℃ for 5min, and then carrying out capillary electrophoresis detection, wherein the injection time is 10s, the injection and running voltage is 15.0kV, the running temperature is 60 ℃, and the running time is 70 min;

8) collecting the fingerprint electrophoresis pattern of the sample, and carrying out Kruskal Wallis Test on the fragment with the length of 310-and 380-bp.

3. The method of claim 1, wherein the bacterial species and corresponding amplified fragments are of:

。

4. an LH-PCR bacterial gene fingerprint map prepared by the preparation method described in claims 1-3, wherein the bacteria is intravaginal bacteria, the gene is 16S rRNA gene, and the map is characterized by performing Kruskal Wallis Test on LH-PCR characteristic peaks of gene fragments with amplification fragment length of 310-380 bp.

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