CN103131661A - Plasmid clone bacterial strain of vibrio cholerae efb-O1 gene, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a plasmid clone bacterial strain of a vibrio cholerae rfb-O1 gene, and a preparation method of the plasmid clone bacterial strain of the vibrio cholerae rfb-O1 gene, and application of the plasmid clone bacterial strain of the vibrio cholerae rfb-O1 gene in the aspect of a non-disease diagnosis purpose. According to the plasmid clone bacterial strain of the vibrio cholerae rfb-O1 gene, an escherichia coli pCC1FOS serves as a carrier, the vibrio cholerae rfb-O1 gene is converted, and a preservation number of the plasmid clone bacterial strain is CGMCC NO.6998. The plasmid clone bacterial strain of the vibrio cholerae rfb-O1 gene is mainly used for being a positive check sample of detection of corresponding genes of vibrio cholerae, and carrying out quality control, also can be used for checking technological level of inspectors to guarantee quality of inspection work, can be used for carrying out inspection and correction on various analytical methods and various identification devices so as to enable the methods and devices to be accurate, uniform, comparable and normative, and has a wide application prospect.

Description

Plasmid clone bacterial strain and the preparation method and application of vibrio cholerae rfb-O1 gene

Technical field

The invention belongs to gene engineering technology field, be specifically related to plasmid clone bacterial strain, its preparation method and the application of the extraction of vibrio cholerae complete genome DNA and rfb-O1 gene.

Background technology

Cholera is the severe intestinal transmissible disease that is caused by vibrio cholerae (Vibrio cholerae), the vibrio cholerae that can cause Cholera Epidemic mainly contains two kinds of serotypes, both O1 group and O139 group, the strong pathogenic effects of vibrio cholerae is mainly that the cholera enterotoxin by its secretion causes due to severe diarrhea, therefore detect accurately and rapidly vibrio cholerae, particularly produce malicious vibrio cholerae, infect significant for the prevention vibrio cholerae.Along with the development of molecular biology research, people deepen continuously to the virulence genes such as the invasion of bacterium and pathogenicity island understanding, and the detection method of bacterium has identified that from traditional general phenotypic characteristic in-depth is the evaluation of hereditary feature gene.Vibrio cholerae hlyA genes encoding has haemolysis-cytolytic albumen, is the house-keeping gene of Vibrio high conservative; Vibrio cholerae rfb-O1 gene and rfb-O139 gene are respectively O1 group cholera vibrio and O139 group cholera vibrio O antigen encoding gene, and the ctxAB gene is the virulence sign of secretion cholera enterotoxin, has high conservative.

Due to relevant virulence gene, house-keeping gene and the functional gene of vibrio cholerae being positioned on karyomit(e) of cluster always, generally all at several kb between tens kb, be difficult to increase and clone these genes with general PCR method.Genomic library (genomic library) is the new technology of a kind of isolated genes group gene of growing up the end of the seventies in last century, it has comprised genomic a complete set of genetic information (being full gene), and people can angle the arbitrary specific gene fragment of taking-up to be studied from the library with corresponding gene probe.The application of metagenomics has at present become the existing gene of research and has sought a new way of new gene and product thereof, carrier comprises Cosmid, Fosmid and BAC etc., average Insert Fragment is greater than 40Kb, the Fosmid carrier is the novel vector in a kind of alternative Cosmid vector construction large fragment library, compare with the BAC library construction, Fosmid library stability, randomness are good, technology maturation, build more Simple fast.The Fosmid carrier has been owing to having inserted escherichia coli fertility factor F-factor, thus it exist with single copy form in Host Strains, good stability.And the high copy of a derivable oriV replication origin is arranged on carrier, can induce when needing to reach high copy (50 left and right).In addition, Fosmid library randomness is good, has guaranteed that the frequency that every segment DNA occurs in the library is impartial.

