CN106701915A - Preparation method and application of LH-PCR bacterial gene fingerprint spectrum for bacterial vaginosis - Google Patents
Preparation method and application of LH-PCR bacterial gene fingerprint spectrum for bacterial vaginosis Download PDFInfo
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Abstract
The invention provides a method for preparing a bacterial gene fingerprint spectrum for bacterial vaginosis by applying an LH-PCR technology and application of the bacterial gene fingerprint spectrum to clinical accurate diagnosis of the bacterial vaginosis. The bacterial gene fingerprint spectrum provided by the invention can be used for clinical quick diagnosis of the bacterial vaginosis and accurately and quickly detecting bacterial flora distribution conditions in vagina; the defects of accurate diagnosis, tedious operation, long time consumption, high workload, expensive high-flux sequencing price and the like of a traditional bacteria separation and culture method are overcome.
Description
Technical field
The present invention relates to a kind of preparation method of genomic fingerprint spectrum, more particularly to one kind application LH-PCR skills
The method that art prepares the genomic fingerprint spectrum of bacterial vaginosis BV.
Background technology
It, by bacterial vagina infection, is child-bearing period female that bacterial vaginosis BV (Bacterial Vaginosis, BV) is
The common LGTI disease of property, it can trigger various Averse pregnancy outcomes, such as spontaneous abortion, premature labor, amniotic fluid infection,
Puerperal endometritis and cesarean section infection and peri-natal infant complication etc..Research discovery in recent years, bacterial vaginosis BV
Recurrence and persistent infection can also increase trichomonas vaginitis, candida albicans bacterium property vaginitis, cervical carcinoma and human immunity lack
Fall into the risk of virus (human immunodeficiency virus, HIV).
At present to the cause of disease of bacterial vaginosis BV, the difference for particularly being constituted to the vaginal flora of Patients with Bacterial Vaginosis
It is different, lack further investigation.The cultivation for using in the past can not comprehensively reflect the composition of vaginal flora, cause to bacillary the moon
The pathogen research of road patient is limited to, though patient clinical evaluation sometimes has been healing state, its vaginal flora is constituted
But normal condition is not returned to also, pathogenic bacteria still suffer from, now as only stopped treating by clinical evaluation, then can cause bacterium
Vaginosis recur, and delay disease treatment, increase the recurrence rate of disease.
With the fast development of Protocols in Molecular Biology over nearly 20 years, bacterial vaginosis are studied using molecular biology method
The mechanism and the clinical different bacterium vaginosis hypotype of refinement diagnosis of disease recurrence turn into a kind of trend, and people are to bacterial vaginosis BV
Bacterial community research also deepening continuously.The polymorphic fragment PCR of length (length heterogeneity PCR, LH-PCR) is
The relative populations from different microorganisms extension increasing sequence are determined with the probe of fluorescence labeling, the fragment of tape label passes through hair
Cons electrophoresis is separated, and is then monitored with fluorescent automatic gene sequenator by one.LH-PCR is
A kind of method for analyzing microorganism structure, its principle is that insertion or shearing due to gene or gene operon cause some specific
Gene natural length is presented polymorphism.Therefore, it can determine using this polymorphism the species information of microorganism.LH-PCR is surveyed
Fixed hypervariable region is primarily present in ribosomal small subunit (rrn).
LH-PCR as a kind of PCR-based molecular fingerprint graphical spectrum technology, its compared with high throughput sequencing technologies have skill
Art manual dexterity, quickly and easily, the advantage of the cheap grade of composition.But, not yet have at present and the technology be applied to medical domain, especially
It is as a kind of report of the diagnostic techniques of disease.
The content of the invention
It is an object of the invention to provide one kind application LH-PCR technologies, the genomic fingerprint of bacterial vaginosis BV is prepared
Whether there is bacterial vaginosis BV pathogenic flora in the method for spectrum, and the application genomic fingerprint accurate judgement sample of spectrum
Purposes.Genomic fingerprint spectrum of the present invention can be used for the clinical quick diagnosis of bacterial vaginosis BV, it is to avoid tradition
Bacteria distribution culture diagnosis it is inaccurate, cumbersome, time-consuming, workload is big and high-flux sequence is expensive etc. lacks
Point, can accurately, fast and effeciently detect the distribution of flora of intravaginal.
