CN103266179B - Multiplex PCR (Polymerase Chain Reaction) detection method for salmonella typhimurium and serum variants of salmonella typhimurium - Google Patents
Multiplex PCR (Polymerase Chain Reaction) detection method for salmonella typhimurium and serum variants of salmonella typhimurium Download PDFInfo
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Abstract
The invention discloses a multiplex PCR (Polymerase Chain Reaction) detection method for salmonella typhimurium and serum variants of the salmonella typhimurium and belongs to the technical field of food safety inspection. The detection method comprises the following steps of: (1) designing amplification primers, with specific gene sequences of the salmonella typhimurium as templates; (2) extracting the DNA (Deoxyribonucleic Acid) of a sample, and carrying out amplification by using a PCR method; and (3) detecting amplification products through gel electrophoresis, and judging electrophoresis results. The invention further relates to four pairs of specific identification primers, wherein the sequences of the primers are respectively shown in SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO: 12. When the detection method disclosed by the invention is used to detect the salmonella typhimurium and the serum variants of the salmonella typhimurium, the detection time is short, the cost is low, the practicability is better, the detection results are specific, and the result judgment is simple.
Description
Technical field
The present invention relates to a kind of multi-PCR detection method and primer of Food Safety Analysis technical field, specifically a kind of multi-PCR detection method that detects Salmonella typhimurium and serovar thereof.
Background technology
Salmonella typhimurium (Salmonella Typhimurium) is one of modal Salmonella infection type of China, approximately has 30% Salmonella infection relevant with it.This bacterium can be present in the enteron aisle of the animals such as poultry, domestic animal, muroid, wild birds and human body, and can propagate to each other.Infect the multiple infant that is born in, often cause heating, feel sick, the illness such as vomiting and diarrhoea.
Hegde, N.V., Cook, M.L., Wolfgang, D.R. wait at < < Journal of Clinical Microbiology > > (clinical microbiology periodical) the 43rd volume the 8th phase 4208-4211 page in 2005, deliver be entitled as < < Dissemination of Salmonella enterica subsp.enterica serovar Typhimurium var.Copenhagen clonal types through a contract heifer-raising operation > > (salmonella enteritis subspecies Salmonella typhimurium serovar Copenhagen clonotype is propagated by an a heifer feedlot) literary composition, in literary composition, mention following content " Salmonella typhimurium has multiple serovar, wherein Copenhagen mutation (S.Typhimurium var.Copenhagen) is the mutation of O5 antigen negative, mainly cause dove group's paratyphoid illness, also have cases of infection in other animal and human's body ", in China, also there is the report that causes food poisoning because infecting Copenhagen mutation.4, [5], 12:i:-is the serotype of finding for 20 end of the centurys, very alike with the pathogenesis of Salmonella typhimurium, is likely the single-phase mutation of Salmonella typhimurium.According to < < Europe monitoring > >, compared with 2005, the infection recall rate of this serotype significantly increases, in Britain, increased by 104 examples, in France, increased by 301 examples, in Italy, increased by 582 examples, increasing degree is 221% to 986%.4,12:-:1,2 see the report of Germany in 2009, may be the single-phase mutation of H antigen of Copenhagen mutation, and this serotype is very likely to propagate to cat from wild flock of birds, infects afterwards the mankind again.
Detection to Salmonella typhimurium and mutation thereof, is conducive to review and infects source, probes into route of transmission and preventing disease infection etc.Yet, China is mainly to rely on traditional cultural method (GB/T4789.4-2010) to the detection of Salmonellas, after biochemical reaction is determined subspecies, the O antigen, H antigen and the Vi antigen-reactive that recycle special serum and its thalline surface are known the serotype of bacterial strain.Herrera-Leon, S., Ramiro, R., Arroyo, M. wait " Research in Microbiology " (microbiological research) the 158th volume the 2nd phase 122-127 page in 2007 deliver be entitled as " Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes " (the Blind Test comparation and assessment to Salmonella serogroup qualification of traditional serotype and triple PCR) literary composition in mention " the classifying method that foundation is current, determining of one strain Salmonella serogroup needs 3 days or longer time ", be unfavorable for diagnosing in time the cause of disease, search pathogeny, control spreading of epidemic situation.