cause in order to prevent " Biosafety accident " " public health event ", all consistent the requirement detected the vibrio cholerae suspicious specimen in PIII or PII biocontainment laboratory both at home and abroad, purpose by building vibrio cholerae Fosmid library is exactly to be cloned into the chromosomal full gene of vibrio cholerae in the intestinal bacteria that are easy to cultivate, filter out vibrio cholerae hlyA gene, the rfb-O1 gene, rfb-O139 gene and ctxAB gene monoclonal bacterial strain, the Host Strains of vibrio cholerae clone gene is intestinal bacteria, do not need to cultivate vibrio cholera strain alive, so can operate at the PI biocontainment laboratory, be conducive to biological safety protection, simultaneously be also pathogenesis, the mechanism of hiding, serology Variation Mechanism, the preparation of gene vaccine and the prerequisite of gene structure and function equimolecular biological characteristics of research vibrio cholerae, and be conducive to carry out vibrio cholerae gene function research and satisfy local laboratories reply Cholera and the needs of plague area epidemic situation rapid detection vibrio cholerae when detecting.

Summary of the invention

The object of the present invention is to provide the mono-clonal bacterial strain of vibrio cholerae (Vibrio cholerae) process for extracting complete genome DNA and vibrio cholerae (Vibrio cholerae) rfb-O1 gene, be mainly used in the positive control sample that the vibrio cholerae corresponding gene detects, carry out quality control, also can be used to examine reviewer's state of the art, to guarantee the check work quality; And can test and calibrate different analytical procedures and different identification equipments, make it to have accuracy, unity, comparability and standardization.

An aspect of of the present present invention is to provide the plasmid clone bacterial strain of a kind of vibrio cholerae (Vibrio cholerae) rfb-O1 gene, it is take intestinal bacteria pCC1FOS as carrier, transformed the plasmid clone bacterial strain of the rfb-O1 gene of vibrio cholerae, its deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC NO.6998.

Another aspect of the present invention is to provide a kind of vibrio cholerae rfb-O1 gene detecting kit, it comprise deposit number mentioned above be the plasmid clone bacterial strain of CGMCC NO.6998 as positive control, and upstream and downstream primer and probe: SEQ ID NO.1; SEQ ID NO.2; SEQ ID NO.3.

In addition, can also comprise the reagent such as 2 * PCR Taqman GEx enzyme premixed liquid for pcr amplification in test kit, those skilled in the art can according to the applied PCR reaction type of test kit, determine the reagent type such as required damping fluid or amplification enzyme.

The multi-PCR detection method that is on the one hand again to provide a kind of vibrio cholerae rfb-O1 gene of non-medical diagnosis on disease purpose of the present invention, it utilizes the vibrio cholerae rfb-O1 gene detecting kit of non-medical diagnosis on disease purpose mentioned above, sample DNA is carried out multiple PCR method detect.

In technique scheme, in preferred situation, the condition that described multiple PCR method detects is:

1. the reaction system cumulative volume is 20 μ L, wherein:

2. each component mixing more than, 5000r/min, after centrifugal 10s, carry out multiple PCR method and detect:

95 ℃/10min, 1 circulation; 95 ℃/15s, 60 ℃/1min, 40 circulations.

In technique scheme, described deposit number is that the construction process of the vibrio cholerae plasmid clone bacterial strain of CGMCC NO.6997, CGMCC NO.6998, CGMCC NO.6999, CGMCC NO.7000 is: adopt the low melting point agarose embedding method to extract the vibrio cholerae total genomic dna, build vibrio cholerae Fosmid library, extract glycerol stock DNA and PCR method screening goal gene, specifically comprise the steps:

Utilize the low melting point agarose embedding method, extract respectively two kinds of serotype vibrio cholerae of O1 and O139 (N16961 and MO45) complete genome group, with the random shearing method, genome being carried out fragmentation processes, the DNA of pulse electrophoresis isolated fragment also cuts glue and reclaims DNA fragmentation between 38 ~ 48Kb, then after the DNA fragmentation that reclaims being carried out end smoothing and phosphatizing treatment, be connected with the pCC1FOS plasmid vector again, build vibrio cholerae Fosmid library after packing, transfection.The glycerol stock that takes a morsel from the library of building up carries out the mono-clonal pure culture and extracts DNA, and filter out virulence gene ctxAB, house-keeping gene hlyA and O1 and the O139 O antigen gene rfb of vibrio cholerae with PCR method, finishing screen is selected the mono-clonal bacterial strain of vibrio cholerae hlyA gene, rfb-O1 gene, rfb-O139 gene and ctxAB gene with good stability, and Host Strains is intestinal bacteria p CC1F O S carrier.

