CN110257535A - Mycobacterium tuberculosis (Mtb) MLVA genotyping kit - Google Patents
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Abstract
The present invention relates to mycobacterium tuberculosis (Mtb) MLVA genotyping kits, belong to Genotyping field；Mycobacterium tuberculosis (Mtb) MLVA genotyping kit, including for detecting Yunnan Province HIV/Mtb double infection and 10 sites high-resolution VNTR of the mono- infection study on genotyping of Mycobacterium tuberculosis of Mtb and corresponding 10 primer pairs, Taq enzymes, PCR buffer and DNA relative molecular weight, establish the methods of genotyping of high resolution, high resolution is of great significance to the space structure dynamic model for establishing Network Management System and suitable this area up to 0.999.
Description
Technical field
The present invention relates to Genotyping fields, and in particular to mycobacterium tuberculosis (Mtb) MLVA genotyping kit.
Background technique
Tuberculosis is the chronic infectious disease as caused by mycobacterium tuberculosis infection.Tulase may invade the various devices of Whole Body
Official, but mainly invade lungs, referred to as pulmonary tuberculosis, are still to endanger one of Important Infectious Diseases of human health at present.Tuberculosis is latent
The volt phase 4~8 weeks, wherein 80% occurs in lung, other positions (neck lymph, meninx, peritonaeum, intestines, skin, bone) can also be secondary
Infection.Interpersonal respiratory infectious is the major way that this disease infects, and the infection sources is the lunger for contacting discharge of bacteria.
HIV is a kind of virus that can attack human immune system.It using CD4+T lymphocyte most important in human immune system as
Primary challenge target, the considerable damage cell, makes human body cell immunodeficiency, and therefore, human body is easy to infect various diseases,
And malignant tumour can occur, case fatality rate is higher.HIV was at the intracorporal incubation period average out to of people 8 years, that is to say, that after infected by HIV,
With can not having any symptom life and work many years.Currently, AIDS, which has become, seriously threatens the important of China's public health
Public health problem.With the propagation of environmental pollution and AIDS, incidence of tuberculosis is more strong.Except minority morbidity is rapid
It outside, is clinically in chronic process more.
The famous tubercle bacillus of mycobacterium tuberculosis or tulase, occurred form, bacterium colony, virulence, immunogenicity and
The variation such as drug resistance.Research tuberculosis Molecular Epidemic main method is genotyping technique at present, passes through genotyping technique
Pathogen can be tracked, distinguish tubercle bacillus molecule parting, it is preliminary to understand this area tubercle bacillus Major Epidemic trend.
Since the prevalence of tubercle bacillus has region, aetology, the gene polymorphic of Mtb are infected for HIV/Mtb at present
Property and yielding characteristics it is considerably less, it is therefore, good by experiment screening stability and repeatability and parting index, and be suitble to
There is positive effect in the site VNTR of Yunnan Province tubercle bacillus parting to Yunnan Province tuberculosis prophylaxis and treatment.
Summary of the invention
To solve the above-mentioned problems of the prior art, the purpose of the present invention is to provide the MLVA Genotypings of Mtb to draw
Object and kit, knot of the primer sets provided by the invention in the HIV/Mtb double infection to Yunnan Province and simple infection
Good stability and repeatability are shown in core mycobacteria parting, and shows high parting index.
In order to achieve the above objectives, the technical solution of the present invention is as follows: mycobacterium tuberculosis (Mtb) MLVA Genotyping reagent
Box, it is characterised in that: including for detecting Yunnan Province HIV/Mtb double infection and the mono- infection M. tuberculosis genes of Mtb point
10 sites high-resolution VNTR of type and corresponding 10 primer pairs, Taq enzymes, PCR buffer and DNA average molecular
Amount, wherein
Upstream primer sequence for detection site MIRU26 is as shown in SED IQ NO:1；
Downstream primer sequence for detection site MIRU26 is as shown in SED IQ NO:2；
Upstream primer sequence for detection site Mtub21 is as shown in SED IQ NO:3；
Downstream primer sequence for detection site Mtub21 is as shown in SED IQ NO:4；
Upstream primer sequence for detection site Mtub30 is as shown in SED IQ NO:5；
Downstream primer sequence for detection site Mtub30 is as shown in SED IQ NO:6；
Upstream primer sequence for detection site Mtub29 is as shown in SED IQ NO:7；
Downstream primer sequence for detection site Mtub29 is as shown in SED IQ NO:8；
Upstream primer sequence for detection site Mtub4 is as shown in SED IQ NO:9；
Downstream primer sequence for detection site Mtub4 is as shown in SED IQ NO:10；
Upstream primer sequence for detection site MIRU31 is as shown in SED IQ NO:11；
Downstream primer sequence for detection site MIRU31 is as shown in SED IQ NO:12；
Upstream primer sequence for detection site MIRU16 is as shown in SED IQ NO:13；
Downstream primer sequence for detection site MIRU16 is as shown in SED IQ NO:14；
Upstream primer sequence for detection site Mtub39 is as shown in SED IQ NO:15；
Downstream primer sequence for detection site Mtub39 is as shown in SED IQ NO:16；
Upstream primer sequence for detection site MIRU10 is as shown in SED IQ NO:17；
Downstream primer sequence for detection site MIRU10 is as shown in SED IQ NO:18；
Upstream primer sequence for detection site MIRU40 is as shown in SED IQ NO:19；
Downstream primer sequence for detection site MIRU40 is as shown in SED IQ NO:20.
