CN102851361B - Detection primer kit for salmonella, salmonella enteritidis and salmonella typhimurium by PCR pyrophosphoric acid method and detection method - Google Patents
Detection primer kit for salmonella, salmonella enteritidis and salmonella typhimurium by PCR pyrophosphoric acid method and detection method Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a detection primer kit for salmonella, salmonella enteritidis and salmonella typhimurium by a PCR pyrophosphoric acid method, and a detection method. Based on an aceA gene of salmonella, a specific sequence of salmonella enteritidis, and a conserved domain of a STM4599 sequence of salmonella typhimurium, the invention respectively designs an amplification primer and a sequencing primer; PCR amplification is performed for a target segment; a single-chain template is prepared by the amplification product; pyrophosphoric acid sequencing is performed; if the sequence of the first 30 bases after the 3'-terminal of the sequencing primer is GCCTGTGGGTACTTCTCCTGCCAGTATAAT, the sample is determined to contain salmonella; if the sequence of the first 30 bases after the 3'-terminal of the sequencing primer is CCTGTTGTCTGCTCACCATTCGCCAGCCAC and TACAACCGGAGTGCACATTAATCCCGCAGC, the sample is determined to contain salmonella enteritidis and salmonella typhimurium.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to detection primer, test kit and the detection method of Salmonellas, Salmonella enteritidis and Salmonella typhimurium PCR-tetra-sodium method.
Background technology:
Salmonellas (Salmonella, Sa.) is one of important food-borne pathogens, in the bacterial food poisoning case of countries in the world, and the normal row umber one of food poisoning case or second that Sa causes.Along with molecular biological development, existing is target gene for genes such as Salmonellas invA, 16s rRNA, SpvC, invB, fimA, agfA, SEFl4, sefA, sdf, ssaQ, fimY, detect the report of Salmonellas with regular-PCR or fluorescent PCR, although quick, high-throughout object that these methods can reach, but cannot obtain the golden standard-gene order of molecular diagnosis, therefore occur false-positive possibility.
Tetra-sodium order-checking (pyrosequencing) technology is that of inventing in recent years carries out short segment DNA sequencing technologies in real time, this technology does not need electrophoresis and fluorescent mark in the time of DNA sequence analysis, directly measure primer base sequence below, by being provided, reliable sequence information confirms PCR product, while is due to the sequence of a chain in Main Analysis PCR product two strands, avoid the stutter bands causing due to the secondary structure of DNA chain, make sequencing result more accurate, being applied at present genetic expression virus detects, snp analysis, drug resistant gene detects, fungi qualification, multiple fields such as DNA methylation analysis.Tetra-sodium order-checking is a kind of Novel DNA sequencing technologies based on synthetic, under the guiding of special primer, add respectively four kinds of Nucleotide according to sequence to be measured, in the process of extending, discharge tetra-sodium, the latter discharges fluorescence in the linked reaction of ATP sulfurylase and luciferase, detect corresponding fluorescence intensity by detector, thereby obtain the nucleotide sequence detecting in sample, it is a kind of very practical real-time detection technique of short sequence, quantitatively accurately, be easy to automatization, can realize the rapid detection of high-throughout large sample, there is the incomparable advantage of DNA sequencing method, Classification Identification and the Surveillance on antibiotic resistance of Clinical microorganism are applied to.Li Xiujuan etc. detect the Salmonellas of 159 strain different sourcess in the level by PCR-tetra-sodium sequencing subordinate taking fimy as goal gene, but the method can not be distinguished different serotypes Salmonellas, also 159 strain Salmonellas serotypes of undeclared detection in article, its specificity needs further to be verified (Li Xiujuan, Tian Huifang).
Summary of the invention:
The object of this invention is to provide easy, quick, accuracy is high, detection primer, test kit and the detection method of the Salmonellas of high specificity, Salmonella enteritidis and Salmonella typhimurium PCR-tetra-sodium method.
The present invention is according to the STM4599 sequence (GenBank:AERV01000023.1) of Salmonellas aceA gene (GenBank:U43344.1), Salmonella enteritidis (SE) distinguished sequence (GenBank:AF370707.1), Salmonella typhimurium (ST), design of amplification primers and sequencing primer respectively, set up in conjunction with the order-checking of PCR-tetra-sodium and detected the method for Salmonellas, Salmonella enteritidis and Salmonella typhimurium from DNA sequence dna level, thereby realized object of the present invention.