In the present invention, the primer that uses and probe are respectively that application number is to have very detection primer and the probe (SEQ ID NO.1,2 and 3) of the rfb gene of the O1 group cholera vibrio of high specific (SEQ ID NO.13), the detection primer of the rfb gene (SEQ ID NO.15) of the detection primer of vibrio cholerae hlyA gene (SEQ ID NO.14) and probe (SEQ ID NO.4,5 and 6) and O139 group cholera vibrio and probe (SEQ ID NO.7,8 and 9) and detection method in 201110281129.0; Based on this, the present invention further discloses primer and the probe sequence (SEQ ID NO.10,11 and 12) of vibrio cholerae virulence gene ctxAB (SEQ ID NO.16).

Description of drawings

The total genomic dna pulse electrophoresis figure that the different OD value of Fig. 1 bacteria suspension extracts, wherein M2:Low Range PFGE Marker; 1-3:N16961OD4,6,81/2 blob of viscoses; 4-6:MO45OD4,6,81/2 blob of viscoses.

Fig. 2 Not I restriction analysis Fosmid library Insert Fragment size, DNAMWM XV Marker(200ng);

The common electrophoretic method of Fig. 3 detects positive plasmid clone strain stability

Wherein, M1 is λ-Hind III Marker(100ng); (1.SYFW296-29 0 generation); (2.SYFW296-29 50 generation); (3.SYFW296-29 100 generation); (4.SYFW296-49 0 generation); (5.SYFW296-49 50 generation); (6.SYFW296-49 100 generation); (7.SYFW296-165-1 0 generation); (8.SYFW296-165-1 50 generation); (9.SYFW296-165-1 100 generation); (10.SYFW296-192-1 0 generation); (11.SYFW296-192-1 50 generation); (12.SYFW296-192-1 100 generation); (13.SYFW296-195 0 generation); (14.SYFW296-195 50 generation); (15.SYFW296-195 100 generation);

Four kinds of goal gene positive plasmid clone strains that use screening to obtain, they are hlyA gene masculine mono-clonal bacterium: SYFW296-29 and SYFW296-49, rfb-O1 gene masculine mono-clonal bacterium: SYFW296-165, rfb-O139 gene masculine mono-clonal bacterium: SYFW296-192 and ctxAB gene masculine mono-clonal bacterium: SYFW296-195 is totally five strain bacterium..The positive plasmid clone strain is inoculated in respectively in the 2YT liquid nutrient medium that 3mL contains paraxin, incubated overnight under 37 ℃ of 200r/min, draw again 5 μ l and be inoculated in the 2YT liquid nutrient medium that 3mL contains paraxin from the bacterium liquid of incubated overnight, incubated overnight under 37 ℃ of 200r/min.Repeat above step and be cultured to the 5th day.Extract the mono-clonal bacterial strain DNA in the 1st day (0 generation), the 3rd day (50 generation), the 5th day (100 generation), carry out common electrophoresis and confirm.

Wherein, swimming lane M1 is λ-Hind III Marker, and swimming lane 1 ~ 6 represents that respectively hlyA plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations; Swimming lane 7 ~ 9 represents that respectively rfb-O1 plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations; Swimming lane 10 ~ 12 represents that respectively rfb-O139 plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations; Swimming lane 13 ~ 15 represents that respectively ctxAB plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations.

Fig. 4 pulse electrophoresis method detects positive plasmid clone strain stability

Wherein, M2 is DNAMWM XVMarker(200ng), 1.SYFW296-29 (0 generation); (2.SYFW296-29 50 generation); (3.SYFW296-29 100 generation); (4.SYFW296-49 0 generation); (5.SYFW296-49 50 generation); (6.SYFW296-49 100 generation); (7.SYFW296-165-1 0 generation); (8.SYFW296-165-1 50 generation); (9.SYFW296-165-1 100 generation); (10.SYFW296-192-1 0 generation); (11.SYFW296-192-1 50 generation); (12.SYFW296-192-1 100 generation); (13.SYFW296-195 0 generation); (14.SYFW296-195 50 generation); (15.SYFW296-195 100 generation);