Further, the mycobacterium tuberculosis includes mycobacterium bovis and human-like combination mycobacteria.
Further, the kit is suitable for Yunnan Province HIV/Mtb double infection and the mono- infection tuberculosis branch bar of Mtb
The Genotyping of bacterium.
Further, a method of detection study on genotyping of Mycobacterium tuberculosis, comprising the following steps:
(1) separation mycobacterium tuberculosis gene group DNA is extracted；
(2) it using DNA in step (1) as template, respectively using 10 pairs of primers described in claim 1 as primer, is reacted through PCR
Obtain the product in 10 sites VNTR described in tubercle bacillus gene group；
(3) using 2% Ago-Gel in step (2) product carry out electrophoresis, by the gel after electrophoresis be put into gel at
As in network analysis system, slice result is observed under ultraviolet lamp, save picture and performs record.Picture is imported into Quantity One
In software, the PCR product length in each site of each bacterial strain is calculated, calculates each site copy number；
(4) the calculated copy number that repeats of each bacterial strain is imported into Bio-Numerics (6.6) software progress clustering；It adopts
Clustering is distinguished with UPMGA method and MST method, then experimental result and site databases are compared, and understands the substantially gene of bacterial strain
Type feature and gene pleiomorphism distribution.
Further, the method for separation mycobacterium tuberculosis is that sample 1mL is taken to add 2% sulfuric acid or hydroxide in step (1)
2~4mL of sodium is placed in room temperature 15min, during which shakes 2~3 times, is then centrifuged 10min with 3000r/min, incline supernatant, leaves
Sediment.Sterile 7.0 physiological saline of pH of 0.5mL is drawn every time with 1mL asepsis injector cleans precipitating, continuous 3 times, the 4th time
It injects after 0.5ml physiological saline and stirs precipitating with syringe needle, then draw 0.1ml and be inoculated in Russell medium, 37 DEG C of cultures turn
The kind inclined-plane L-J obtains purebred.
Compared with the existing technology, the invention has the benefit that the present invention provides study on genotyping of Mycobacterium tuberculosis inspections
Primer sets are surveyed, including the primer pair especially suitable for 10 sites VNTR in Yunnan Province detection tubercle bacillus gene group；Experiment
The result shows that carrying out the detection of Yunnan Province tubercle bacillus with primer sets provided by the invention, high resolution is up to 0.999；HGI is total
Resolution ratio is 99.9%.In terms of genotypic results, Yunnan Mtb genotype has polymorphism, 122 plants of Dali clinical lung knot
Core isolated strains and 1 plant of reference culture (clinical separation strain including 61 plants of simple infection persons and 61 plants of double infection persons and 1 plant
H37Rv digital information) carries out clustering, generates dendrogram, sees that Fig. 2, genotypic results are shown, Dali 122
Strain Clinical isolation and 1 plant of reference culture are at least divided into 7 gene groups (I, II, III, IV, V, VI, VII), wherein I group accounts for
65.0% (80/123), including 80 genotype；It includes 19 genotype that II group, which accounts for 15.4% (19/123),；III group accounts for 8.1%
(10/123), including 10 genotype；IV group accounts for 5.7% (7/123), including 7 genotype；V group accounts for 2.4% (3/123),
Including 3 genotype；VI group accounts for 1.6% (2/123), including 2 genotype；VII group accounts for 1.6% (2/123), including 2 genes
Type.Wherein I group is the main popular gene group in Dali.Therefore based on I group of tuberculosis patient of prophylactic treatment, pass through
UPGMA method or MST method, the space structure dynamic model for establishing Network Management System and suitable this area are of great significance.