For can be quick, identify accurately Salmonella typhimurium, the people such as Lim are according to the serotype O4:Hi:1 of Salmonella typhimurium, 2 corresponding antigen encoding gene rfbJ, fliC and fljB, form multiplex PCR system and detect Salmonella typhimurium, as Lim, Y.H., Hirose, K., Izumiya, H. wait being entitled as described in < < Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar Typhimurium > > (specific detection of multiple PCR method to a Salmonella typhimurium) literary composition of delivering at < < Japanese Journal of Infectious Diseases > > (Japanese transmissible disease magazine) the 56th volume the 4th phase 151-155 page in 2003.The method can only be for detection of Salmonella typhimurium, and for the mutation of Salmonella typhimurium, not only can not distinguish, also likely by 4, [5], the mutation such as 12:i:-think it is non-Salmonella typhimurium.Therefore,, in order further to distinguish Salmonella typhimurium mutation and non-Salmonella typhimurium, except these antigen genes, the method also needs to add other target gene.Yet target gene too much can aggravate the interference between multi-primers, and bring difficulty for the judgement of net result.Therefore, the specific target gene of Salmonella typhimurium and O antigen and H antigen encoding gene thereof the new multiplex PCR detection system that has been Foundation is take in this research, has reduced the quantity of target spot, has simplified the decision process of result.
Through the literature search of prior art is found, not yet find the report relevant with multiple PCR method detection Salmonella typhimurium of the present invention and serovar thereof.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the multi-PCR detection method of a kind of Salmonella typhimurium and serovar thereof is provided.Adopt method of the present invention to detect Salmonella typhimurium and serovar thereof, detection time is short, and cost is low, more has practicality, and detected result is special, and result is judged simple.
The present invention realizes by following technical scheme.
Salmonella typhimurium and a serovar multi-PCR detection method thereof, comprise the steps:
Step 1, design of amplification primers;
Step 2, extracts sample DNA, the amplification of PCR method;
Step 3, detected through gel electrophoresis amplified production, and electrophoresis result is judged;
In described step 1, the template of design of amplification primers foundation is: the conserved sequence in the sequence of the 1800-2930 section of the specific gene STM4495 of Salmonella typhimurium as shown in SEQ ID NO:1, the DNA sequence dna of the O5 antigen encoding gene oafA of Salmonella typhimurium as shown in SEQ ID NO:2, H
1the DNA sequence dna of antigen encoding gene fliC is as shown in SEQ ID NO:3 and H
2the DNA sequence dna of antigen encoding gene fljB is as shown in SEQ ID NO:4.
Preferably, in described step 1, described primer is specially: specific gene STM4495 forward primer sequence is as shown in SEQ ID NO:5, and reverse primer sequence is as shown in SEQ ID NO:6; O5 antigen encoding gene oafA forward primer sequence is as shown in SEQ ID NO:7, and reverse primer sequence is as shown in SEQ ID NO:8; H1 antigen encoding gene fliC forward primer sequence is as shown in SEQ ID NO:9, and reverse primer sequence is as shown in SEQ ID NO:10; H2 antigen encoding gene fljB forward primer sequence is as SEQ ID NO:11, and reverse primer sequence is as SEQ ID NO:12.
In described step 3, determination methods is:
(1) there is the corresponding amplified band of specific gene STM4495 in electrophoresis result, in interpret sample, contains Salmonella typhimurium; There is not the corresponding amplified band of specific gene STM4495, in interpret sample, do not contain Salmonella typhimurium;
(2) detect and confirm to contain after salmonella typhimurium strain in sample,
1. there is not the corresponding amplified band of O5 antigen encoding gene oafA in electrophoresis result, is Copenhagen mutation; Occur the corresponding amplified band of O5 antigen encoding gene oafA, explanation is not Copenhagen mutation;
2. there is not H in electrophoresis result
1the corresponding amplified band of antigen encoding gene fliC, is 4,12:-:1,2 serological type strains; There is H
1the corresponding amplified band of antigen encoding gene fliC, explanation is not 4,12:-:1,2 serological type strains;
3. there is not H in electrophoresis result
2the corresponding amplified band of antigen encoding gene fljB, is 4, [5], 12:i:-serological type strain; There is H
2the corresponding amplified band of antigen encoding gene fljB, explanation is not 4, [5], 12:i:-serological type strain.