Four kinds of goal gene positive plasmid clone strains that use screening to obtain, they are hlyA gene masculine mono-clonal bacterium: SYFW296-29 and SYFW296-49, rfb-O1 gene masculine mono-clonal bacterium: SYFW296-165, rfb-O139 gene masculine mono-clonal bacterium: SYFW296-192 and ctxAB gene masculine mono-clonal bacterium: SYFW296-195 is totally five strain bacterium.The positive plasmid clone strain is inoculated in respectively in the 2YT liquid nutrient medium that 3mL contains paraxin, incubated overnight under 37 ℃ of 200r/min, draw again 5 μ l and be inoculated in the 2YT liquid nutrient medium that 3mL contains paraxin from the bacterium liquid of incubated overnight, incubated overnight under 37 ℃ of 200r/min.Repeat above step and be cultured to the 5th day.Extract the mono-clonal bacterial strain DNA in the 1st day (0 generation), the 3rd day (50 generation), the 5th day (100 generation), the DNA of extraction runs pulsed field gel electrophoresis after Not I enzyme is cut.

Wherein, swimming lane M2 is DNAMWM XV Marker(200ng), swimming lane 1 ~ 6 represents that respectively hlyA plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations; Swimming lane 7 ~ 9 represents that respectively rfb-O1 plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations; Swimming lane 10 ~ 12 represents that respectively rfb-O139 plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations; Swimming lane 13 ~ 15 represents that respectively ctxAB plasmid positive colony cultivated for 0 generation, cultivated for 50 generations and cultivate the mono-clonal bacterial strain DNA in 100 generations.

Embodiment

The below is specific embodiments of the invention, and foundation and the application thereof of technical solution of the present invention is further described, and following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.The chemical reagent that uses in the embodiment of the present invention or solvent solution if no special instructions, all can or be obtained by commercial sources by the ordinary method preparation.

If without specified otherwise, the present invention's biological sample used is from Dandong Microbiological Lab of Entry-Exit Inspection and Quarantine Bureau.

Cell suspension (CSB): 10ml1M Tris-HCl(pH8.0) and 20ml0.5M EDTA(pH8.0), with the sterilization ultrapure water be diluted to 100mL.

Library construction uses the copycontrol fosmid library production kit of Epicentre company, and carrier is pCC1FOS, and Host Strains is intestinal bacteria;

Low melting-point agarose gel (SeaPlague GTG Agarose) is available from U.S. Lonza company;

Xho I, Barn H I, λ-Hind III, Proteinase K, DNA MWM XV Marker, DNA purification kit and flat end phosphorate test kit all available from the precious biotech firm in Dalian;

The sarcosine sodium salt likes that available from ladder is uncommon (Shanghai) changes into industrial development company limited;

The PMSF(phenylmethylsulfonyl fluoride) available from U.S. sigma company;

TSA (soybean casein agar substratum) is available from U.S. Oxoid company;

PFGE glue and disposable glue mould are all available from U.S. Bio-Rad(Bole) company;

2YT (Tryptones yeast extract meat soup) is available from U.S. Oxoid company;

Extraction of plasmid DNA uses AxyPrep Easy-96 plasmid DNA test kit available from U.S. Axygen Bioscience company;

Primer and probe are synthetic by Applied biosystems;

2 * PCR Taqman GEx enzyme premixed liquid is available from Applied biosystems;

Key instrument has pulsed field gel electrophoresis instrument and imager, ABI7300 real-time fluorescence PCR instrument and the ABI PRISMTM3730XL DNA Analyzer of BioMerieux Vitek Colorimeter bacteria concentration determinator, Bio-Rad company.