Detailed description of the invention
PCR amplification result (including 61 plants of simple infection persons and 61 plants of double infection persons in the 10 high-resolution sites Fig. 1
Clinical separation strain and 1 plant of H37Rv attenuated strain)；
122 plants of mycobacterium tuberculosis BioNumerics6.6 clustering figures (UPGMA) of Fig. 2；
Fig. 3 merges I PFGE finger-print of HIV double infection person mycobacterium tuberculosis clinical separation strain Dra.H:H37Rv subtracts
Strain；S1-s13: clinical separation strain；
Fig. 4 PFGE fingerprint map carries out processing with Bionumerics6.6 and clustering (UPGMA) is generated
Gene development tree
Specific embodiment
Technical solution of the present invention is described in further detail with reference to the accompanying drawings and detailed description:
1 collection of specimens of embodiment and strain idenfication
Select to merge in HIV Serologic detection positive and AIDS patient Mtb the infected as research object.According to tuberculosis
Sick diagnostic criteria, in conjunction with the sings and symptoms of patient, imaging data acquires the clinical samples of corresponding site；Predominantly phlegm mark
This (under trained medical staff's guidance, is advised patient first to gargle, by deep lung expectoration 5~10mL of sputum after deep breathing, sets
In the sterile Sputum cup with screw lid, first smear for microscopic examination confirmatory sample quality after sample is received, qualified sputum sample is in low power lens
Visible squamous cell ﹤ 10 in every visual field, leucocyte ﹥ 25.Saliva or saliva are considered as unqualified sample, resurvey,
Then acid-fast stain microscopy is carried out.It takes sample 1mL that 2% sulfuric acid or 2~4mL of sodium hydroxide is added to be placed in room temperature 15min, during which shakes
2~3 times, 10min is then centrifuged with 3000r/min, incline supernatant, leaves sediment.It is drawn every time with 1mL asepsis injector
The sterile pH7.0 physiological saline cleaning precipitating of 0.5mL, continuous 3 times, to remove soluble impurity and soda acid object in precipitating, makes to sink
Shallow lake is in neutrality, and is stirred and is precipitated with syringe needle after the 4th injection 0.5mL physiological saline, is then drawn 0.1mL and is inoculated in Roche culture
In base, 37 DEG C of cultures are observed daily, and size, shape, edge, surface, the face of (a) observation bacterium colony are carried out if having bacterium colony growth
Color etc.；(b) form and dyeability of bacterium；(c) inclined-plane transferred species L-J obtains purebred.Suspicious sample is routinely raw using mycobacteria
Change reaction, automatically cultivates instrument and matched BacT/ using the BacT/ALERT 3D 360 of Mei Liai company, France when necessary
ALERT MP mycobacterium tuberculosis culture bottle is cultivated and is identified.
The mycobacterium tuberculosis being separated to uses paranitrobenzoic acid (PNB) and thiophene-2-carboxylic acid hydrazine (TCH) to identify culture again
Base carries out strain idenfication.Two kinds of culture mediums are not grown then for mycobacterium bovis, are grown to non-tuberculous mycobacteria；
TCH growth and PNB is not grown, if bacterium colony slow growth, drying, cheese color cauliflower-shaped are human-like Mtb.Qualification result see the table below
1。
1 M. tuberculosis mycobacteria of table, mycobacterium bovis BCG and non-tuberculous mycobacteria identify table
Note: "+" growth, "-" are not grown
More than the 2 number of loci variable number tandem repeats of embodiment analyze (Multiple Locus Variable Number
Of Tandem Repeats Analysis, MLVA) Genotyping
21 sites VNTR have been screened for the first time, and design primer sequence, see Table 2 for details for sequence.With type strain H37Rv in experiment
DNA relative molecular weight is reference standard.
The primer sequence in 2 21 sites VNTR of table
Note: F refers to upstream primer, and R refers to downstream primer.