To reach above-mentioned purpose, the present invention provides a kind of system of the LH-PCR genomic fingerprints spectrum of bacterial vaginosis BV
Preparation Method, wherein the bacterium is intravaginal bacterium, the gene is 16S rRNA genes.
Further, the preparation method of the LH-PCR genomic fingerprints spectrum of the bacterial vaginosis BV, is to amplification
Fragment length is analyzed for the gene segment of 310-380bp.
Further, the preparation method of the LH-PCR genomic fingerprints spectrum of the bacterial vaginosis BV, wherein when
When the relative peak area of any one segment in fragment length 340-374bp is more than 10%, it is defined as having bacterial vaginosis BV
Pathogenic flora is present；When the relative peak area of all fragments in fragment length 340-374bp is both less than 10%, it is defined as normal bacterium
Group, there is no bacterial vaginosis BV pathogenic flora.
Preferably, the preparation method of the LH-PCR genomic fingerprints spectrum of the bacterial vaginosis BV, wherein LH-PCR
Primer be：5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-ATTACCGCGGCTGCTGG-3', wherein 5'-
The 5' ends FAM fluorescence labelings of ATTACCGCGGCTGCTGG-3'.
Further, the preparation method of the LH-PCR genomic fingerprints spectrum of the bacterial vaginosis BV, including：
1. the bacterial genomes DNA of vaginal fluid is extracted；
2. the 16S rRNA genes in couple bacterial genomes DNA enter performing PCR amplification：
Primer：5'-AGAGTTTGATCCTGGCTCAG-3', and
5'-ATTACCGCGGCTGCTGG-3', wherein 5' ends FAM fluorescence labelings；
The reaction system (50 μ L) of PCR：10 × buffer solution 5 μ L, dNTPs1 μ L, each 1.5 μ L of primer, the μ L of archaeal dna polymerase 1,
BSA2.5 μ L, template DNA 1 μ L, ddH2O supplies 50 μ L；Negative control without template is set simultaneously；
Response procedures：95 DEG C of predegenerations 5min, 94 DEG C of denaturation 45s, 55 DEG C of annealing 1min, 72 DEG C of extension 2min, 30 are followed
Ring, most preserves after 4 DEG C of constant temperature；
3. Capillary Electrophoresis LH-PCR products：
The PCR primer is enriched with 1.0% agarose gel electrophoresis, the segment of 300-400bp is collected；Then use
Qiagen gel purifications, and mix with Hi-Di formamides and GS500 Liz inside dimension standards, mixture is in PCR instrument
5min is denatured at 95 DEG C, then carries out capillary electrophoresis detection, injection time is 10s, injection and working voltage are 15.0kV, fortune
Trip temperature is 60 DEG C, and run time is 70min；
4. the analysis of Capillary Electrophoresis collection of illustrative plates
The fingerprint electrophoresis pattern of sample is collected with GeneScan 3.7, LH-PCR data applications BioNumerics
6.0 softwares are processed, to expanding fragment length for the segment of 310-380bp is analyzed.
Preferably, the preparation method of the LH-PCR genomic fingerprints spectrum of the bacterial vaginosis BV, wherein described thin
The species of bacterium be selected from Mycobacterium, Ureaplasma, Mycoplasma, Sneathia, Corynebacterium,
Atopobium、Gardnerella、Mobiluncus、Prevotella、Actinomyces、Staphylococcus、
Anaerococcus、Peptoniphilus、Megasphaera、Lactobacillus。
Preferably, the preparation method of the LH-PCR genomic fingerprints spectrum of the bacterial vaginosis BV, wherein described thin
Strain class and corresponding expanding fragment length are：
Composed in diagnosing bacterial present invention also offers a kind of genomic fingerprint for applying above-mentioned preparation method to prepare
Purposes in vaginopathy.
Composed in judgement sample present invention also offers a kind of genomic fingerprint for applying above-mentioned preparation method to prepare
With the presence or absence of the purposes in bacterial vaginosis BV pathogenic flora.