Embodiment 1

The extraction of vibrio cholerae complete genome DNA:

The DNA extraction method has a lot, but only have the low melting point agarose embedding of employing method minimum to the DNA physical abuse, segment is the most complete and can once extract in a large number, the DNA that extracts can prolonged preservation can not be degraded, and builds the Fosmid genomic library so the present invention adopts the DNA of low melting point agarose embedding method extraction to be suitable for most purchasing.Concrete operation step is as follows:

Scraping is cultivated vibrio cholerae (Vibrio cholerae) MO45 and the N16961 thalline (be so kind as to give national disease prevention and control center) of 18h～24h from the TSA substratum, evenly outstanding turbid in 2ml cell suspension (CSB) respectively, measure bacteria concentration with BioMerieux Vitek Colorimeter and be respectively 4,6,8 three OD value gradients, optimize optimum cell suspension concentration.get 400 μ l bacterial suspensions in corresponding 1.5ml Eppendorf pipe, and add 2% the low melting-point agarose of the 400 μ l that are placed in 45 ℃ of water-baths, add immediately in the disposable tool that can be made into blob of viscose after mixing, be placed on 4 ℃ and solidify 30min, take out blob of viscose, be placed in the ESP damping fluid (0.4mol/L EDTA contains the Proteinase K that final concentration is 2%N-methylglycine sodium salt and 1mg/ml) of 10 times of volumes, cracking 48h left and right in 50 ℃ of water-baths, an ESP damping fluid is changed in the 24h left and right therebetween, observe blob of viscose after cracking, can first cut the fritter gel when being transparence and run common electrophoresis observation cracking situation, continue to be placed on cracking in 50 ℃ if electrophoresis result is undesirable, after finishing, cracking bathes 2h with isopyknic 1 * TE room temperature temperature that contains 0.1mmol/L PMSF, use again twice of 1 * TE washing embedded block, embedded block is stored in 0.5mol/L EDTA(pH8.0) in, 4 ℃ of preservations, at this moment, the total genomic dna that comprises purifying in gel piece.

The thalline suspension liquid of three OD value gradients runs pulsed field gel electrophoresis after entrapping method extracts genomic dna, applied sample amount is 1/2 blob of viscose, upper Low Range PFG Marker, its electrophorogram such as Fig. 1.As shown in Figure 1, for the vibrio cholerae O of two kinds of serotypes ₁And O ₁₃₉, the OD value is 8 thalline suspension liquid, and the Genome DNA content that extracts after cracking 48h is maximum, and the DNA that therefore selects this OD value to extract builds the Fosmid library.

Embodiment 2

The structure in Fosmid library (identifying and end sequencing)

With shearing method immediately, genomic dna is carried out fragmentation and process, run the DNA of isolated fragment after pulse electrophoresis, the pulse electrophoresis condition is 14 ℃, 6V/cm, pulse 1～10s, 16h.After finishing, cuts electrophoresis the DNA fragmentation between glue recovery 38 ~ 48kb, the DNA fragmentation that reclaims is confirmed by common electrophoresis and pulse electrophoresis, utilize the reagent in copycontrol Fosmid library production kit to fill sticky end, carry out phosphatizing treatment with the flat end test kit that phosphorates again, DNA fragmentation after processing is connected on the pCC1FOS carrier, 4 ℃ are spent the night, the method that provides according to test kit with connecting fluid pack, transfection, spread plate.

Choose at random 16 single bacterium colonies of white from the flat board that is obtained by aforesaid method, cultivate and extract FosmidDNA, carry out enzyme with Not I and cut, because Not I can identify 8 bases, approximately have restriction enzyme site in the 40Kb left and right by probability, but also might in Insert Fragment, a more than restriction enzyme site be arranged.Can find out from Fig. 2 (Not I restriction analysis Fosmid library Insert Fragment size), the enzyme of 16 positive bacterium colonies of white is cut product an onesize band, this band namely represents the pCC1FOS carrier, Insert Fragment has between the fragment 35Kb of a restriction enzyme site ~ 40Kb, there is the fragment of a plurality of restriction enzyme sites to add with also more than 35Kb, the suitableeest intubating length of Fosmid all meets the requirements so enzyme is cut result in the 35Kb left and right.8 Fosmid DNA carry out two end sequencings to above 16 clone's random chooses, and sequencing result confirms to be the vibrio cholerae correlated series after BLAST, illustrate that the library preparation is correct.

The positive white colony of picking is kept on 96 orifice plates respectively, makes with one, every hole mono-clonal, and 37 ℃ of incubated overnight suspend the plate after cultivating and move in new 96 orifice plates with the LB substratum that contains 30% glycerine, stores the glycerol stock that obtains under-70 ℃.