Preferably, in described step 3, the amplified production of described specific gene STM4495 is 413bp; The amplified production of described O5 antigen encoding gene oafA is 195bp; Described H
1the amplified production of antigen encoding gene fliC is 963bp; Described H
2the amplified production of antigen encoding gene fljB is 789bp.
Realize concrete steps of the present invention as follows:
Step 1 is stencil design amplimer according to the DNA sequence dna SEQ ID NO:4 of the DNA sequence dna SEQ ID NO:2 of O5 antigen encoding gene oafA of the conserved sequence SEQ ID NO:1 in the sequence of the 1800-2930 section of the specific gene STM4495 of Salmonella typhimurium (gene sequence number is from Salmonella typhimurium LT2 whole genome sequence), Salmonella typhimurium and the DNA sequence dna SEQ ID NO:3 of H1 antigen encoding gene fliC and H2 antigen encoding gene fljB;
Step 2, extracts sample DNA, the amplification of PCR method;
Step 3, detected through gel electrophoresis amplified production, whether judgement sample contains Salmonella typhimurium, if contain this bacterium, judges whether it belongs to its serovar again; Described judgement is specially: (1), if the corresponding amplified band of specific gene STM4495 appears in electrophoresis result, contains Salmonella typhimurium in interpret sample; If no, do not contain Salmonella typhimurium in interpret sample; (2) detecting and turn out to be after salmonella typhimurium strain, if 1. the corresponding amplified band of O5 antigen encoding gene oafA does not appear in electrophoresis result, is Copenhagen mutation; If there is, explanation is not Copenhagen mutation; If there is not the corresponding amplified band of H1 antigen encoding gene fliC in electrophoresis result 2., be 4,12:-:1,2 serological type strains; If there is, explanation is not 4,12:-:1,2 serological type strains; If there is not the corresponding amplified band of H2 antigen encoding gene fljB in electrophoresis result 3., be 4, [5], 12:i:-serological type strain; If there is, explanation is not 4, [5], 12:i:-serological type strain.
In step 1, described primer is specially: specific gene STM4495 forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6; O5 antigen encoding gene oafA forward primer SEQ ID NO:7 and reverse primer SEQ ID NO:8; H1 antigen encoding gene fliC forward primer SEQ ID NO:9 and reverse primer SEQ ID NO:10; H2 antigen encoding gene fljB forward primer SEQ ID NO:11 and reverse primer SEQ ID NO:12.
In step 2, in described PCR method, PCR detection system is specially: 25 μ L reaction systems are specially, 10 * PCR reaction buffer, 2.5 μ L, the Mg of 25mmol/L
2+2.0 μ L, the dNTP2.0 μ L of 2.5mmol/L, each 1.0 μ L of the primer pair of 5 μ mol/L, the TaqDNA polysaccharase 1.0 μ L of 1.0U/ μ L, template solution 5.0 μ L, last moisturizing to 25 μ L.
In step 2, in described PCR method, PCR detection system Amplification is specially: first 94 ℃ of denaturation 5min, start afterwards amplification cycles, and the program of each circulation is: 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ are extended 60s; Totally 35 of circulations; After loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
In step 3, described judgement is specially: whether (1) detected through gel electrophoresis amplified production exists amplified band in 413bp position, if existed, in interpret sample, contains Salmonella typhimurium; If no, do not contain Salmonella typhimurium in interpret sample; (2) electrophoresis detection exists after amplified band on 413bp position, if 1. gel electrophoresis result does not have amplified band on 195bp position, is Copenhagen mutation; If there is, explanation is not Copenhagen mutation; If 2. gel electrophoresis result does not have amplified band on 963bp position, be 4,12:-:1,2 serological type strains; If there is, explanation is not 4,12:-:1,2 serological type strains; If 3. gel electrophoresis result does not have amplified band on 789bp position, be 4, [5], 12:i:-serological type strain; If there is, explanation is not 4, [5], 12:i:-serological type strain.