The detection primer of Salmonellas of the present invention, Salmonella enteritidis and Salmonella typhimurium PCR-tetra-sodium method, is characterized in that comprising PCR primer and sequencing primer:
PCR primer for Salmonellas:
5 '-TCACCGAAAGACCAACAGAA-3 '; (as shown in SEQ ID NO.1)
5 '-GGTGGAACTCGCTGAAATGA-3 '; (as shown in SEQ ID NO.2)
Sequencing primer: 5 '-CGAAAGACCAACAGAAG-3 '; (as shown in SEQ ID NO.3)
PCR primer for Salmonella enteritidis:
5 '-GCATGTTCTGGAAAGCCTCTTTAT-3 '; (as shown in SEQ ID NO.4)
5 '-TCGTTCTTCTGGTACTTACGATGA-3 '; (as shown in SEQ ID NO.5)
Sequencing primer: 5 '-AAAAAGGTTTAGTAAATCAG-3 '; (as shown in SEQ ID NO.6)
PCR primer for Salmonella typhimurium:
5 '-AAGCTAAGTGTGGGGGCTAGTAT-3 '; (as shown in SEQ ID NO.7)
5 '-GGGTTTGTTTTTGATGATACAGG-3 '; (as shown in SEQ ID NO.8)
Sequencing primer: 5 '-AGTATTGATAAATAATGGTT-3 '; (as shown in SEQ ID NO.9).
Second object of the present invention is to provide the detection kit of Salmonellas, Salmonella enteritidis and Salmonella typhimurium PCR-tetra-sodium method, comprise DNA extraction reagent, PCR reaction reagent, single-stranded template is prepared reagent, tetra-sodium sequencing reagent, PCR primer and sequencing primer, is characterized in that, described PCR primer and sequencing primer are:
PCR primer for Salmonellas:
5 '-TCACCGAAAGACCAACAGAA-3 '; (as shown in SEQ ID NO.1)
5 '-GGTGGAACTCGCTGAAATGA-3 '; (as shown in SEQ ID NO.2)
Sequencing primer: 5 '-CGAAAGACCAACAGAAG-3 '; (as shown in SEQ ID NO.3)
PCR primer for Salmonella enteritidis:
5 '-GCATGTTCTGGAAAGCCTCTTTAT-3 '; (as shown in SEQ ID NO.4)
5 '-TCGTTCTTCTGGTACTTACGATGA-3 '; (as shown in SEQ ID NO.5)
Sequencing primer: 5 '-AAAAAGGTTTAGTAAATCAG-3 '; (as shown in SEQ ID NO.6)
PCR primer for Salmonella typhimurium:
5 '-AAGCTAAGTGTGGGGGCTAGTAT-3 '; (as shown in SEQ ID NO.7)
5 '-GGGTTTGTTTTTGATGATACAGG-3 '; (as shown in SEQ ID NO.8)
Sequencing primer: 5 '-AGTATTGATAAATAATGGTT-3 '; (as shown in SEQ ID NO.9).
The 3rd object of the present invention is to provide the detection method of a kind of Salmonellas, Salmonella enteritidis and Salmonella typhimurium of non-diagnostic purpose, it is characterized in that, comprises the following steps:
(1) sample is increased to bacterium and cultivate, extract the genomic dna of thalline in enrichment liquid as template;
(2) use respectively above-mentioned PCR primer to carry out respectively PCR reaction, reclaim PCR product, prepare strand masterplate, then apply above-mentioned sequencing primer corresponding PCR product is carried out to tetra-sodium order-checking, first base after sequencing primer 3 ends starts order-checking, when current 30 base sequences are GCCTGTGGGTACTTCTCCTGCCAGTATAAT, in judgement sample, contain Salmonellas, when current 30 base sequences are CCTGTTGTCTGCTCACCATTCGCCAGCCAC, in judgement sample, contain Salmonella enteritidis, when current 30 base sequences are TACAACCGGAGTGCACATTAATCCCGCAGC, in judgement sample, contain Salmonella typhimurium.
Described sample can be food, meat product, and as pork, and seafood, as the prawn of selling.