Embodiment 3

Screening positive clone bacterial strain from the glycerol stock that embodiment 2 obtains, working method is as follows:

Getting 2～10 μ L glycerol stocks, to add the final concentration that contains paraxin be in the 100 μ L LB of 12.5ng/ μ L, after 37 ℃ of 1800rpm shaken overnight are cultivated, adding the final concentration that 500 μ L2YT(contain paraxin in the nutrient solution is 12.5ng/ μ L) inductor (1000 * Induction), 37 ℃ of 1800rpm shaking culture 5h of 1/1000 amount.Use test kit AxyPrep Easy-96 plasmid DNA test kit to carry out DNA extraction, the plasmid DNA of extracting is carried out the amplification of real-time fluorescence multiplex PCR, filter out the mono-clonal bacterial strain of hlyA gene, rfb-O1 gene, rfb-O139 gene and ctxAB gene, concrete PCR method operation steps is as follows:

1, the PCR of each gene detects and uses following primer and probe

Rfb-O1: forward primer: 5 '-GCCCGTTTTGCATTATGGAT-3 ' (SEQ ID NO.1)

Reverse primer: 5 '-CCTTTTACCGCCGCAATACGA-3 ' (SEQ ID NO.2)

Probe: 5 ' FAM-TGATCCGACAAGCCCAAATGCCACTA (Eclipse)-3 ' (SEQ ID NO.3)

HlyA: forward primer: 5 '-GAGAAATTTGAGCGCAAAGAGGTTT-3 '; (SEQ ID NO.4)

Reverse primer: 5 '-ACTTCCACCCCACCAGTCA-3 ' (SEQ ID NO.5)

Probe: 5 ' NED-CCAAGCTCAAAACCTG (MGB)-3 ' (SEQ ID NO.6)

Rfb-O139: forward primer: 5 '-AGTTACCTGTTATGTACGATGAACC-3 ' (SEQ ID NO.7)

Reverse primer: 5 '-CCATCACCAGACAAGCATACAG-3 ' (SEQ ID NO.8)

Probe: 5 ' VIC-TGCTGACGCCTCTCAAGTGCCTACG (Eclipse)-3 ' (SEQ ID NO.9)

CtxAB: forward primer: 5 '-CTCATCCAGATGAACAAGAAGTTTCTG-3 ' (SEQ ID NO.10)

Reverse primer: 5 '-CTATCTCTGTAGCCCCTATTACGATGT-3 ' (SEQ ID NO.11)

Probe: 5 ' FAM-AAGCCCCCAAAATGA (MGB)-3 ' (SEQ ID NO.12)

2, multi-PRC reaction system

Cumulative volume 20ul, comprise the template DNA solution 2 μ L that 3. step makes, 2 * PCR Taqman GEx enzyme premixed liquid, 10 μ L, rfb-O1 gene and ctxAB gene upstream and downstream primer each 0.8 μ L, probe 0.4 μ L, hlyA gene and rfb-O139 gene upstream and downstream primer each 0.4 μ L, probe 0.2 μ L, according to the experiment needs select different primers to the probe experiment of increasing, replenishing ultrapure water to the cumulative volume of sterilizing is 20 μ L; Wherein the concentration of primer and probe is 10 μ M.

Above each component is added in 0.2mL PCR reaction tubes, mixing, 5000r/min, centrifugal 10s;

Above-mentioned reaction system is carried out PCR by following condition and is detected:

95 ℃/10min, 1 circulation; 95 ℃/15s, 60 ℃/1min, 40 circulations;

Collect fluorescence during each annealing that circulates, after detecting end, according to amplification curve and Ct value result of determination.

Ct value 〉=40 can judge that this genescreen result is negative; Ct value＜40 can judge that this genescreen result is positive.

