CN103757110A - Vibrio cholera analysis typing kit - Google Patents
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Abstract
The invention relates to a vibrio cholera analysis typing kit. A fluorescently-marked composite primer is used for amplifying six VNTR (variable number of tandem repeats) loca of vibrio cholera respectively. According to the vibrio cholera analysis typing kit provided by the invention, the six loca are divided into three groups, and four fluorescent markers are involved in total; after capillary electrophoresis detection, by comparing with an allele typing standard substance, the type of vibrio cholera can be obtained quickly and accurately.
Description
Technical field
The present invention relates to a kind of vibrio cholerae molecule parting detection kit, specifically relate to a kind of fluorescence labeling composite amplification system and allelic ladder of simultaneously analyzing 6 vibrio cholerae VNTR locus, this system can be applied to tracing to the source of vibrio cholerae.
Background technology
Bacteriosis is endangering the sound development of human health and livestock industry always, day by day frequent along with growth in the living standard, movement of population, and the food-safety problem that food borne bacteria causes has become the social public health problem that the whole world is faced jointly.And the factor such as universal of the growth of population, scientific and technological develop rapidly, interventional medical apparatus is remained high ward infection incidence, become a clinical the most thorny difficult problem at present.
Evaluation, the typing method of bacterium can, according to the biochemical character of bacterium or genomic constitution, be analyzed the relation between different sources bacterial strain.It understanding the propagation kinetics of bacterium in crowd, search the source of infection of food poisoning, and judge breaking out, find the source of infection and identify the aspects such as some special pathogenic bacterium and having important effect of ward infection, so become the powerful of epidemiology survey.
Vibrio cholerae (V.cholera) is the pathogenic agent of mankind's cholera, and cholera is a kind of ancient and popular one of deadly infectious disease widely.Once caused repeatedly and be very popular in the world, main manifestations is violent vomiting, diarrhoea, and dehydration, mortality ratio is very high.Belong to international quarantine transmissible disease.The technology that Typing of Vibrio Cholerae detects has a lot, using at present maximum detection techniques is that pulsed field gel electrophoresis detects (pulsed field gel electrophoresis, PFGE), this detection method requires high to operator, complex operation, do not accomplish automatization, high throughput testing, so promote, be difficult for, only have the tool somatotype monitoring capability in regional center laboratory, provincial disease prevention and control center.Heidelberg JF etc. were in 2000, take bacterial strain N16961 as template, completed the mensuration of vibrio cholerae complete sequence, genome sequence Vibrio cholerae O1biovar El Tor str.N16961RefSeq Genome(Heidelberg JF, et al.DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae.Nature2000Aug3).
6 vibrio cholerae VNTR locus of two composite amplification system amplifications for Jaran S.Olsen etc. (Olsen et al.Journal of Microbiological Methods78 (2009) 271 – 285), by ABI310 automatic sequencer, amplification is detected.Data results shows, 6 VNTR locus somatotype results and PFGE somatotype result are similar, the good somatotype ability of tool, and operation is easier, quick.It is MLVA(multiple locus VNTR analysis, MLVA that multiple VNTR composite amplifications detect english abbreviation).
(the Yael Danin-Poleg et al.Vibrio cholerae Strain Typing and PhylogenyStudy Based on Simple Sequence Repeats Journal of Clinical Microbiology such as Yael Danin-Poleg, Mar.2007, p.736 – 746) the VNTR locus of cholera vibrio gene group is analyzed to wherein 9 VNTR locus tool polymorphisms.
Vibrio cholerae VNTR locus and the polymorphism of the statistics such as table 1Yael Danin-Poleg
By the test of comparison by Internet and actual sample, find that definite allelotrope scope such as Yael Danin-Poleg is less than normal, utilize former scope design composite amplification system, may cause the overlapping of amplified production size, cause result erroneous judgement.
Summary of the invention
The object of the invention is to provide the test kit that a kind of VNTR of utilization locus carries out fluorescence labeling composite amplification to vibrio cholerae and carries out somatotype, and it is overlapping and can cause the problem of erroneous judgement to solve the vibrio cholerae VNTR locus allelotrope scope amplified production causing less than normal in prior art.The present invention has selected in 9 locus, 6 VNTR locus that polymorphism is the highest, and according to the allelotrope sequence of each locus, design primer, thus make test kit there is good somatotype ability.Technical scheme is:
A kind of vibrio cholerae is analyzed parting kit, includes the designed primer of VNTR locus of following vibrio cholerae: VC1650, VC0437, VC0147, VCA0171, VCA0283 and VC1457.