The principle of cost can be reduced again in line with guarantee resolution ratio, subsequent bacterial strain passes through optimum choice wherein 15 sites
(i.e. MIRU10, MIRU26, MIRU40, Mtub21, Mtub30, Mtub39, Mtub29, MIRU39, MIRU27, ETR-B,
MIRU23, MIRU2, MIRU4, MIRU31, Mtub4) it is expanded, electrophoresis is carried out using 2% Ago-Gel, after electrophoresis
Gel is put into gel imaging system analysis system, and slice result is observed under ultraviolet lamp, is saved picture and is performed record.Picture is led
Enter in Quantity One software, calculate the PCR product length in each site of each bacterial strain, is copied by calculating each site
Number.The calculated copy number that repeats of each bacterial strain is imported into Bio-Numerics (6.6) software progress clustering.Using UPMGA
Method and MST method distinguish clustering, then experimental result and site databases are compared, understand the substantially yielding characteristics of bacterial strain with
Gene pleiomorphism distribution.
The resolving power in each site MLVA indicates that HGI value is directly proportional to the resolving power in the site with HGI value, and HGI value is bigger,
The site resolving power is stronger, and result of study shows the resolving power in 21 sites, and there are larger difference, the HGI index highests in 21 sites
It is 0.999, minimum 0.301.Referring to the research such as SOLAC, resolving power is divided into high, medium and low three kinds, compared with high resolution (> 0.6) point
Not are as follows: MIRU26, Mtub21, Mtub30, Mtub39, Mtub4, MIRU31, MIRU16, Mtub29, MIRU10, MIRU40 etc. 10
A site；Medium resolving power (0.3≤HGI≤0.6) be respectively as follows: MIRU27, MIRU39, MIRU2, ETR-B, ETR-A,
9 sites such as MIRU26, MIRU23, MIRU4, Mtub34；It is respectively 2 positions such as MIRU24, ETR-C compared with low resolution (< 0.3)
Point.MIRU26, Mtub21, Mtub30, Mtub39, Mtub4, MIRU31, MIRU16, Mtub29, MIRU10, MIRU40 this 10
Site can be used as the preferred site of this area study on genotyping of Mycobacterium tuberculosis.
The mycobacterium tuberculosis and 1 plant of reference culture that HIV/Mtb double infection and simple Mtb infection are clinically separated are preferred
10 pairs of primers in site carry out PCR amplification result as shown in Figure 1, different strains DNA fingerprinting same site molecular weight
It has differences, showing mycobacterium tuberculosis, there are polymorphisms.
The number of repetition of each specific site of strain to be tested: electrophoresis picture is converted into tiff format and imports Quantity One
Software is arranged swimming lane, test strip value, to Marker assignment etc. under VNTR Quick Guide, is positive right with H37Rv
According to calculating the repetition copy number of strain to be tested, lead according to the clip size of the flanking sequence in each site and each specific site
Excel table storage is spare out.
The number of repetition for surveying bacterial strain 122 and 1 plant reference culture is converted into text file, and it is soft to import Bionumerics (6.6)
In part, to above 122 plants of bacterium (clinical separation strain and 1 plant of H37Rv including 61 plants of simple infection persons and 61 plants of double infection persons)
Digital information carry out clustering, generate dendrogram, see Fig. 2.It is shown according to UPGMA method dendrogram genotypic results,
122 plants of Dali Clinical isolation and 1 plant of reference culture are divided into 7 gene groups (I, II, III, IV, V, VI, VII), wherein
I type accounts for 65.0% (80/123), including 80 genotype；It includes 19 genotype that II type, which accounts for 15.4% (19/123),；III type accounts for
8.1% (10/123), including 10 genotype；IV type accounts for 5.7% (7/123), including 7 genotype；V type accounts for 2.4% (3/
, including 3 genotype 123)；VI type accounts for 1.6% (2/123), including 2 genotype；VII type accounts for 1.6% (2/123), including 2
A genotype.Wherein I type is the main popular genotype in Dali, and H37Rv standard attenuated strain is then in II type.HGI overall resolution
Rate is 99.9%.In terms of genotypic results, Yunnan Mtb genotype has polymorphism, is at least divided into 7 gene group.
The dominant strain of this area is concentrated mainly on I type, therefore (based on tuberculosis patient, is passed through with I type of prophylactic treatment
UPGMA method, the space structure dynamic model for establishing Network Management System and suitable this area are of great significance.