The invention still further relates to a kind of primer system for Salmonella typhimurium and the detection of serovar multiplex PCR thereof, wherein, described primer system comprises at least two kinds in primer as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.Preferably, described primer system comprises 4 pairs of primers, and its base sequence is respectively as SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; Shown in SEQ ID NO:11 and SEQ ID NO:12.
Compared with prior art, the present invention has following beneficial effect: adopt detection method of the present invention to detect Salmonella typhimurium and serovar thereof, detection time is short, owing to need not adopting the antiserum(antisera) that must use in traditional detection method, has reduced testing cost; Meanwhile, detection method of the present invention also can not detected bacterial strain for detection of some antiserum(antisera), as the bacterial strain that antigen is not expressed, makes up the defect of immunodetection, more has practicality; Detection target spot of the present invention has single specificity, and detected result is special, and result is judged simple.
Accompanying drawing explanation
Fig. 1 is PCR detection method primer pair stm4495-2f/r sensitivity evaluation experimental gel electrophoresis result figure in embodiment 1;
Fig. 2 is PCR detection method primer pair O5-3f/r sensitivity evaluation experimental gel electrophoresis result figure in embodiment 1;
Fig. 3 is PCR detection method primer pair Flic-2f/r sensitivity evaluation experimental gel electrophoresis result figure in embodiment 1;
Fig. 4 is PCR detection method primer pair Fljb-4f/r sensitivity evaluation experimental gel electrophoresis result figure in embodiment 1;
Fig. 5 is multi-PCR detection method sensitivity evaluation experimental gel electrophoresis result figure in embodiment 1;
Fig. 6 is multi-PCR detection method Evaluation on specificity experiment gel electrophoresis result figure in embodiment 1;
Fig. 7 is multi-PCR detection method Evaluation on specificity experiment gel electrophoresis result figure in embodiment 1;
Fig. 8 is that in embodiment 2, multi-PCR detection method is identified isolated strains experiment gel electrophoresis result figure.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The foundation of Salmonella typhimurium and serovar multi-PCR detection method thereof
Step 1, according to the conserved sequence design of amplification primers in the genomic dna sequence of Salmonella typhimurium
From Salmonella typhimurium genomic dna sequence, find the 1800-2930 section of specific gene STM4495 (gene sequence number is from Salmonella typhimurium LT2 whole genome sequence), detection target gene using it as Salmonella typhimurium, gene order is as shown in SEQ ID NO:1, simultaneously, choose the detection target gene that the O5 antigen encoding gene oafA of Salmonella typhimurium and the DNA sequence dna of H1 antigen encoding gene fliC and H2 antigen encoding gene fljB are its serovar, gene order is as SEQ ID NO:2, shown in SEQ ID NO:3 and SEQ ID NO:4,
The DNA sequence dna of selected gene is input in primer-design software Primer Premier5.0 and designs primer, it is 40～60% that GC% scope is set, product magnitude range is 150～1000bp, from alternative primer pair, select primer, primer sequence following (primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd):
stm4495-2f：5’-AAAAGCAGGCATGTCCACCG-3’(SEQ?ID?NO：5)；
stm4495-2r：5’-ATCCCGCAGCGTAAAGCAAC-3’(SEQ?ID?NO：6)；
O5-3f：5’-CAGACAACAAGCCTACTACGGTA-3’(SEQ?ID?NO：7)；
O5-3r：5’-CGGTCCTTCCGGAATTAATC-3’(SEQ?ID?NO：8)；
Flic-2f：5’-GGCTACGGGCAGAAGTCA-3’(SEQ?ID?NO：9)；
Flic-2r：5’-TCAACGGCGTGAAAGTCC-3’(SEQ?ID?NO：10)；
Fljb-4f：5’-ACAGATTGTTTACGGTATTGC-3’(SEQ?ID?NO：11)；
Fljb-4r：5’-CTACACTGGATGTATCGGGTC-3’(SEQ?ID?NO：12)；
Step 2, DNA profiling preparation
The Salmonellas bacterial strain (as shown in table 1) of the various serotypes such as Salmonella typhimurium, Salmonella enteritidis and Salmonella choleraesuls is connect respectively to bacterium to the LB liquid nutrient medium of 50mL, at 37 ℃, increase after bacterium 8h, get 1mL bacterium liquid, put into 1.5mL centrifuge tube;
Afterwards, the centrifugal 10min of 3,000r/min, gets supernatant liquor, then at the centrifugal 5min of 12,000r/min, collects thalline.With aseptic double-distilled water Eddy diffusion thalline, after centrifuge washing, add the aseptic ultrapure water of 100 μ L, in boiling water bath, boil 15min, take out immediately, at-20 ℃, place 30min; 37 ℃ thaw after, the centrifugal 5min of 12,000r/min, get supernatant liquor place-20 ℃ standby.