Further, for the result that makes somatotype is not prone to erroneous judgement, the allelotrope that each locus is corresponding can be detected exactly, preferably will carry out packet marking to the primer of each locus, grouping is: the 1st group, and VC1650, VC0437; The 2nd group, VC0147, VCA0171; The 3rd group, VCA0283, VC1457.
Further, described each group is used different fluorescence color to carry out mark, is more excellently the 1st group and uses FAM mark, uses HEX mark for the 2nd group, uses TAMRA mark for the 3rd group.
Further, the corresponding allelotrope scope of above each locus is larger, for fear of the erroneous judgement in somatotype testing process, raising accuracy rate, needs optimization design primer.Between fluorescently-labeled primer of the same race, leave more than 20 interval more than base, the amplification of avoiding unknown size allelotrope to cause is crossed the border.Preferred primer sequence is as follows:
Table 2 vibrio cholerae composite amplification primer, allelotrope scope and product scope
Further, the most preferable concentrations of above-mentioned each primer in final amplification system is:
The preferred concentration of each primer of table 3
Further, in described test kit, also include allelic ladder.In test kit of the present invention, described allelic ladder is to be mixed by the allelic ladder of each locus; Allelic ladder refers to the DNA fragmentation group for the known array of gene type, it is the contrast of somatotype while carrying out result detection, as long as allelic ladder is synchronizeed to electrophoresis with sample pcr amplification product, different laboratories, different equipment, different electrophoresis methods, all can obtain consistent repeatable result.In test kit of the present invention, by the situation of each locus grouping, prepare allelic ladder, utilize this locus of each primer pair to increase to each allelotrope; PCR product cloning; DNA sequencing confirms size and the structure of Insert Fragment; After naming by international standard, after enlarged culturing, amplification and identifying again; Mix and all allelotrope of balancing, make the allelic ladder equivalent of each locus of preparing, the gram molecular weight that is the allelic ladder of each locus all equates, the above-mentioned method of preparing allelic ladder by those skilled in the art known.
Further, in test kit, also include reaction mixture 10.0 μ L, warm start Taq enzyme 0.5 μ L, sdH
2o complements to 25.0 μ L; In described reaction mixture, include: MgCl
27.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg/ml.
Beneficial effect
1, by an individual system, detect 6 vibrio cholerae VNTR locus, somatotype effect is consistent with PFGE simultaneously.
2, experimental implementation is easy, in conjunction with automatization sequenator, can realize automatization, high-throughput that Typing of Vibrio Cholerae detects.
3, invention has been proposed in typing of bacteria, to use allelic ladder first, utilized software, somatotype is easier, accurate.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of genotyping standard thing, the information of the corresponding table 8 of each locus allelotrope.
Fig. 2 is the somatotype result figure of vibrio cholera strain N16961.
VCA0171 No. 3 primer amplification failure sample sequencing results (part) in Fig. 3 embodiment 1, drawn area is the calmodulin binding domain CaM of non-marked downstream primer, by two bases of some mark, is wherein the mutating alkali yl different from primer.
Embodiment
Determining of the design of embodiment 1 test kit and amplification condition
1. the selection of locus
Choose 6 vibrio cholerae VNTR locus that polymorphism is high, its allelic relevant information is in Table 3.Template sequence is downloaded, and from NCBI genebank, has downloaded locus sequence, has determined the position of sequence iteron.By the sequence of the homologous genes seat different strains of vibrio cholerae in comparison Genebank, determined allelotrope scope.Result is as follows:
Table 4 locus and allelotrope information
| Locus | No. Genebank, sequence | Position, iteron | Allelotrope scope |
| VC1650 | CP002555.1 | 1646667-1646729 | 2-14 |
| VC0437 | CP003330.1 | 466934-467328 | 2-24 |
| VC0147 | CP003330.1 | 137105-137159 | 2-17 |
| VCA0171 | CP003331.1 | 187738-187875 | 6-26 |
| VCA0283 | CP003331.1 | 303918-303989 | 3-30 |
| VC1457 | KF664581.1 | 2702-5730 | 2-15 |
2. the design of composite amplification primer
6 locus divide three groups, every group of two locus, locus and a locus that iteron is little that iteron is large.VCA1650 and VC0437, VC0147 and VCA0171, VCA0283 and VC1457 are respectively one group.Small one and large one collocation, is divided into three kinds of fluorescent marks, makes the fragment of every kind of color as far as possible little.