Composed in judgement sample present invention also offers a kind of genomic fingerprint for applying above-mentioned preparation method to prepare
With the presence or absence of Mycobacterium, Ureaplasma, Mycoplasma, Sneathia, Corynebacterium,
Atopobium、Gardnerella、Mobiluncus、Prevotella、Actinomyces、Staphylococcus、
Application in Anaerococcus, Peptoniphilus, Megasphaera, Lactobacillus bacterium.
Any fingerprint pattern technology is provided to provide an overall condition for flora, single without being to discriminate between each
Strain.Therefore, it is mainly the differentiation or difference for reflecting Bacterial community in environment, to analyze morbid state and Bacterial community
Correlation.LH-PCR is changed by analyzing peak shape, is easier to track the change of Bacterial community, can accurately analyze vagina
The Bacterial community of secretion, can be classified and be identified to Main Pathogenic Bacteria.And the method operates relatively easy, less operation
Step reduces error and produces probability, therefore reappearance is very good.Additionally, LH-PCR and high-flux sequence and other finger-prints
Technology such as T-RFLP (detection of terminal restriction digestion polymorphism, Terminal restriction fragment length
Polymorphism) compare, PCR primer need not carry out restricted digestion, can directly carry out genetic analysis, you can obtain micro-
The configuration information of biotic population, with low cost, quick advantage easy to operate, while relative to Amsel standards and Nugent
The Bacterial community of vaginal fluid can be accurately analyzed again for scoring, therefore can be applied to as Protocols in Molecular Biology
Clinical analysis vaginal flora changes, and clinically can carry out classification to morbid state by building different LH-PCR collection of illustrative plates examines
It is disconnected.
Brief description of the drawings
Fig. 1：D0 groups (0), SG-D7 groups (1), FG-D7 groups (2), SG-D30 groups (3), (4) 5 groups of LH- of sample of FG-D30 groups
The principal component analysis result (Principal Component Analysis, PCA) of PCR characteristic peaks.
Fig. 2：D0 groups (0), SG-D7 groups (1), FG-D7 groups (2), SG-D30 groups (3), (4) 5 groups of LH- of sample of FG-D30 groups
PCR characteristic peaks；Transverse axis is fragment length, and the longitudinal axis is the relative peak area (unit %) of each fragment length.
Fig. 3：The LH-PCR collection of illustrative plates of (D0) before the medication of 2 patient of embodiment 1；Transverse axis is fragment length, and the longitudinal axis is each piece
The relative peak area (unit %) of segment length.
Fig. 4：The LH-PCR collection of illustrative plates of 7 days (D7) after the medication of 2 patient of embodiment 1；Transverse axis is fragment length, and the longitudinal axis is each
The relative peak area (unit %) of fragment length.
Fig. 5：The LH-PCR collection of illustrative plates of 30 days (D30) after the medication of 2 patient of embodiment 1；Transverse axis is fragment length, and the longitudinal axis is each
Plant the relative peak area (unit %) of fragment length.
Fig. 6：The LH-PCR collection of illustrative plates of (D0) before the medication of 2 patient of embodiment 2；Transverse axis is fragment length, and the longitudinal axis is each piece
The relative peak area (unit %) of segment length.
Fig. 7：The LH-PCR collection of illustrative plates of 7 days (D7) after the medication of 2 patient of embodiment 2；Transverse axis is fragment length, and the longitudinal axis is each
The relative peak area (unit %) of fragment length.
Fig. 8：The LH-PCR collection of illustrative plates of 30 days (D30) after the medication of 2 patient of embodiment 2；Transverse axis is fragment length, and the longitudinal axis is each
Plant the relative peak area (unit %) of fragment length.
Specific embodiment
The present invention is described in more detail for example below, artisan will appreciate that not departing from this hair
The change made in the case of bright scope and spirit, belongs to the scope of the present invention.
Bacterial genomes DNA extraction kit (DP302) is purchased from：Tiangeng biochemical technology Co., Ltd.
All data are analyzed using SPSS15.0 (Chicago, IL, USA), if P<0.05, illustrate that data are deposited
In significant difference.