Preferably, described PCR reaction, its amplification system is 50 μ L, PCR Mix Buffer 25 μ l, Taq enzyme 5 μ l, MgCl
21 μ l, the each 2 μ l of 10 μ M upstream and downstream primer, DNA profiling 1 μ l, deionized water 14 μ l, PCR reaction conditions is 95 DEG C of 5min of denaturation; 95 DEG C of 15s, 65 DEG C of 30s,, 72 DEG C of 30s, 45 circulations; 72 DEG C of 12min.
The present invention is according to the conservative region in the STM4599 sequence of Salmonellas (Sa) aceA gene, Salmonella enteritidis (SE) distinguished sequence, Salmonella typhimurium (ST), design of amplification primers and sequencing primer respectively, set up PCR-tetra-sodium order-checking, detected the method for Sa, SE and ST from DNA base sequence level.The present invention first carries out pcr amplification to target fragment, ensures the specificity of amplified fragments, then prepares single-stranded template with amplified production, carries out tetra-sodium order-checking under the guiding of sequencing primer.The gained base sequence and the known array contrast judgement result that detect, be that after sequencing primer, front 30 base sequences are GCCTGTGGGTACTTCTCCTGCCAGTATAAT, in judgement sample, contain Salmonellas, after sequencing primer, front 30 base sequences are for being CCTGTTGTCTGCTCACCATTCGCCAGCCAC and TACAACCGGAGTGCACATTAATCCCGCAGC, and judgement contains Salmonella enteritidis (SE) and Salmonella typhimurium (ST) respectively.Pcr amplification experimental result, aceA, SE and ST amplimer have good specificity, and 24 strain different serotypes Sa, 16 strain SE and 6 strain ST amplify respectively the DNA fragmentation of big or small 81bp, 176bp and 131bp, and other control strains do not amplify DNA band.Tetra-sodium sequencing result shows, the order-checking effective dna base number of 24 strain Sa, 16 strain SE and 6 strain ST is respectively 31bp-36bp, 31bp-39bp, 34bp-36bp, all exceed the 30bp DNA base sequence that needs detection, and other control strains are not measured DNA base sequence.Analog sample detected result, tetra-sodium sequencing is consistent with traditional detection method, but in sense cycle, traditional method needs to cultivate, line separates, biochemical identification, serotype etc., need 5d-6d, and tetra-sodium sequence measurement BP increases bacterium 10h for the first time, TTB increases bacterium 18h for the second time, nucleic acid extraction, pcr amplification and tetra-sodium order-checking 3h, whole test 31h completes, simple and efficient, and can be to its somatotype by detecting Salmonellas in the level of DNA base sequence subordinate when, judge whether detection Sa is SE and ST serotype, avoid simple pcr amplification to detect and easily polluted the false positive results causing, accuracy is high.Therefore the present invention has good application prospect.
Brief description of the drawings:
Fig. 1 is the aceA amplified production electrophorogram of 24 strain Sa, wherein M:50bp marker, 1:SE ATCC13076,2:ST ATCC14028,3-24:Sa1-Sa22;
Fig. 2 is the aceA specificity experiment amplified production electrophorogram of Sa, wherein M:50bp maker, 1:SE ATCC13076,2:ST ATCC14028,3: Enterobacter sakazakii ATCC29544,4: intestinal bacteria ATCC25922,5: yersinia entero-colitica CMCC52221,6: streptococcus aureus ATCC6538,7: shigella dysenteriae NICPBP51252,8: Listeria monocytogenes ATCC19115,9: Klebsiella pneumonia CMCC46102,10: deionized water;
Fig. 3 is the amplified production electrophorogram of SE, wherein M:50bpmaker, 1:SE ATCC13076,2-16:SE1-SE15,17: deionized water;
Fig. 4 is SE specificity experiment amplified production electrophorogram, wherein M:50bp maker, 1:SE ATCC13076,2:ST ATCC14028,3: Enterobacter sakazakii ATCC29544,4: intestinal bacteria ATCC25922,5: yersinia entero-colitica CMCC52221,6: streptococcus aureus ATCC6538,7: shigella dysenteriae NICPBP51252,8: Listeria monocytogenes ATCC19115,9: Klebsiella pneumonia CMCC46102,10: deionized water;