In the process of the design of primer, main principle is the general 20-30bp of length, and in oligo6 software, Tm value is between 68～72 ℃, and mispairing initiation rate is less than 150.Primer self avoids forming secondary structure hairpin structure, and mark is arranged on the conventional requirement of the design of primers such as primer 5 ' end.
Take VCA0171 locus as example, with OLIGO6 software automatic search primer, search coverage is set, Tm value scope, at 68～72 ℃, is therefrom selected 8 pairs of primers that mispairing initiation rate is little, dimer is few.Through NCBI primer blast comparison, rejecting PBR has the primer of sudden change, remaining 3 pairs of primers, as following table:
The design of primers of table 5VCA0171 locus
Primer is synthetic by the raw work in Shanghai, and list does annealing temperature gradient amplification to primer.
3. Amplification determines
Amplification system is as follows:
Table 6 amplification system composition
Wherein said Reaction Mix is MgCl
27.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg/ml.
Amplification thermal cycling
(l) pcr amplification pipe is placed on thermal cycler;
(2) program that selection is recommended below increases;
(3) sample after amplification should keep in Dark Place;
Table 7 amplification program
Same sample, the identical amplification system of configuration 18 pipe, point 6 groups of cycle of annealing amplifications, every group 3 pipe repeats.Wherein annealing temperature is respectively 54 ℃, and 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, all the other step temperature are identical, does gradient test.Selection is from all good primers of 54 ℃～64 ℃ specific amplifications, amplification efficiency.Wherein 54～56 ℃ of No. 1 primer pair low temperature there will be non-specific amplification.Although No. 2 primer pairs are without non-specific amplification, 62 ℃ there will be amplified peak step-down above, and 64 ℃ of amplifications are lost.3, No. 4 primer thermograde test compound requirement.While further doing more multiple sample test, No. 3 primer pair part sample there will be amplification to lose, and through order-checking, proving template has two base mutations in downstream primer land, and primer binding ability declines, and causes amplification to be lost, and sequencing result is shown in accompanying drawing 3.
Re-start design of primers, through a large amount of repetition tests, find following No. 4 primers, through thermograde test and large sample test, do not occur amplification loss and non-specific amplification.
The design of primers of table 8VCA0171 locus
All the other 5 pairs of design of primers and primer screening are as the step of VCA0171.By single primer concentration that expands test, be configured to the test of composite amplification primer, finally determined 6 pairs of primers, annealing temperature tolerance, specificity are all good, and each other without non-specific amplification.In primer test process, because locus number in composite amplification system is more, have each other competitive influence, therefore the relative equilibrium control difficulty of each locus strengthens, and by repeatedly experiment repeatedly, regulates primer concentration and proportioning, reach eventually balance, finally determined each primer concentration.
Each locus sequence and primer concentration are as follows:
The primer that the each locus of table 9 is corresponding
Synthesized simultaneously and be not with fluorescently-labeled primer, for product sequence verification.Primer is synthetic by the raw work in Shanghai.By above-mentioned concentration, configuration 5 × composite amplification primer, does thermograde test, and amplification system and amplification program are as follows:
Amplification system application of sample:
Table 10 amplification system composition
| Component | Volume |
| Reaction Mix | 10.0μL |
| Genomic dna | 0.1～10 μ l content is 0.125～5ng |
| Primer mixture | 5.0μL |
| Warm start Taq enzyme (5U/ μ L) | 0.5μL |
| sdH 2O | Complement to 25.0 μ L |
Amplification program:
Table 11 amplification program
Same sample, the identical amplification system of configuration 18 pipe, point 6 groups of cycle of annealing amplifications, every group 3 pipe repeats.Wherein annealing temperature is respectively 54 ℃, and 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, all the other step temperature are identical, does gradient test.Test result shows, when 58～62 ℃ of annealing, amplification peak height is best, considers each PCR instrument control temperature temperature difference, 60 ℃ of annealing for recommendation.