Embodiment 3 pulsed field gel electrophoresis (PFGE) method carries out Genotyping
It takes the bacterium colony of appropriate well-grown above-mentioned bacterial strains to move in mill tube in the Russell medium newly cultivated to be made
Bacteria suspension, 85 DEG C of inactivation 30min.350 μ L bacteria suspensions are taken to move in 1.5mL EP pipe, 12000/rpm, 5min.Supernatant is removed, is precipitated
It is resuspended in 300 μ L TEN buffer (100mM EDTA, 0.15MNaCl, 100mM TrisPH7.5), is centrifuged 4500rpm/
10min.Supernatant is removed, precipitating is resuspended in 150 μ L EC buffer (100mM EDTA, 1M NaCl, 6mM Trish-HCl
PH7.6,0.2%deoxycholate, 0.5%Sarkosyl, 0.5%Brij-58) in, 15min is heated in boiling water.After cooling
Lysozyme, which is added, makes its final concentration of 25mg/ml, and it is (spare in 56 DEG C of water-baths) mixed that isometric 2% low melting-point agarose is added later
It is even.80 μ L mixtures are taken to move in plug mold, 4 DEG C of solidification 5min are spare.By the glue bolt containing Mtb genomic DNA of preparation
It moves in the 50ml centrifuge tube containing 10ml37 DEG C of cell dissolution buffer I (0.5mM EDTA, 10%Sarkosyl), 37 DEG C
Water-bath concussion is overnight.Topple over cell dissolution buffer I, be added 9800 μ L cell dissolution buffer II (0.5mM EDTA, 1%
Sarkosyl), 200 μ L Proteinase K (20mg/ml) solution are added later, 50 DEG C of water-bath concussions are for 24 hours.Topple over cell dissolution
Buffer II is added 10ml TE buffer (10mM Tris-HCl, 1mM EDTA, PH8.0) shaking table and washes 3 times, each 30min,
Glue bolt is moved in sterile 10ml test tube after 3rd washing.The digestive juice containing 30U Dra I, predigestion in 30 DEG C is added
30min is moved back to digesting 16h in 37 degrees Celsius.Electrophoresis in 1% Ago-Gel made of 0.5 × TBE, electric field setting
(CHEF DR III: 6V/cm, 120 ° of corners, 14 DEG C；1s~15s is arranged in preceding 16h pulse, and 60s~70s is arranged in rear 8h pulse).It will
Blob of viscose after electrophoresis, which moves in 0.5mg/mL EB dye liquor, dyes 20min, and water cleans 20min.Gel imaging passes through pulse
Double sense mycobacterium tuberculosis clinical separation strain DNA fingerprintings that field gel electrophoresis system electrophoresis obtains are shown in Fig. 3.PFGE method is obtained
The finger image obtained carries out clustering with BioNumerics (6.6) software UPGMA method and generates gene development tree, is detailed in Fig. 4.
Calculate resolving power Simpson's diversity index (Simpson ' the s index of of resolving index PFGE classifying method
Diversity it) indicates, formula is as follows:
Note: N indicates that total bacterial strain number, nj indicate strain typing number.
PFGE and the two different classifying methods of VNTR point are obtained by the comparison to two kinds of genotyping results of VNTR and PFGE
The result of type is roughly the same, and the bacterial strain distribution of the secondary advantage gene group of two kinds of different classifying methods is also roughly the same, but
VNTR method further by protogene group A group obtained by PFGE method parting still further be divided into two it is independent
Gene group I group and II group, it follows that the genotyping result of VNTR method is more fine, low in cost, therefore can be used as Yunnan
The prefered method of Mtb/HIV double infection person's Mtb Genotyping.
4 Mtb of embodiment is to isoniazid, rifampin, ethambutol, the sensitivity Detection of four kinds of drugs of streptomysin
Drug sensitive test is pressed with antituberculotic isoniazid (INH), the biological value of rifampin (RFP), ethambutol (EMB)
Its unit of weight calculates, and is 1000ug/mg, need to only consider its purity when calculating dosage.Streptomycin sulfate (SM-SO4)
Paraaminosalicylic acid sodium salt (PAS-Na) etc. is while considering its purity, it should calculate its salt form according to the milligram potency of manufacturer
Dosage.It is such as calculated without calibration potency gram by following reference value: streptomycin sulfate (SM-SO4)780ug/mg.Except RFP diformazan
The dissolution of base first thiamines, then it is outer with sterile purified water dilution, other drugs can use aseptic distillation salt water dissolved dilution.Every kind of drug is pressed
According to high concentration medical fluid is made shown in following table, low concentration medical fluid is diluted to according still further to corresponding proportion.Drug is whole in rule of three culture medium
Concentration are as follows: isoniazid 0.2ug/ml, streptomysin 4ug/mL, rifampin 40ug/mL, ethambutol 2ug/mL.It is real that culture medium is added
The calculating of border dose is with reference to following standard: actual drug amount=(drug final concentration in culture medium × culture volume need to be prepared)/
(pharmaceutical purity × drug titers × 1000).