Step 3, sensitivity evaluation test
After measured, the concentration of Salmonella typhimurium reference culture CMCC50115 genomic dna solution is about 22 μ g/mL, with sterilized water, make 10 times of gradient dilutions, dilute altogether 9 gradients, each dilution gradient is got respectively 5 μ L and is added PCR reaction system, detected through gel electrophoresis amplified production is observed gel electrophoresis result in gel imaging instrument.Primer pair stm4495-2 as shown in Figure 1, primer pair O5-3f/r as shown in Figure 2, primer pair Flic-2f/r as shown in Figure 3, primer pair Fljb-4f/r as shown in Figure 4, multiplex PCR as shown in Figure 5, in figure: swimming lane 1～10:DNA template concentrations is respectively 11ng, 1.1ng, 110pg, 11pg, 1.1pg, 110fg, 11fg, 1.1fg, 0.11fg/ reaction, sterilized water; Swimming lane M:200bp molecular weight standard.In Fig. 1-4, the 6th swimming lane can be seen band clearly, and institute's corresponding DNA concentration is 110fg/PCR, and after the 7th swimming lane, can't see amplified band; In Fig. 5, the 5th swimming lane can be seen band clearly, and institute's corresponding DNA concentration is 1.1pg/PCR, and can't see amplified band after the 6th swimming lane.Therefore, judging single is 110fg/PCR to the detection sensitivity of primer PCR, and the detection sensitivity of multiplex PCR is 1.1pg/PCR, has higher sensitivity.
Step 4, the experiment of PCR detection method Evaluation on specificity
PCR detection method is as follows: first add 8.5 μ L sterilized waters in reaction tubes, then add successively 10 * PCR reaction buffer, 2.5 μ L, the Mg of 25mmol/L
2+2.0 μ L, the dNTP1.0 μ L of 2.5mmol/L, each 1.0 μ L of 5 μ mol/L primers, 2.5U/ μ L TaqDNA polysaccharase 1.0 μ L, finally add template solution 5 μ L, and using sterilized water as the negative control of template as reaction.Then after reaction tubes is centrifugal, put into PCR reaction instrument, according to following PCR loop parameter, carry out: at 94 ℃ of denaturation 5min, then do 35 circulations, the program of each circulation comprises 94 ℃ of sex change 30s, 60 ℃ of annealing temperatures, annealing time is 45s, then at 72 ℃, extends 60s, after loop ends, at 72 ℃, extends 10min, finally be cooled to 12 ℃, finish all operations program.
Get each 17 strains of Salmonellas and non-Salmonellas reference culture (as shown in table 1, bacterial strain shown in table is those skilled in the art and can is bought and be obtained by disclosed channel), according to DNA profiling extracting method, extract respectively genomic dna.The DNA solution of every strain bacterial strain is all got 5 μ L and is added in PCR reaction system as PCR reaction template, by aforementioned PCR detection method, carries out amplified reaction.