Fig. 5 is SE specificity experiment amplified production electrophorogram, wherein M:50bp maker, 1:SE ATCC13076,2-23:Sa1-Sa22,24: deionized water;
Fig. 6 is ST amplified production electrophorogram, wherein M:50bp maker, 1:ST ATCC14028,2-6:ST1-ST5,7: deionized water;
Fig. 7 is ST specificity experiment amplified production electrophorogram, wherein M:50bp maker, 1:ST ATCC14028,2:SE ATCC13076,3: Enterobacter sakazakii ATCC29544,4: intestinal bacteria ATCC25922,5: yersinia entero-colitica CMCC52221,6: streptococcus aureus ATCC6538,7: shigella dysenteriae NICPBP51252,8: Listeria monocytogenes ATCC19115,9: Klebsiella pneumonia CMCC46102,10: Vibrio parahemolyticus ATCC33847,11: deionized water;
Fig. 8 is ST specificity experiment amplified production electrophorogram, wherein M:50bp maker, 1:ST ATCC14028,2-23:Sa1-Sa22,24: deionized water;
Fig. 9 is Sa3 tetra-sodium sequencing result figure;
Figure 10 is negative control (single listeria spp that increases) tetra-sodium sequencing result figure;
Figure 11 is SE2 tetra-sodium sequencing result figure;
Figure 12 is ST2 tetra-sodium sequencing result figure;
Embodiment:
Following examples are to further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
1, materials and methods
1.1 bacterium source
24 kinds of different serotypes Salmonellas 44 strains, wherein SE, each 1 strain of ST reference culture, the non-SE separating in different foods, non-ST different serotypes Sa25 strain, 15 strains of SE strain isolated, 5 strains of ST strain isolated, bacterial strain information is in table 1.8 strains of negative control bacterial strain, are respectively Listeria monocytogenes ATCC19115, streptococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, intestinal bacteria ATCC25922, shigella dysenteriae NICPBP51252, yersinia entero-colitica CMCC52221, Klebsiella pneumonia CMCC46102, Vibrio parahemolyticus ATCC33847.Above-mentioned bacterial strains is from Chinese common micro-organisms culture presevation administrative center, Guangdong Prov. Disease Prevention-control Center, China Inst. of Quarantine Inspection Sciences, Guangdong Entry-Exit Inspection and Quarantine Bureau technique center.All bacterial strains are all confirmed through VITEK2 and API reagent strip and Serologic test.
Table 1: Salmonellas (Sa) bacterial strain information
1.2 key instruments and reagent
Biotage company of tetra-sodium order-checking PyroMark lD system-Sweden; Pcr amplification instrument-U.S. Bole 2400 types; Instrument for extracting nucleic acid-Japanese PSS company; Buffer, dNTP, agarose, PCR molecular weight marker (50-500bp) Taq enzyme-Bao biotechnology (Dalian) company limited for PCR; Buffered peptone water (BP), TTB enrichment liquid-Beijing overpass company limited.
1.3 primers and probe design
The Sa aceA gene(sequence number of announcing according to GenBank: GenBank:U43344.1), SE distinguished sequence (GenBank:AF370707.1), conservative region in the STM4599 sequence (GenBank:AERV01000023.1) of ST, with PyroMark Assay Design2.0 software design PCR primer and sequencing primer, every pair of downstream primer 5/ end mark vitamin H, entrusts the precious biological company limited in Dalian synthetic.Salmonellas (Sa) aceA gene, SE and ST amplification PCR primer and sequencing primer design in table 2.
Table 2: Salmonellas primer and sequencing primer sequence
1.4 template preparations
All bacterial strains are all cultivated 10h with 37 DEG C of BP buffered peptone waters, get 10ml and be inoculated in TTB enrichment liquid, cultivate 18h for 44.5 DEG C, get 1ml bacteria suspension and move into centrifuge tube, 12000r/min is centrifugal, and 8min removes supernatant, by the floating precipitation of 1ml deionized water, 12000r/min is centrifugal, and 5min removes supernatant, repeat twice, finally add 200 μ l deionized waters and on instrument for extracting nucleic acid, extract DNA, for pcr amplification.