Embodiment 1：The genomic fingerprint for preparing bacterial vaginosis BV using LH-PCR technologies is composed
1. research object and standard：
1.1 research objects：63 Female in child bearing period for being diagnosed as bacterial vaginosis BV are chosen, Normalization rule metronidazole coagulates
Glue is treated 5 days, and 30 days before medication 7 days after (0 day), medication, after medication sample.
1.2 inclusion criterias：Using Amsel clinical criterias and the bacillary the moon of Nugent laboratory diagnosis standard diagnostics
Road disease (Bacterial Vaginosis, BV).All of secretion smear is by the experienced micro- life in 2, Tiny ecosystem laboratory
Thing expert's interpretation.
1.3 exclusion standards：
1. the gestational period, nursing period and menopausal woman
2. because of other diseases Long-term Hormone class medicine person
3. immunodepressant person is taken
4. intentionally, liver, kidney, the internal disease person (mainly by interrogation) such as endocrine
5. with other vagina infections persons such as trichomonad, vulvovaginal candidiasis
6. metronidazole allergy sufferers
7. compliance difference person
1.4 follow up time：Follow-up twice is carried out to enrolled BV patient, 7 days respectively after medication, 30 days after medication.Note
Symptom, sign, pH value and Nugent scorings when record patient per is medical.
1.5 treatment methods and criterion of cure：Enrolled BV patient uses Metrogel (37.5mg, qd, on vagina
Medicine), successive administration 5 days.The 7th day and difference follow-up in the 30th day after medication, evaluate therapeutic effect.Criterion of cure：Meet simultaneously
Tiny ecosystem evaluation recovery from illness standard and clinical evaluation recovery from illness standard are healing.Tiny ecosystem evaluation recovery from illness standard：Nugent scorings 0-3
Point.Clinical recovery standard：Vaginal fluid proterties is normal, Whiff negatives, and clues cell disappears, vaginal fluid pH value<
4.5。
2. sample preparation：
2.1. sample collection：Obtain vagina point from 1/3 on research object vaginal sidewall with two sterile cotton swabs respectively
Secretion, rapid being put into contains 1ml PBS (pH:7.4) in sterile centrifugation tube, -80 DEG C of ultra low temperature freezers are put in so as to gene
Group is extracted.Another is evenly coated on slide, row Grain stain, and oily Microscopic observation carries out Nugent scorings.
2.2. the extraction of bacterial genomes DNA is (using the bacterial genomes extractant box of Tiangeng biochemical technology Co., Ltd
And its interior reagent)：
1) take and freeze vaginal fluid, 10000rpm (about 11500 × g) centrifugation 1min, exhaust supernatant as far as possible.
2) 180 μ l buffer solutions (20mM Tris, pH 8.0 are added；2mMNa2-EDTA；1.2%Triton)；It is final concentration of
The lysozyme (lysozyme is dissolved in buffer solution with lysozyme dry powder and is prepared) of 20mg/ml, 37 DEG C for the treatment of more than 30min.
3) to 20 μ l Proteinase K Solutions are added in pipe, mix.
4) 220 μ l buffer solution GB are added, 15sec is vibrated, 70 DEG C of placement 10min, solution strain is limpid, and brief centrifugation is going
Except the globule of cap wall.
5) plus 220 μ l absolute ethyl alcohols, fully vibration mixes 15sec, flocculent deposit occurs, and brief centrifugation is removing in lid
The globule of wall.
6) previous step resulting solution and flocculent deposit all add in an adsorption column CB3 (adsorption column is put into collecting pipe
In), 12000rpm (about 13400 × g) centrifugation 30sec outwell waste liquid, and adsorption column CB3 is put into collecting pipe.
7) to adding 500 μ l buffer solutions GD (please first checked whether before and added absolute ethyl alcohol) in adsorption column CB3,
12000rpm (about 13400 × g) is centrifuged 30sec, outwells waste liquid, and adsorption column CB3 is put into collecting pipe.
8) to adding 600 μ l rinsing liquids PW (please first checked whether before and added absolute ethyl alcohol) in adsorption column CB3,
12000rpm (about 13400 × g) is centrifuged 30sec, outwells waste liquid, and adsorption column CB3 is put into collecting pipe.