Fig. 6 is multi-PCR detection method Evaluation on specificity experiment gel electrophoresis result figure, detected result for Salmonellas reference culture, in figure: swimming lane 1～17 is respectively: Salmonella Typhimurium AS1.1174, Salmonella Typhimurium ATCC14028, Salmonella Typhimurium ATCC13311, Salmonella Typhimurium ATCC51812, Salmonella Typhimurium CMCC50115, Salmonella Paratyphi AATCC9150, Salmonella Paratyphi B CMCC50004, Salmonella Hirschfeldii CMCC50017, Salmonella Choleraesuis AS1.1190, Salmonella Typhi CMCC50180, Salmonella Enteritidis ATCC13076, Salmonella Gallinarum CMCC50770, Salmonella Anatum ATCC9270, Salmonella Vellore ATCC15611, Salmonella Tallahassee ATCC12002, Salmonella Poona NCTC4840, Salmonella Abony NCTC6017, swimming lane M:200bp molecular weight standard.Swimming lane 1～5 is salmonella typhimurium strain, swimming lane 6～17 is non-salmonella typhimurium strain, as can be seen from the figure, there is the feature band of 413bp in swimming lane 1～5, and in swimming lane 6～17, there is not the feature band of 413bp, result proof present method has good specificity in detecting salmonella typhimurium strain.
Fig. 7 is the detected result of non-Salmonellas reference culture, and its strain number refers to accompanying drawing explanation and table 1.In figure: swimming lane 1～17:Vibrio parahaemolyticus ATCC17802, Listeria monocytogenes ATCC27708, Staphylococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, Enterobacter cloacae ATCC13047, Proteus mirabilis ATCC12453, Proteus vulgaris ATCC33420, Klebsiella peneumoniae ATCC27336, Shigella dysenteriae CMCC51335, Enterococcus avium ATCC14025, Pseudomonas aeruginosa CDC B32116, Pseudomonas putida ATCC17485, Serratia marcescens ATCC27592, Bacillus subtilis ATCC6633, Escherichia coli ATCC25922, Citrobacter freundii ATCC8090, Bacillus cereus ATCC1220, swimming lane M:200bp molecular weight standard.
Known from Fig. 6,7, only take Salmonella typhimurium genomic dna during as template, can amplify the product of 413bp size, as swimming lane 1～5; The special product of target sequence of the amplified 195bp of the Salmonellas that contains O5 antigen in some serologic group B, as swimming lane 7 and 17; Other salmonella gene groups DNA of take can not amplify the special product of 413bp and 195bp during as template, but some Salmonellass can amplify the 963bp of Flic-2f/r and Fljb-4f/r and the special product (Fig. 6) of 789bp, as swimming lane 8～10; The genomic dna template of non-Salmonellas, almost without any amplified production, only has a few strain bacterium to amplify non-specific product (Fig. 7).
Table 1 Evaluation on specificity bacterial strain uses therefor
Note: in table, listed bacterial strain is purchased from Chinese microorganism strain preservation center.
Embodiment 2
The evaluation of the doubtful bacterial strain of Salmonellas
Utilize the Salmonella typhimurium of implementation column 1 foundation and the multi-PCR detection method of serovar thereof, detected the doubtful bacterial strain of Salmonellas of 150 strains separation from food samples, food samples is collected in supermarket, outskirts of a town and the country fair in Shanghai, and the separation of sample preparation and doubtful bacterial strain is referring to GB GB/T4789.4-2010.150 strain strain isolateds extract after genomic dna, first with the universal primer of 16S rDNA, verify the DNA profiling of all extractions, then detect by the multiplex PCR system of embodiment 1, identify Salmonella typhimurium, Copenhagen mutation, 4, [5], 12:i:-and 4,12:-:1,2 serotypes.
Meanwhile, all bacterial strains all line on BHI flat board, cultivate 12h for 37 ℃, adopt slide agglutination to identify the serotype (serological diagnosis step refers to product description and GB GB/T4789.4-2008) of bacterial strain.60 kinds of diagnostic serums (Lanzhou Institute of Biological Products) and 79 kinds of diagnostic serums (S & A, Ltd, Thailand) that the serum using is salmonella.