1.5PCR amplification system and electrophoresis parameter
1.6 single-stranded template preparations and tetra-sodium order-checking
15 μ l PCR products are transferred in 96 hole PCR plates, add 2 μ l magnetic beads (sepharose beads), 20 μ l binding buffer liquid (Binding Buffer) and 43 μ l pure water, the mixed 20min of normal temperature concussion; Open vacuum pump, pre-vacuum holding tool prep tool is cleaned 30 seconds in high purity water, then prep tool is moved on in 96 hole PCR plates, capture magnetic bead wherein, treat, after magnetic bead captures substantially, the prep tool that carries magnetic bead is put into 70% ethanol 5 seconds, then in sex change damping fluid Denaturation Buffer and lavation buffer solution Washing Buffer, respectively clean 20s～30s respectively.Prep tool is placed on and annealing premixed liquid PSQ96 is housed (adds in advance 43 μ l annealing buffer Annealing Buffer and 2 μ l 10 μ M sequencing primers again, corresponding corresponding sequencing primer for PCR product) top of plate, turn off vacuum pump, shake discharges magnetic bead gently, PSQ96 plate is placed to 5min at 80 DEG C, naturally cool to room temperature.Order-checking program adopts SQA pattern, put in order by the base of ATCG, circulation adds 15 times successively, the dosage providing according to software adds substrate mixture, enzyme mixture and 4 kinds of deoxyribonucleotides in agent bin, PSQ96 orifice plate and agent bin is put into PyroMark lD system and carry out tetra-sodium order-checking.
1.7 analog samples detect
Weigh respectively 25g pork, prawn with homogenizing bag, in every duplicate samples, add 225ml buffering protein to freeze water, add respectively 10 of the modulated good concentration of 1ml
2cfu/ml SE ATCC13076 and ST ATCC14028 bacteria suspension, pat 20min with beater, cultivate 10h for 37 DEG C, get 10ml and move into 90ml TTB enrichment liquid, cultivate 18h for 44.5 DEG C, then get a part and carry out the detection of PCR-tetra-sodium method by above-mentioned steps extraction DNA, another part is proceeded to detect by GB4789.4-2010 " food sanitation Micro biological Tests Salmonellas ".
2 results and analysis
2.1PCR amplification
The pcr amplification result of Sa aceA gene, 24 strain different serotypes Salmonellass, be the DNA fragmentation (Fig. 1) that each 1 strain of SE, ST type strain, 22 strain different serotypes Salmonellas Sa1-Sa22 all amplify 81bp size, and Listeria monocytogenes ATCC19115, streptococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, intestinal bacteria ATCC25922, shigella dysenteriae NICPBP51252, yersinia entero-colitica CMCC52221, Klebsiella pneumonia CMCC46102 7 strain control strains have no amplified band (Fig. 2), the pcr amplification result of SE, 1 strain SE type strain and SE1-SE15, totally 16 strain SE all amplify the DNA fragmentation (Fig. 3) of 176bp size, and ST ATCC14028, Listeria monocytogenes ATCC19115, streptococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, intestinal bacteria ATCC25922, shigella dysenteriae NICPBP51252, yersinia entero-colitica CMCC52221, Klebsiella pneumonia CMCC46102 7 strain control strains and Sa1-Sa22 all do not go out to see amplified band (Fig. 4, Fig. 5), the pcr amplification result of ST, 1 strain ST type strain and ST1-ST5, 6 strain ST all amplify the DNA fragmentation (Fig. 6) of 131bp size, and SE ATCC13076, Listeria monocytogenes ATCC19115, streptococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, intestinal bacteria ATCC25922, shigella dysenteriae NICPBP51252, yersinia entero-colitica CMCC52221, Klebsiella pneumonia CMCC46102, Vibrio parahemolyticus ATCC33847 8 strain contrast strains and Sa1-Sa22 are showed no amplified band (Fig. 7, Fig. 8), show Sa aceA, the PCR primer of SE and ST has good specificity.