9) step 8 is repeated.
10) adsorption column CB3 is put back in collecting pipe, 12000rpm (about 13400 × g) centrifugation 2min outwell waste liquid.To inhale
Attached column CB3 is placed in room temperature and places several minutes, and thoroughly to dry remaining rinsing liquid in sorbing material, (purpose of this step is to inhale
Remaining rinsing liquid removal in attached column, the residual of ethanol can influence follow-up enzyme reaction (digestion, PCR etc.) to test in rinsing liquid).
11) adsorption column CB3 is transferred in a clean centrifuge tube, 50-200 is vacantly added dropwise to the middle part of adsorbed film
μ l elution buffer TE, room temperature places 2-5min, 12000rpm (about 13400 × g) centrifugation 2min, and solution is collected into centrifuge tube
In.
12) 4 μ l RNase A (100mg/ml) solution are added, 15sec is vibrated, room temperature places 5min.
2.3. DNA concentration and purity detecting：
The concentration and purity of total genomic dna are detected with agarose gel electrophoresis and ultraviolet spectrophotometry respectively, is measured
All sample DNAs have notable absworption peak at OD260, and DNA concentration is more than 10ng/ μ l, and total amount is more than 0.1 μ g and OD260/
OD280 ratios illustrate that DNA concentration that step 2.1 extracted and purity meet PCR and Capillary Electrophoresis between 1.8-2.0
Requirement of experiment.
3. the step of preparing genomic fingerprint using LH-PCR technologies and compose
3.1. bacteria PCR amplification
From primer
FD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and
5'FAM-PRUN518r(5'-ATTACCGCGGCTGCTGG-3')
Amplification bacterial 16 S rRNA genes, the 5' ends FAM fluorescence labelings of wherein 5'FAM-PRUN518r.
The reaction system (50 μ L) of PCR：10 × buffer solution 5 μ L, dNTPs1 μ L, each 1.5 μ L of primer, the μ L of archaeal dna polymerase 1,
BSA2.5 μ L, template DNA 1 μ L, ddH2O supplies 50 μ L.
Negative control without template is set simultaneously.
Response procedures are：95 DEG C of predegenerations 5min, 94 DEG C of denaturation 45s, 55 DEG C of annealing 1min, 72 DEG C extend 2min, 30
Circulation, most preserves after 4 DEG C of constant temperature.
3.2. Capillary Electrophoresis LH-PCR products
The PCR primer of step 3.1 is enriched with 1.0% agarose gel electrophoresis, the segment of 300-400bp is collected.
Then purified with Qiagen gel purification kit gel extractions, and with Hi-Di formamides and GS500Liz inside dimension standards
Mixing, mixture is first put into PCR instrument (Peltier Thermal Cycler DNAEngine), 5min is denatured at 95 DEG C, stands
Quarter is put into ice chest.Place into and carry out capillary in the genetic analyzers of ABI PRISM 310 (Applied Biosystems, the U.S.)
Electrophoresis tube detects that injection time is 10s, and injection and working voltage are 15.0kV, and running temperature is 60 DEG C, and run time is
70min。
3.3. the analysis of Capillary Electrophoresis collection of illustrative plates
The fingerprint electrophoresis pattern of each sample is collected with GeneScan 3.7.LH-PCR data applications
The softwares of BioNumerics 6.0 are further processed.Electrophoresis pattern is converted to BioNumerics curve formats, and operating specification
The inside dimension standard of change, to expanding fragment length for the segment of 310-380bp is analyzed.
4. genomic fingerprint is composed standby
4.1. sample data packet：
By the research object successive administration 5 days of step 1.5 pair 63, the follow-up in the 7th day after medication, clinical evaluation is to be controlled
More；The follow-up in the 30th day after medication, 42 healings are not recurred, 21 recurrences, and cure rate is 66.67%.