It is inoculated on neutral Russell medium with single bacterium colony in oese picking Russell medium, is cultivated in 37 DEG C of incubators
6 weeks, continuous passage was three times.Then the bacterium of logarithmic growth phase carries out mill bacterium (mill tube method): bacterium on careful scraping culture medium
It falls and (avoids blowing down medium component), mill tube (A pipe) is added.It puts to be shaken on the oscillator to most of fungus block and be broken up, when
Between should control within 1min, in order to avoid influence bacterial action.2mL~10mL sterile saline is added, stands 10min to bacterium
Bulky grain or fungus block precipitating in liquid, are made into suspension.Supernatant in A pipe is drawn to be added dropwise in B pipe, at the same with the pipe of Maxwell 1
Carry out than turbid, if bacterial concentration is lower in A pipe, solution in B pipe can be also sucked out in advance, by A supernatant be added B pipe, with
Physiological saline carries out adjustment, or adds bacterium colony in A pipe and carry out mill bacterium operation.50ul bacterium solution is taken out from B pipe adds to preparation
In pipe C pipe, mixes, that is, be made into 10-2Mg/mL bacterium solution.It adds to from 50ul bacterium solution is taken out in C pipe in tubulation D pipe, mixes,
10-4Mg/mL bacteria suspension.
The inoculation of rule of three medicament sensitivity test: 10uL 10 is taken respectively with micropipettor-2Mg/mL and 10-4Mg/mL's
Bacteria suspension is inoculated into neutral control tetra- kinds of drug sensitive culture mediums of Russell medium and INH, RFP, EMB, SM with uniform inocalation method of crossing
In.Final quantity of microorganism inoculated is 10-4Mg and 10-6Mg, the culture medium after inoculation are placed in 37 DEG C of cultures, and report is as a result, susceptibility after 6 weeks
It the results are shown in Table 4.
Quality control: if high dilution bacterium night (10-4Mg/mL the clump count) grown in control medium is less than 20 bacterium
It falls, then should repeat to test from control tube secondary culture.Every batch of is tested with Mycobacterium tuberculosis Reference Strains (H37Rv type strain) 10- 3The quality of mg detection pastille culture medium.
It calculates drug resistance percentage: being grown in the bacterium colony/control medium grown in drug resistance percentage=pastille culture medium
Bacterium colony.If drug resistance percentage > 1%, then it is assumed that tested bacterium is to the anti-tubercular drug drug resistance.
4 drug sensitivity tests (10 of table-2Mg/mL bacteria suspension)
27 plants of mycobacterium tuberculosis are to the drug resistance of 4 kinds of antituberculotics as a result, being shown in Table 5
5 Mtb. drug resistance situation of table
Occur 5 plants of multi-drug resistant bacterial strains in 27 plants of mycobacterium tuberculosis altogether, wherein there is resistance to INH+RFP, resistance to INH+RFP+SM and
Tri- kinds of situations of resistance to INH+RFP+SM+EMB, the results are shown in Table 6.
6 27 plants of multi-drug resistance of Mycobacterium tuberculosis bacterial strain distribution tables of table
Simple mycobacterium tuberculosis infects and the drug resistance of Mycobacterium tuberculosis rate comparison for merging HIV double infection and 4 kinds
The comparison of the resistant rate of antituberculotic the results are shown in Table 7,8.
Table 7 merges the bis- sense bacterial strains of HIV compared with the resistant rate of the mono- sense bacterial strain of Mtb.
Statistical analysis, Chi-square Test, χ 2=10.316, P=0.001 are carried out through SPSS11.0 software.P < 0.05, merely
Mycobacterium tuberculosis infected patient is statistically significant with the resistant rate difference for merging HIV double infection patient.
Comparison of the table 8 to 4 kinds of antituberculotic antibody-resistant bacterium
The infection of simple mycobacterium tuberculosis divides with the tuberculosis for merging HIV/Mtb double infection as can be seen from the above results
During branch bacillus resistant rate compares, double infection is higher than single sense to the resistant rate of four kinds of anti-tubercular drugs.Illustrate the knot for merging HIV infection
Core mycobacteria is easier drug resistance occur, it should carry out keypoint control.