2.2 tetra-sodium sequencing results
24 strain Sa tetra-sodium sequencing results, be SE, each 1 strain of ST type strain and Sa1-Sa25, measure respectively the DNA base sequence (table 3 of 33bp-36bp size, Fig. 9, the present invention only lists the tetra-sodium sequencing result figure of Sa3), and Listeria monocytogenes ATCC19115, streptococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, intestinal bacteria ATCC25922, shigella dysenteriae NICPBP51252, yersinia entero-colitica CMCC52221, DNA base sequence (Figure 10 is not measured in Klebsiella pneumonia CMCC46102 7 strain contrast strains, only list single listeria spp sequencer map that increases).SE tetra-sodium sequencing result is measured the DNA base sequence (table 4, Figure 11 only list the tetra-sodium sequencing result figure of SE2) of 39bp-43bp size, ST tetra-sodium sequencing result is measured the DNA base sequence (table 5 of 36bp-38bp size, Figure 12, only list the tetra-sodium sequencing result figure of ST2), and find in SE and the order-checking of ST tetra-sodium, 23 strain different serotypes Sa and Listeria monocytogenes ATCC19115, streptococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, intestinal bacteria ATCC25922, shigella dysenteriae NICPBP51252, yersinia entero-colitica CMCC52221, Klebsiella pneumonia CMCC46102, DNA sequence dna is not all measured in Vibrio parahemolyticus ATCC33847 8 strain contrast strains.Sa, SE, ST sequenced dna base sequence respectively with table 3, Fig. 4, DNA sequence dna comparison in Fig. 5 after sequencing primer, find that the DNA base number that comparison result fits like a glove is respectively 31bp-36bp, 31bp-39bp, 34bp-36bp, all sequencing results all exceed 30 bases, the specificity that shows 3 sequencing primers is all better, and the GCCTGTGGGT A CTTCTCCTG CCAGTATAAT after available sequencing primer sequence, CCTGTTGTCTGCTCACCATTCGCCAGCCAC and TACAACCGGA GTGCACATTA AT CCCGCAGC base sequence detect Sa specifically, SE and ST.
Tetra-sodium order-checking be sequencing primer 3 first base after end start order-checking, discovery while repeatedly comparison with the BLAST function of NCBI, 30 DNA base sequences after Sa aceA, SE and ST sequencing primer can be identified respectively Sa, SE and ST.Judgment principle is: base sequence is that GCCTGTGGGTACTTCTCCTGCCAGTATAAT is judged as Sa, and base sequence is CCTGTTGTCTGCTCACCATTCGCCAGCCAC and TACAACCGGA GTGCACATTA ATCCCGCAGC, is judged as SE and ST.
Table 3: Salmonellas order-checking detected result
Table 4:SE tetra-sodium sequencing result
Table 5:ST tetra-sodium sequencing result
2.3 analog sample tetra-sodium order-checking detected results, add SE ATCC13076, the pork of ST ATCC14028, shrimp samples, use respectively the PCR primer for Salmonellas: 5 '-TCACCGAAAGACCAACAGAA-3 '; 5 '-GGTGGAACTCGCTGAAATGA-3 '; Sequencing primer: 5 '-CGAAAGACCAACAGAAG-3 '.PCR primer for Salmonella enteritidis: 5 '-GCATGTTCTGGAAAGCCTCTTTAT-3 '; 5 '-TCGTTCTTCTGGTACTTACGATGA-3 '; Sequencing primer: 5 '-AAAAAGGTTTAGTAAATCAG-3 '.PCR primer for Salmonella typhimurium: 5 '-AAGCTAAGTGTGGGGGCTAGTAT-3 '; 5 '-GGGTTTGTTTTTGATGATACAGG-3 '; Sequencing primer: 5 '-AGTATTGATAAATAATGGTT-3 '.Carry out according to the method described above respectively the reaction of PCR-tetra-sodium.Result shows, the sample (pork and shrimp samples) that adds SE ATCC13076 amplifies CCTGTTGTCT GCTCACCATT CGCCAGCCAC CACCTCGAG(for SE) and GCCTGTGGGT ACTTCTCCTG CCAGTATAAT TTCAGG(for Sa), the sample (pork and shrimp samples) of interpolation ST 14028 amplifies TACAACCGGA GTGCACATTA ATCCCGCAGC GTAAAGAC(for ST) and GCCTGTGGGT ACTTCTCCTG CCAGTATAAT TTCAGG(for Sa).As can be seen here, in sample, detect the DNA base sequence of SE in line and ST, be CCTGTTGTCTGCTCACCATTCGCCAGCCAC and TACAACCGGA GTGCACATTA AT CC CGCAGC, in 4 duplicate samples, also detect Sa aceA gene DNA base sequence, GCCTGTGGGT ACTTCTCCTG CCAGTATAAT simultaneously.The analog sample detecting according to GB4789.4-2010 " inspection of food microbiological analysis Salmonellas ", all separates and identifies the SE, the ST that conform to the bacterial strain adding, and result is consistent with the result of PCR-tetra-sodium method of the present invention.