Above-mentioned 63 research objects, every takes 3 samples the 0th day, the 7th day, the 30th day respectively, totally 189 samples.According to
Above-mentioned treatment results, sample data is divided into 5 groups：
0 group：D0 groups (sample that patient samples before medication, for 63),
1 group：SG-D7 groups (sample that the patient that 30 days do not recur after medication sampled at 7 days, totally 42),
2 groups：FG-D7 groups (sample that the patient that 30 days recur after medication sampled at 7 days, totally 21),
3 groups：SG-D30 groups (sample that the patient that 30 days do not recur after medication sampled at 30 days, totally 42),
4 groups：FG-D30 groups (sample that the patient that 30 days recur after medication sampled at 30 days, totally 21).
4.2. LH-PCR characteristic peaks
LH-PCR amplified fragments are carried out by 189 (63 patients, each patient samples 3 times) vaginal fluid samples
It is analyzed, it is found that 340-375bp is mainly concentrated at the scanning peak of LH-PCR, but the LH-PCR characteristic peaks of different sample data groups
It is different.
D0 groups that Fig. 1 shows, SG-D7 groups, FG-D7 groups, SG-D30 groups, the LH-PCR features of FG-D30 groups this 5 groups of samples
The principal component analysis result (Principal Component Analysis, PCA) at peak, as can be seen from Figure 1：
For the sample of D30, the vagina microorganism of SG-D30 groups (solid circles) and FG-D30 groups (empty circles)
Group significantly divide into two clusters, illustrate that the colony of healing group (not recurring group) and recurrence group is significantly different；
FG-D30 groups (empty circles) overlap with microbiologic population's distribution of D0 groups (hollow square), illustrate recurrence group
The colony of non-treatment group has similitude；
The sample distribution presence of SG-D7 groups (triangles) and FG-D7 groups (open triangles) partly overlaps, but not exclusively
Identical, clinical evaluation is all healing when illustrating even the 7th day, and sample colony that is follow-up recurrence and not recurring is in intermediateness
In there is also difference.
Therefore, the LH-PCR characteristic peaks for being formed based on the present invention, can be by BV and healthy women intravaginal microorganism
The principal component analysis of flora, hence it is evident that the difference of vaginal flora when distinguishing different time points, can be by under disease and health status
Vaginal flora significantly makes a distinction.
Kruskal Wallis Test inspections are further carried out to LH-PCR characteristic peaks, Fig. 2 and Biao 1 is as a result seen.Fig. 2 shows
What is shown is the LH-PCR characteristic peaks of D0 groups, SG-D7 groups, FG-D7 groups, SG-D30 groups, FG-D30 groups this 5 groups of samples.At each group
In peak spectrum, the longitudinal axis is the relative peak area of each fragment, and transverse axis is 14 to be had after Kruskal Wallis Test are checked
The fragment length of significant difference, be followed successively by from left to right 340bp, 341bp, 343bp, 344bp, 345bp, 347bp, 349bp,
353bp、354bp、356bp、361bp、363bp、368bp、375bp.Table 1 is shown the relative peak face of this 14 fragment lengths
Long-pending interquartile range.
Table 1 has the interquartile range of the relative peak area of the fragment length of significant difference
From the result of Fig. 2 and Biao 1 it can be calculated that relative when any one segment in fragment length 340-374bp
When peak area (interquartile range) is more than 10%, it is defined as with the presence of bacterial vaginosis BV pathogenic flora；Work as fragment length
The relative peak area (interquartile range) of all fragments is both less than 10% in 340-374bp, is defined as normal flora, without thin
Bacterium vaginosis pathogenic flora is present.
In order to determine to produce the bacterial species corresponding to the genetic fragment length of significant difference, applicant is by LH-PCR's
Analysis result has carried out comparing with high-flux sequence result.