According to cluster analysis result and drug sensitivity assay result, obtaining does not have phase between bacterial resistance and gene pleiomorphism
Guan Xing.Therefore it only needs to carry out keypoint control for dominant microflora, and can not consider the factor of gene pleiomorphism in medication.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
The change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the invention
Protection scope should be determined by the scope of protection defined in the claims.
Sequence table
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<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
caacatcgcc tggttctgta 20
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cttgaagccc cggtctcatc tgt 23
<210> 6
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
acttgaaccc ccacgcccat tagta 25
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gccagccgcc gtgcataaac ct 22
<210> 8
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
agccacccgg tgtgccttgt atgac 25
<210> 9
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cttggccggc atcaagcgca ttatt 25
<210> 10
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ggcagcagag cccgggattc ttc 23
<210> 11
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
actgattggc ttcatacggc ttta 24
<210> 12
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gtgccgacgt ggtcttgat 19
<210> 13
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tcggtgatcg ggtccagtcc aagta 25
<210> 14
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
cccgtcgtgc agccctggta c 21
<210> 15
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
cggtggaggc gatgaacgtc ttc 23
<210> 16
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tagagcggca cgggggaaag cttag 25
<210> 17
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gttcttgacc aactgcagtc gtcc 24
<210> 18
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gccaccttgg tgatcagcta cct 23
<210> 19
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gggttgctgg atgacaacgt gt 22
<210> 20
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
gggtgatctc ggcgaaatca gata 24
<210> 21
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
aaatcggtcc catcaccttc ttat 24
<210> 22
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cgaagcctgg ggtgcccgcg attt 24
<210> 23
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
cgagagtggc agtggcggtt atct 24
<210> 24
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
aatgacttga acgcgcaaat tgtga 25
<210> 25
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
cgcatcgaca aactggagcc aaac 24
<210> 26
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
cggaaacgtc tacgccccac acat 24
<210> 27
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
tcgaaagcct ctgcgtgcca gtaa 24
<210> 28
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gcgatgtgag cgtgccactc aa 22
<210> 29
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
atggccaccc gataccgctt cagt 24
<210> 30
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cgacgggcca tcttggatca gctac 25
<210> 31
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ggtgcgcacc tgctccagat aa 22
<210> 32
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
ggctctcatt gctggagggt tgtac 25
<210> 33
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
ctgtcgatgg ccgcaacaaa acg 23
<210> 34
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
agctcaacgg gttcgccctt ttgtc 25
<210> 35
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
tcggagagat gcccttcgag ttag 24
<210> 36
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ggagaccgcg accaggtact tgta 24
<210> 37
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
tggacttgca gcaatggacc aact 24
<210> 38
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
tactcggacg ccggctcaaa at 22
<210> 39
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
cgaccaagat gtgcaggaat acat 24
<210> 40
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
gggcgagttg agctcacaga a 21
<210> 41
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
gcgcgagagc ccgaactgc 19
<210> 42
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
gcgcagcaga aacgccagc 19
Claims (5)
- Mycobacterium tuberculosis 1. (Mtb) MLVA genotyping kit, it is characterised in that: including for detecting Yunnan Province HIV/ 10 sites high-resolution VNTR and corresponding 10 of Mtb double infection and the mono- infection study on genotyping of Mycobacterium tuberculosis of Mtb A primer pair, Taq enzyme, PCR buffer and DNA relative molecular weight, whereinUpstream primer sequence for detection site MIRU26 is as shown in SED IQ NO:1；Downstream primer sequence for detection site MIRU26 is as shown in SED IQ NO:2；Upstream primer sequence for detection site Mtub21 is as shown in SED IQ NO:3；Downstream primer sequence for detection site Mtub21 is as shown in SED IQ NO:4；Upstream primer sequence for detection site Mtub30 is as shown in SED IQ NO:5；Downstream primer sequence for detection site Mtub30 is as shown in SED IQ NO:6；Upstream primer sequence for detection site Mtub29 is as shown in SED IQ NO:7；Downstream primer sequence for detection site Mtub29 is as shown in SED IQ NO:8；Upstream primer sequence for detection site Mtub4 is as shown in SED IQ NO:9；Downstream primer sequence for detection site Mtub4 is as shown in SED IQ NO:10；Upstream primer sequence for detection site MIRU31 is as shown in SED IQ NO:11；Downstream primer sequence for detection site MIRU31 is as shown in SED IQ NO:12；Upstream primer sequence for detection site MIRU16 is as shown in SED IQ NO:13；Downstream primer sequence for detection site MIRU16 is as shown in SED IQ NO:14；Upstream primer sequence for detection site Mtub39 is as shown in SED IQ NO:15；Downstream primer sequence for detection site Mtub39 is as shown in SED IQ NO:16；Upstream primer sequence for detection site MIRU10 is as shown in SED IQ NO:17；Downstream primer sequence for detection site MIRU10 is as shown in SED IQ NO:18；Upstream primer sequence for detection site MIRU40 is as shown in SED IQ NO:19；Downstream primer sequence for detection site MIRU40 is as shown in SED IQ NO:20.