Claims (7)
1. a detection primer sets compound for Salmonellas, Salmonella enteritidis and Salmonella typhimurium PCR-tetra-sodium method, is characterized in that comprising PCR primer and sequencing primer:
PCR primer for Salmonellas:
5’-TCACCGAAAGACCAACAGAA-3’；
5’-GGTGGAACTCGCTGAAATGA-3’；
Sequencing primer: 5 '-CGAAAGACCAACAGAAG-3 ';
PCR primer for Salmonella enteritidis:
5’-GCATGTTCTGGAAAGCCTCTTTAT-3’；
5’-TCGTTCTTCTGGTACTTACGATGA-3’；
Sequencing primer: 5 '-AAAAAGGTTTAGTAAATCAG-3 ';
PCR primer for Salmonella typhimurium:
5’-AAGCTAAGTGTGGGGGCTAGTAT-3’；
5’-GGGTTTGTTTTTGATGATACAGG-3’；
Sequencing primer: 5 '-AGTATTGATAAATAATGGTT-3 '.
2. the detection kit of a Salmonellas, Salmonella enteritidis and Salmonella typhimurium PCR-tetra-sodium method, comprise DNA extraction reagent, PCR reaction reagent, single-stranded template is prepared reagent, tetra-sodium sequencing reagent, PCR primer and sequencing primer, is characterized in that, described PCR primer and sequencing primer are:
PCR primer for Salmonellas:
5’-TCACCGAAAGACCAACAGAA-3’；
5’-GGTGGAACTCGCTGAAATGA-3’；
Sequencing primer: 5 '-CGAAAGACCAACAGAAG-3 ';
PCR primer for Salmonella enteritidis:
5’-GCATGTTCTGGAAAGCCTCTTTAT-3’；
5’-TCGTTCTTCTGGTACTTACGATGA-3’；
Sequencing primer: 5 '-AAAAAGGTTTAGTAAATCAG-3 ';
PCR primer for Salmonella typhimurium:
5’-AAGCTAAGTGTGGGGGCTAGTAT-3’；
5’-GGGTTTGTTTTTGATGATACAGG-3’；
Sequencing primer: 5 '-AGTATTGATAAATAATGGTT-3 '.
3. a detection method for the Salmonellas of non-diagnostic purpose, Salmonella enteritidis and Salmonella typhimurium, is characterized in that, comprises the following steps:
(1) sample is increased to bacterium and cultivate, extract the genomic dna of thalline in enrichment liquid as template;
(2) right to use requires the PCR primer described in 1 to carry out respectively PCR reaction respectively, reclaim PCR product, prepare strand masterplate, then application rights requires the sequencing primer described in 1 to carry out tetra-sodium order-checking to corresponding PCR product, first base after sequencing primer 3 ends starts order-checking, when current 30 base sequences are GCCTGTGGGTACTTCTCC TGCCAGTATAAT, in judgement sample, contain Salmonellas, when current 30 base sequences are CCTGTTGTC TGCTCACCATTCGCCAGCCAC, in judgement sample, contain Salmonella enteritidis, when current 30 base sequences are TACAACCGGAGTGCACATTAATCCCGCAGC, in judgement sample, contain Salmonella typhimurium.
4. detection method according to claim 3, is characterized in that, described sample is food.
5. detection method according to claim 4, is characterized in that, described food is meat product.
6. detection method according to claim 5, is characterized in that, described meat product is pork.
7. detection method according to claim 3, is characterized in that, described PCR reaction, and its amplification system is 50 μ L, PCR Mix Buffer25 μ l, Taq enzyme 5 μ l, MgCl
21 μ l, the each 2 μ l of 10 μ M upstream and downstream primer, DNA profiling 1 μ l, deionized water 14 μ l, PCR reaction conditions is 95 DEG C of 5min of denaturation; 95 DEG C of 15s, 65 DEG C of 30s,, 72 DEG C of 30s, 45 circulations; 72 DEG C of 12min.
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