Embodiment 4

One. the positive monoclonal strain stability detects

Use four kinds of goal gene positive plasmid clone strains that in embodiment 3, screening obtains, they are hlyA gene masculine mono-clonal bacterium: SYFW296-29 and SYFW296-49, rfb-O1 gene masculine mono-clonal bacterium: SYFW296-165, rfb-O139 gene masculine mono-clonal bacterium: SYFW296-192 and ctxAB gene masculine mono-clonal bacterium: SYFW296-195 is totally five strain bacterium, be inoculated in respectively in the 2YT liquid nutrient medium that 3mL contains paraxin incubated overnight under 37 ℃ of 200r/min.Draw respectively 5 μ l and be inoculated in the 2YT liquid nutrient medium that 3mL contains paraxin from the bacterium liquid of incubated overnight, incubated overnight under 37 ℃ of 200r/min repeats above step and is cultured to the 5th day.Extract the mono-clonal bacterial strain DNA in the 1st day (0 generation), the 3rd day (50 generation), the 5th day (100 generation), carry out common electrophoresis and confirm, carry out the agarose pulse electrophoresis after Not I enzyme is cut, detect clone's stability.

The common electrophoresis result of positive monoclonal bacterial strain DNA of extracting is seen Fig. 3 (the common electrophoresis of Fosmid DNA of extraction); Result shown in figure is the collection of illustrative plates no significant difference as can be known, does not find loss or the rearrangement of Insert Fragment, illustrates that constructed positive plasmid clone strain is stable.

Positive monoclonal bacterial strain DNA cuts through Not I enzyme, and enzyme is cut the product pulsed field gel electrophoresis and be the results are shown in Figure the Fosmid DNA Not I enzyme that 4(extracts and cut pulsed field gel electrophoresis).Result shown in figure is the collection of illustrative plates no significant difference as can be known, does not find loss or the rearrangement of Insert Fragment, illustrates that constructed positive plasmid clone strain is stable.

Two. culture presevation

With the screening the positive plasmid clone strain on December 17th, 2012, be preserved in respectively China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101, specifying information is as follows:

Intestinal bacteria pCC1FOS carrier, transformed the plasmid clone bacterial strain SYFW296-192 of the rfb-O139 gene of vibrio cholerae, namely transformed the intestinal bacteria of vibrio cholerae rfb-O139 gene clone plasmid, called after VC-rfb-O139, preserving number is: CGMCC NO.6997;

Intestinal bacteria pCC1FOS carrier has transformed the plasmid clone bacterial strain SYFW296-165 of the rfb-O1 gene of vibrio cholerae, has namely transformed the intestinal bacteria of vibrio cholerae rfb-01 gene clone plasmid, called after VC-rfb-O1, and preserving number is: CGMCCNO.6998;

Intestinal bacteria pCC1FOS carrier has transformed the plasmid clone bacterial strain SYFW296-29 of vibrio cholerae hlyA gene, has namely transformed the intestinal bacteria of vibrio cholerae hlyA gene clone plasmid, called after VC-hlyA, and preserving number is: CGMCC NO.6999;

Intestinal bacteria pCC1FOS carrier has transformed the plasmid clone bacterial strain SYFW296-195 of the ctxAB gene of vibrio cholerae, has namely transformed the intestinal bacteria of ctxAB-containing Vibrio cholerae cloned plasmids, called after VC-ctxAB.Preserving number is: CGMCC NO.7000.

Embodiment 5

One. the experiment grouping

The rfb-O139(CGMCC NO.6997 of vibrio cholerae that utilized respectively conversion that the described method of embodiment 4 prepares), rfb-O1(CGMCC NO.6998), hlyA(CGMCC NO.6999), ctxAB(CGMCC NO.7000) the plasmid clone bacterial strain of gene, do following experiment.

Detected sample is distinguished mark:

The known positive control sample that contains vibrio cholerae N16961 and MO45: be labeled as PC;

Sample water: be labeled as NC;

The plasmid clone bacterial strain freeze-dried vaccine CGMCC NO.6997 of the rfb-O139 gene of vibrio cholerae: be labeled as A;

The plasmid clone bacterial strain freeze-dried vaccine CGMCC NO.6998 of the rfb-O1 gene of vibrio cholerae: be labeled as B;

The plasmid clone bacterial strain freeze-dried vaccine CGMCC NO.6999 of the hlyA gene of vibrio cholerae: be labeled as C;

The plasmid clone bacterial strain freeze-dried vaccine CGMCC NO.7000 of the ctxAB gene of vibrio cholerae: be labeled as D;

The detected sample of mark respectively as blind sample, is given the inspector and detected, and detection method and result are as follows:

The inspector 1: utilize primer and probe SEQ ID NO.1,2 and 3, do following experiment.