- 2. kit according to claim 1, it is characterised in that: the mycobacterium tuberculosis includes bovine tuberculosis branch bar Bacterium and human-like combination mycobacteria.
- 3. the application of kit according to claim 1, it is characterised in that: the kit is suitable for Yunnan Province The Genotyping of HIV/Mtb double infection and the mono- infection mycobacterium tuberculosis of Mtb.
- 4. a kind of method for detecting study on genotyping of Mycobacterium tuberculosis, it is characterised in that: the following steps are included:(1) separation mycobacterium tuberculosis gene group DNA is extracted；(2) it using DNA in step (1) as template, respectively using 10 pairs of primers described in claim 1 as primer, reacts and obtains through PCR The product in 10 sites VNTR described in tubercle bacillus gene group；(3) electrophoresis is carried out to product in step (2) using 2% Ago-Gel, the gel after electrophoresis is put into gel imaging system In system analysis system, slice result is observed under ultraviolet lamp, is saved picture and is performed record.Picture is imported into Quantity One software In, the PCR product length in each site of each bacterial strain is calculated, each site copy number is calculated；(4) the calculated copy number that repeats of each bacterial strain is imported into Bio-Numerics (6.6) software progress clustering；Using UPMGA method and MST method distinguish clustering, then experimental result and site databases are compared, and understand the substantially genotype of bacterial strain Feature and gene pleiomorphism distribution.
- 5. according to the method described in claim 4, it is characterized by: the method for separation mycobacterium tuberculosis is to take in step (1) Sample 1mL adds 2% sulfuric acid or 2~4mL of sodium hydroxide to be placed in room temperature 15min, during which shakes 2~3 times, then with 3000r/min It is centrifuged 10min, incline supernatant, leaves sediment.The sterile pH7.0 physiological saline of 0.5mL is drawn every time with 1mL asepsis injector Cleaning precipitating continuous 3 times, is stirred after the 4th injection 0.5ml physiological saline with syringe needle and is precipitated, and then absorption 0.1ml is inoculated in In Russell medium, 37 DEG C of cultures, the inclined-plane transferred species L-J obtains purebred.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647647A (en) * | 2020-06-03 | 2020-09-11 | 皖南医学院 | Mycobacterium tuberculosis MIRU-VNTR gene multi-copy number rapid detection and analysis method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102094077A (en) * | 2009-12-15 | 2011-06-15 | 复旦大学 | Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly |
| TW201142038A (en) * | 2010-05-31 | 2011-12-01 | Univ Nat Cheng Kung | Method for identifying Mycobacterium |
| CN103834743A (en) * | 2014-03-25 | 2014-06-04 | 中华人民共和国吉林出入境检验检疫局 | Genotype detection primer group and kit for mycobacterium tuberculosis |
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2019
- 2019-06-27 CN CN201910564575.9A patent/CN110257535A/en active Pending
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| CN102094077A (en) * | 2009-12-15 | 2011-06-15 | 复旦大学 | Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly |
| TW201142038A (en) * | 2010-05-31 | 2011-12-01 | Univ Nat Cheng Kung | Method for identifying Mycobacterium |
| CN103834743A (en) * | 2014-03-25 | 2014-06-04 | 中华人民共和国吉林出入境检验检疫局 | Genotype detection primer group and kit for mycobacterium tuberculosis |
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| 刘开明等: "中国南方3省173例HIV/MTB双重感染病例分子流行特征研究", 《中国国境卫生检疫杂志》 * |
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| CN111647647A (en) * | 2020-06-03 | 2020-09-11 | 皖南医学院 | Mycobacterium tuberculosis MIRU-VNTR gene multi-copy number rapid detection and analysis method |
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