Wherein high-flux sequence is that the 454/Roche GS-FLX schemes for using standard are completed to 16S rRNA genes V1-V3
The examining order in area.High-flux sequence generates 1,622,359 high-quality reads altogether, and each sample mean has 8071
Sequence (3424-18517).All samples detect 8967 OTU (activity classification unit, operation taxonomy altogether
Units), and each sample OTUs numbers between 54-706.After being screened to original series, by comparing RDP numbers
The comparing of bacterial species is completed according to storehouse.The final bacterial species confirmed corresponding to differential fragment length are for shown in table 2：
The bacterial species of table 2 and corresponding fragment length
Embodiment 2：Bacterial vaginosis BV diagnosis is carried out using LH-PCR collection of illustrative plates of the present invention
Patient 1：
(0 day) LH-PCR collection of illustrative plates (Fig. 3) shows relative peak area at 343bp, 353bp, 366bp tri- and is more than before medication
10%, therefore be diagnosed as with bacterial vaginosis BV.Carry out Tiny ecosystem evaluation simultaneously and clinical criteria evaluation is verified, its
Nugent scorings are 9, and vaginal fluid is thin, there is fish bad smell, and Whiff experiments are positive, and clues cell is positive, vaginal fluid
PH value is 5.1, and clinical diagnosis is with bacterial vaginosis BV, with the result diagnosed by LH-PCR gene fingerprints of the present invention
Unanimously.
Patient's Normalization rule Metrogel is treated 5 days, it is analyzed again within 7 days after medication, now the trouble
The Tiny ecosystem evaluation of person and clinical criteria evaluation show that its BV has cured, and are analyzed by LH-PCR collection of illustrative plates (Fig. 4), show piece segment length
The relative peak area of all fragments in degree 340-374bp<10%, according to the atlas analysis of embodiment 1, illustrate that now patient has controlled
More, and flora returns to normal condition, there is no pathogenic bacteria, predict that it does not have risk of recurrence.
30 days after medication, LH-PCR analyses are carried out for the 3rd time, own in collection of illustrative plates (Fig. 5) display fragment length 340-374bp
The relative peak area of fragment<10%, it is diagnosed as its bacterial vaginosis BV and does not recur, cure.Simultaneously to sample at 30 days
Carry out Tiny ecosystem evaluation and clinical criteria evaluation is verified, its Nugent scorings are 1, and vaginal fluid proterties is normal, Whiff
Negative, clues cell is negative, and vaginal fluid pH value is 4.1, and clinical diagnosis and passes through not recur to cure similarly
The result of LH-PCR gene fingerprints of the present invention diagnosis is consistent, and with medication after the LH-PCR analysis predictions of 7 days it is correct.
Patient 2：
(0 day) LH-PCR collection of illustrative plates (Fig. 6) shows relative peak area at 343bp, 353bp two and, more than 10%, examines before medication
Break is with bacterial vaginosis BV.Carry out Tiny ecosystem evaluation simultaneously and clinical criteria evaluation is verified, its Nugent scorings are
9, vaginal fluid proterties is thin, there is fish bad smell, and Whiff experiments are positive, and clues cell is positive, vaginal fluid pH value 4.8,
Clinical diagnosis be with bacterial vaginosis BV, it is consistent with the result diagnosed by LH-PCR gene fingerprints of the present invention.
Patient's Normalization rule Metrogel is treated 5 days, it is analyzed again within 7 days after medication, now the trouble
The Tiny ecosystem evaluation of person and clinical criteria evaluation show that its BV has cured, but are analyzed by LH-PCR collection of illustrative plates (Fig. 7), and display is still
There is at 364bp, 368bp two relative peak area be more than 10%, according to the atlas analysis of embodiment 1, now flora does not recover
To normal, abnormal flora state is still belonged to, pathogenic bacteria still suffer from, predict that it may subsequently have risk of recurrence.
30 days after medication, LH-PCR analyses are carried out for the 3rd time, collection of illustrative plates (Fig. 8) shows relative peak at 353bp, 366bp two
Area is more than 10%, is diagnosed as it and still suffers from bacterial vaginosis BV.Simultaneously sample at 30 days is also carried out Tiny ecosystem evaluation and
Clinical criteria evaluation is verified that its Nugent scorings are 8, and vaginal fluid proterties is thin, there is fish bad smell, Whiff experiments
The positive, clues cell is positive, vaginal fluid pH value 5.2, and clinical diagnosis is to be similarly, with bacterial vaginosis BV, and to pass through
The result of LH-PCR gene fingerprints of the present invention diagnosis is consistent, and with medication after the LH-PCR analysis predictions of 7 days it is correct.
Claims (10)
1. the preparation method that a kind of LH-PCR genomic fingerprints are composed, wherein the bacterium is intravaginal bacterium, the gene
It is 16S rRNA genes.