The inspector 2: utilize primer and probe SEQ IDNO.4,5 and 6, do following experiment.

The inspector 3: utilize primer and probe SEQ ID NO.7,8 and 9, do following experiment.

The inspector 4: utilize primer and probe SEQ ID NO.10,11 and 12, do following experiment.

Two. the method for inspection

To above-mentioned sample to be checked, extract respectively DNA, method is as follows:

1. take sample that 300mg prepared in the 2mL centrifuge tube, adding 1.5mL CTAB damping fluid and 10 μ L concentration is 20mg/ μ L Proteinase K, 65 ℃ of 30min vibration mixings; The centrifugal 5min of 12000r/min draws in the new centrifuge tube of supernatant liquor to, and the volume ratio that adds 400 μ L is the trichloromethane of 24:1: primary isoamyl alcohol, fully mixing;

2. the centrifugal 5min of 12000r/min, draw in the new centrifuge tube of supernatant liquor to, adds 0.8 times of volume Virahol, precipitates 1～2h under room temperature;

3. the centrifugal 10min of 12000r/min, abandon supernatant liquor; 70% washing with alcohol is once dried; Add 50 μ L TE, the dissolving DNA precipitation;

4. the mensuration of DNA concentration and purity: get the DNA solution thin up that 3. step obtains, to concentration be 10 μ g/mL ~ 100 μ g/mL, A260/A280 ratio is between 1.7 ~ 1.9, and is standby.

Three. assay

Utilize the PCR in real time screening method described in embodiment 3, respectively the sample that extracts DNA is detected, each sample repeats for 4 times, and its result is as follows:

?

PC

NC

A

B

C

D

The inspector 1

+

-

+

-

-

-

The inspector 2

+

-

-

+

-

-

The inspector 3

+

-

-

-

+

-

The inspector 4

+

-

-

-

-

+

Wherein ,+positive result;-negative result.

The NC group: the demonstration of negative control detected result, FAM passage, NED passage and VIC passage all detect without fluorescent signal, illustrate that reaction result is normal, and reaction system is pollution-free;

The PC group: the demonstration of positive control detected result, FAM passage, NED passage and VIC passage all have fluorescent signal to detect, and reaction result is normal;

Sample ABCD detected result all conforms to expection.

In addition, in real work, adopt aforesaid method to detect actual sample, take traditional detection method as contrast (SN/T1022-2010).Actual test sample comprises 260 parts of food samples, 310 parts of water body samples, such as totally 570 parts of Jiang Yaobei, sea snail meat, chilled beef and water samples etc., by a large amount of actual detected results as seen, use the inventive method detection different serotypes vibrio cholerae and have application result preferably.

Claims (4)

1. the plasmid clone bacterial strain of a vibrio cholerae rfb-O1 gene, its deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC NO.6998.

2. a vibrio cholerae rfb-O1 gene detecting kit, is characterized in that: comprise that deposit number claimed in claim 1 is the plasmid clone bacterial strain of CGMCC NO.6998, reach upstream and downstream primer and probe: SEQ ID NO.1; SEQ ID NO.2; SEQ ID NO.3.

3. the multi-PCR detection method of the vibrio cholerae rfb-O1 gene of a non-medical diagnosis on disease purpose, is characterized in that: utilize the described vibrio cholerae rfb-O1 of claim 2 gene detecting kit, sample DNA is carried out multiple PCR method detect.

4. the multi-PCR detection method of the vibrio cholerae rfb-O1 gene of non-medical diagnosis on disease purpose according to claim 3 is characterized in that: the condition that described multiple PCR method detects is:

1. the reaction system cumulative volume is 20 μ L, wherein:

2. each component mixing more than, 5000r/min, after centrifugal 10s, carry out multiple PCR method and detect:

95 ℃/10min, 1 circulation; 95 ℃/15s, 60 ℃/1min, 40 circulations.

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