Preferred Amplification and test kit composition are as follows respectively:
Table 12 test kit configuration composition
| Component | Volume |
| Reaction Mix | 10.0μL |
| Genomic dna | 0.1～10 μ l content is 0.125～5ng |
| Primer mixture | 5.0μL |
| Warm start Taq enzyme (5U/ μ L) | 0.5μL |
| sdH 2O | Complement to 25.0 μ L |
Wherein said Reaction Mix is MgCl
27.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg/ml.Wherein genome DNA sample can be bacterium liquid, can be also to extract DNA.
The step that somatotype detects is:
(l) pcr amplification pipe is placed on thermal cycler;
(2) program of option table 13 increases;
The amplification program of table 13 thermal cycler
(3) sample after amplification should keep in Dark Place;
(4) amplified production fluoroscopic examination on genetic analyzer
By marking AGCU Marker SIZ-500 in molecular weight in deionized formamide and system, form loading mixture ((0.5 μ lAGCU Marker SIZ-500(Zhongde Meilian Biotech Co., Ltd. Wuxi)) × (sample introduction number)+(12 μ l deionized formamide) × (sample introduction number)).By 12.5 μ l loading mixture and PCR products, avoid producing bubble.95 ℃ of sex change 3 minutes, ice bath 3 minutes, and electrophoresis as early as possible; With genetic analyzer, detect and analyze;
(5) with genetic analyzer in fragment analysis software GeneMapper analytical procedure (4), detect the data of collecting.Electrophoresis adopts multiple tracks or single track capillary electrophoresis.
According to above design of primers, test kit composition and amplification condition, to the detection of bacterial strain N16961.
Cholera reference culture N16961 is purchased from American Type Culture Collecti (ATCC), after 100 ℃ of boiling water boilings of bacterium liquid 20 minutes, gets 1 μ l as template, for amplification.
With genetic analyzer in fragment analysis software GeneMapper analytical procedure (4), detect the data of collecting.Amplification is as accompanying drawing 2.
To N16961 bacterial strain, use respectively that the 6 pairs of sequences are identical, the primer amplification of tape label not, PCR product send raw work order-checking.Sequencing result is consistent with N16961 genome sequence (Vibrio cholerae O1biovar El Tor str.N16961RefSeq Genome) result, has proved the reliability of result.
The preparation of embodiment 2 allelic ladders
Vibrio cholera strain to Zhejiang Province's disease control collection detects, and selects same gene seat, the sample of the different sizes of allelotrope, and following table is the allelic composition of each locus:
Table 14 allelotrope information
Use respectively VC1650, VC0437, VC0147, VCA0171, VCA0283 and VC1457 single locus amplimer (primer concentration 10 μ M) amplification.Wherein genome DNA sample can be bacterium liquid, can be also to extract DNA.
Primer sequence is as follows:
Table 15 primer sequence
A, amplification system are as follows:
Table 16 test kit configuration composition
Wherein said Reaction Mix is MgCl
27.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg/ml.
B, amplification thermal cycling
The amplification program of table 17 thermal cycler
The fluoroscopic examination on genetic analyzer of C, amplified production
By marking AGCU Marker SIZ-500 in molecular weight in deionized formamide and system, form loading mixture ((0.5 μ lAGCU Marker SIZ-500(Zhongde Meilian Biotech Co., Ltd. Wuxi)) × (sample introduction number)+(12 μ l deionized formamide) × (sample introduction number)).By 12.5 μ l loading mixture and PCR products, avoid producing bubble.95 ℃ of sex change 3 minutes, ice bath 3 minutes, and electrophoresis as early as possible; With genetic analyzer, detect and analyze;
D, phenotypic analysis
With genetic analyzer in fragment analysis software GeneMapper analytical procedure C, detect the data of collecting.
Electrophoresis adopts multiple tracks or single track capillary electrophoresis.
E, according to detected peaks at high proportion, lacking of peak height adds, and what peak was low adds, and each amplified allele product is mixed to composition allelic ladder.Electrophoresis detection again, detects figure as accompanying drawing 1.
F, each allelic use tape label PCR primer amplification not accordingly, amplification program is identical with table 11, and PCR product is served Hai Shenggong order-checking, according to sequencing result, determines allelotrope repeat number.
Allelotrope information in the corresponding table 14 in peak going out below the each locus of accompanying drawing, the allelotrope zero lap of different genes seat.