2. preparation method according to claim 1, it is the LH- of the gene segment to expanding fragment length 310-380bp
PCR characteristic peaks carry out Kruskal Wallis Test inspections.
3. preparation method according to claim 2, wherein when the phase of any one segment in fragment length 340-374bp
When being more than 10% to peak area, it is defined as with the presence of bacterial vaginosis BV pathogenic flora；When all in fragment length 340-374bp
The relative peak area of fragment is both less than 10%, is defined as no bacterial vaginosis BV pathogenic flora and exists.
4. the preparation method according to any one of claim 1-3, the wherein primer of LH-PCR is：5'-
AGAGTTTGATCCTGGCTCAG-3' and 5'-ATTACCGCGGCTGCTGG-3', wherein 5'-ATTACCGCGGCTGCTGG-3'
5' ends FAM fluorescence marks.
5. preparation method according to claim 4, comprises the following steps：
1) the bacterial genomes DNA of vaginal fluid is extracted；
2) performing PCR amplification is entered to the 16S rRNA genes in bacterial genomes DNA：
3) primer：5'-AGAGTTTGATCCTGGCTCAG-3', and
4) 5'-ATTACCGCGGCTGCTGG-3', wherein 5' ends FAM fluorescence labelings；
5) reaction system (50 μ L) of PCR：10 × buffer solution 5 μ L, dNTPs1 μ L, each 1.5 μ L of primer, the μ L of archaeal dna polymerase 1,
BSA2.5 μ L, template DNA 1 μ L, ddH2O supplies 50 μ L；Negative control without template is set simultaneously；
6) response procedures：95 DEG C of predegenerations 5min, 94 DEG C of denaturation 45s, 55 DEG C of annealing 1min, 72 DEG C of extension 2min, 30 circulate,
Most preserved after 4 DEG C of constant temperature；
7) Capillary Electrophoresis LH-PCR products：
8) PCR primer is enriched with 1.0% agarose gel electrophoresis, is collected the segment of 300-400bp；Then use
Qiagen gel purifications, and mix with Hi-Di formamides and GS500Liz inside dimension standards, mixture is in PCR instrument
5min is denatured at 95 DEG C, then carries out capillary electrophoresis detection, injection time is 10s, injection and working voltage are 15.0kV, fortune
Trip temperature is 60 DEG C, and run time is 70min；
9) the fingerprint electrophoresis pattern of sample is collected, to expanding fragment length for the segment of 310-380bp carries out Kruskal
Wallis Test are checked.
6. the preparation method according to any one of claim 1-3, wherein the species of the bacterium is selected from
Mycobacterium、Ureaplasma、Mycoplasma、Sneathia、Corynebacterium、Atopobium、
Gardnerella、Mobiluncus、Prevotella、Actinomyces、Staphylococcus、Anaerococcus、
Peptoniphilus、Megasphaera、Lactobacillus。
7. preparation method according to claim 6, wherein the bacterial species and corresponding expanding fragment length are：
8. a kind of LH-PCR genomic fingerprints are composed, wherein the bacterium is intravaginal bacterium, the gene is 16S rRNA
Gene, the collection of illustrative plates is to carry out Kruskal to the LH-PCR characteristic peaks of the gene segment of expanding fragment length 310-380bp
Wallis Test are checked.
9. a kind of LH-PCR genomic fingerprints prepared by preparation method of claim 1-7 compose in judgement sample whether
There is the purposes in bacterial vaginosis BV pathogenic flora.
10. a kind of LH-PCR genomic fingerprints of claim 8 are composed and whether there is bacterial vaginosis BV in judgement sample
Purposes in pathogenic flora.
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| GREGORY T. SPEAR ET AL.: "Comparison of the Diversity of the Vaginal Microbiota in HIV-Infected and HIV-Uninfected Women with or without Bacterial Vaginosis", 《THE JOURNAL OF INFECTIOUS DISEASES》 * |
| 孙庆华等: "DGGE、T-RFLP、LH-PCR对两种活性污泥的微生物种群多样性分析的比较", 《环境工程学报》 * |
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