Fig. 8 is the identification with multi-plex PCR result of strain isolated.In figure: swimming lane 1～5:S.Typhimurium, 4,5,12:i:-, 4,5,12:i:-, S.Typhimurium var.Copenhagen, ddH
2o, swimming lane M:200bp molecular weight standard.
150 strain strain isolateds are carried out respectively to PCR detection and serological diagnosis, and both detected results are compared, result shows that both are consistent.
When PCR detects, if there is the clear band of 413bp size in PCR product, pattern of descriptive parts DNA derives from Salmonella typhimurium.Detection turns out to be after salmonella typhimurium strain, if be Copenhagen mutation while there is not 195bp product after amplification, does not occur that 963bp product is 4,12:-:1, and 2 bacterial strains, do not occur that 789bp product is 4, [5], 12:i:-serotype.Through PCR, detect, in 150 strain strain isolateds, having 31 strains is that Salmonella typhimurium is positive, it is 4 that 2 strains (SJTUF10421 and SJTUF10548) bacterium detects, [5], 12:i:-serotype, 1 strain (SJTUF10419) is the mutation of Salmonella typhimurium Copenhagen, as shown in Figure 8.
Serological diagnosis result shows, in 150 strain strain isolateds, has 36 strain bacterium and O4 antiserum(antisera) generation agglutination reaction.Wherein the H1 phase antigen of 2 strains reacts with Hf and Hg antiserum(antisera), and 2 strains are reacted with Hf, Hg and Hs antiserum(antisera), and 1 strain is reacted with Hb antiserum(antisera), and all the other 31 strains can be reacted with Hi antiserum(antisera).And the H2 phase antigen of this 31 strain bacterial strain, except 2 strains (SJTUF10421 and SJTUF10548) be 4,5, outside 12:i:-serological type strain, can with H
2antiserum(antisera) generation aggegation.Remain in 29 strain Salmonella typhimuriums, only have bacterial strain SJTUF10419 can not with O5 antigen-reactive.
By the present embodiment, prove, the multiple PCR method that detects Salmonella typhimurium and serovar thereof has extreme high reliability.
More than describe preferred embodiment of the present invention in detail.The ordinary skill that should be appreciated that this area just can design according to the present invention be made many modifications and variations without creative work.Therefore, all technician in the art, all should be in the determined protection domain by claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (7)
1. for Salmonella typhimurium and the serovar multi-PCR detection method thereof of non-medical diagnosis on disease therapeutic purpose, comprise the steps:
Step 1, design of amplification primers;
Step 2, extracts sample DNA, utilizes the amplimer in step 1 to carry out the amplification of PCR method;
Step 3, detected through gel electrophoresis amplified production, and electrophoresis result is judged;
In described step 1, the template of design of amplification primers foundation is: the conserved sequence in the sequence of the 1800-2930 section of the specific gene STM4495 of Salmonella typhimurium as shown in SEQ ID NO:1, the DNA sequence dna of the O5 antigen encoding gene oafA of Salmonella typhimurium as shown in SEQ ID NO:2, H
1the DNA sequence dna of antigen encoding gene fliC is as shown in SEQ ID NO:3 and H
2the DNA sequence dna of antigen encoding gene fljB is as shown in SEQ ID NO:4;
Described primer is specially: specific gene STM4495 forward primer sequence is as shown in SEQ ID NO:5, and reverse primer sequence is as shown in SEQ ID NO:6; O5 antigen encoding gene oafA forward primer sequence is as shown in SEQ ID NO:7, and reverse primer sequence is as shown in SEQ ID NO:8; H1 antigen encoding gene fliC forward primer sequence is as shown in SEQ ID NO:9, and reverse primer sequence is as shown in SEQ ID NO:10; H2 antigen encoding gene fljB forward primer sequence is as SEQ ID NO:11, and reverse primer sequence is as SEQ ID NO:12.