Embodiment 3:19 strain bacterial strain MLVA detects
The vibrio cholerae bacterial classification that bacterium source is preserved in Zhejiang Province Disease Control and Prevention Center, has randomly drawed the 19 strain bacterial strains that different time is collected.These samples, Disease Control and Prevention Center, with classical PFGE method somatotype, is divided into 12 kinds of types, is for No. 10-14 same type, is for 15,16,17, No. 19 same type, and other bacterial classification is unique type.
Press experimental system, the amplification program of embodiment 1, detection method detects, and 19 strain strain typing results are as follows:
Table 18 strain typing result
Result from table, 19 strain bacterium are divided into 17 kinds of types, and wherein experiment numbers 12 and 13,17 and 19 is respectively identical type, has shown good somatotype ability.
SEQUENCE LISTING
<110> Zhongde Meilian Biotech Co., Ltd. Wuxi
Zhejiang Center For Disease Control and Prevention
<120> vibrio cholerae is analyzed parting kit
<130> without
<160> 12
<170> PatentIn version 3.5
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Claims (7)
1. vibrio cholerae is analyzed a parting kit, it is characterized in that, includes the designed primer of VNTR locus of the following vibrio cholerae of amplification: VC1650, VC0437, VC0147, VCA0171, VCA0283 and VC1457.
2. vibrio cholerae according to claim 1 is analyzed parting kit, it is characterized in that: the primer that each locus is corresponding is to carry out packet marking, and the 1st group, VC1650, VC0437; The 2nd group, VC0147, VCA0171; The 3rd group, VCA0283, VC1457.
3. vibrio cholerae according to claim 2 is analyzed parting kit, it is characterized in that: each group is used respectively the one in FAM, HEX, TAMRA to carry out mark, and the mark look difference between each group.
4. vibrio cholerae according to claim 1 is analyzed parting kit, it is characterized in that, the primer that each locus is corresponding is: VC1650, SEQ ID NO.1～2; VC0437, SEQ ID NO.3～4; VC0147, SEQ ID NO.5～6; VCA0171, SEQ ID NO.7～8; VCA0283, SEQ ID NO.9～10; VC1457, SEQ ID NO.11～12.
5. vibrio cholerae according to claim 4 is analyzed parting kit, it is characterized in that, the concentration of primer corresponding to each locus in final amplification system is: SEQ ID NO.1～2,0.10 μ M; SEQ ID NO.3～4,0.15 μ M; SEQ ID NO.5～6,0.12 μ M; SEQ ID NO.7～8,0.21 μ M; SEQ ID NO.11～12,0.15 μ M.
6. vibrio cholerae according to claim 1 is analyzed parting kit, it is characterized in that: also include allelic ladder.
7. vibrio cholerae according to claim 1 is analyzed parting kit, it is characterized in that: also include reaction mixture 10.0 μ L, warm start Taq enzyme 0.5 μ L, sdH
2o complements to 25.0 μ L; In described reaction mixture, include: MgCl
27.5mM, Tris-HCl buffer 125mM, KCl 125mM, dNTPs 7.5mM, BSA 2mg/ml.
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| CN105177133A (en) * | 2015-09-07 | 2015-12-23 | 南开大学 | Nucleotide specific to vibrio cholerae O6, O4, O7 and O15 and application of nucleotide |
| CN110578017A (en) * | 2019-07-30 | 2019-12-17 | 深圳市百迈生命科学有限公司 | Kit for synchronously detecting twenty-three respiratory pathogens and detection method thereof |
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2014
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| Title |
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| OLSEN ET AL.: "Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177133A (en) * | 2015-09-07 | 2015-12-23 | 南开大学 | Nucleotide specific to vibrio cholerae O6, O4, O7 and O15 and application of nucleotide |
| CN105177133B (en) * | 2015-09-07 | 2018-08-17 | 南开大学 | The nucleotide special to comma bacillus O6, O4, O7 and O15 and its application |
| CN110578017A (en) * | 2019-07-30 | 2019-12-17 | 深圳市百迈生命科学有限公司 | Kit for synchronously detecting twenty-three respiratory pathogens and detection method thereof |
| CN110578017B (en) * | 2019-07-30 | 2023-07-25 | 深圳市百迈生命科学有限公司 | Kit for synchronously detecting twenty-three respiratory pathogens and detection method thereof |
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