2. the Salmonella typhimurium for non-medical diagnosis on disease therapeutic purpose as claimed in claim 1 and serovar multi-PCR detection method thereof, wherein,
In described step 3, determination methods is:
(1) there is the corresponding amplified band of specific gene STM4495 in electrophoresis result, in interpret sample, contains Salmonella typhimurium; There is not the corresponding amplified band of specific gene STM4495, in interpret sample, do not contain Salmonella typhimurium;
(2) detect and confirm to contain after salmonella typhimurium strain in sample,
1. there is not the corresponding amplified band of O5 antigen encoding gene oafA in electrophoresis result, is Copenhagen mutation; Occur the corresponding amplified band of O5 antigen encoding gene oafA, explanation is not Copenhagen mutation;
2. there is not H in electrophoresis result
1the corresponding amplified band of antigen encoding gene fliC, is 4,12:-:1,2 serological type strains; There is H
1the corresponding amplified band of antigen encoding gene fliC, explanation is not 4,12:-:1,2 serological type strains;
3. there is not H in electrophoresis result
2the corresponding amplified band of antigen encoding gene fljB, is 4, [5], 12:i:-serological type strain; There is H
2the corresponding amplified band of antigen encoding gene fljB, explanation is not 4, [5], 12:i:-serological type strain.
3. the Salmonella typhimurium for non-medical diagnosis on disease therapeutic purpose as claimed in claim 1 and serovar multi-PCR detection method thereof, wherein,
In described step 2, PCR reaction system is: in 25 μ L reaction systems, and 10 * PCR reaction buffer, 2.5 μ L, the Mg of 25mmol/L
2+2.0 μ L, the dNTP2.0 μ L of 2.5mmol/L, each 1.0 μ L of the primer pair of 5 μ mol/L, the TaqDNA polysaccharase 1.0 μ L of 1.0U/ μ L, template solution 5.0 μ L, last moisturizing to 25 μ L.
4. the Salmonella typhimurium for non-medical diagnosis on disease therapeutic purpose as claimed in claim 1 and serovar multi-PCR detection method thereof, wherein,
In described step 2, PCR reaction parameter is: first 94 ℃ of denaturation 5min, start afterwards amplification cycles, and the program of each circulation is: 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ are extended 60s; Totally 35 of circulations; After loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
5. the Salmonella typhimurium for non-medical diagnosis on disease therapeutic purpose as claimed in claim 2 and serovar multi-PCR detection method thereof, wherein,
In described step 3,
The amplified production of described specific gene STM4495 is 413bp;
The amplified production of described O5 antigen encoding gene oafA is 195bp;
Described H
1the amplified production of antigen encoding gene fliC is 963bp;
Described H
2the amplified production of antigen encoding gene fljB is 789bp.
6. the Salmonella typhimurium for non-medical diagnosis on disease therapeutic purpose as claimed in claim 1 and serovar multi-PCR detection method thereof, wherein,
In described step 3, determination methods is:
(1) whether detected through gel electrophoresis amplified production there is amplified band in 413bp position, if existed, in interpret sample, contains Salmonella typhimurium; If no, do not contain Salmonella typhimurium in interpret sample;
(2) electrophoresis detection exists after amplified band on 413bp position,
If 1. gel electrophoresis result does not have amplified band on 195bp position, it is Copenhagen mutation; If there is, explanation is not Copenhagen mutation;
If 2. gel electrophoresis result is haunted and is occurred amplified band at 963bp, be 4,12:-:1,2 serological type strains; If there is, explanation is not 4,12:-:1,2 serological type strains;
If 3. gel electrophoresis result does not have amplified band on 789bp position, be 4, [5], 12:i:-serological type strain; If there is, explanation is not 4, [5], 12:i:-serological type strain.
7. the primer system detecting for Salmonella typhimurium and serovar multiplex PCR thereof, wherein, described primer system comprises at least three pairs in 4 pairs of primers as shown in SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
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| US11946107B2 (en) | 2016-06-16 | 2024-04-02 | Bio-Rad Europe Gmbh | Method of detecting Salmonella typhimurium |
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| CN109486975B (en) * | 2018-12-29 | 2022-02-25 | 深圳华大生命科学研究院 | Molecular typing method and specific SNP site combination of salmonella typhimurium |
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