CN101705307B - PCR detection method of Salmonella typhimurium, nucleic acid and primer pair thereof - Google Patents
PCR detection method of Salmonella typhimurium, nucleic acid and primer pair thereof Download PDFInfo
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- CN101705307B CN101705307B CN2009103112281A CN200910311228A CN101705307B CN 101705307 B CN101705307 B CN 101705307B CN 2009103112281 A CN2009103112281 A CN 2009103112281A CN 200910311228 A CN200910311228 A CN 200910311228A CN 101705307 B CN101705307 B CN 101705307B
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Abstract
The invention relates to a PCR detection method of Salmonella typhimurium, nucleic acid and a primer pair thereof, belonging to the technical field of the safety inspection of foods; the detection method comprises the following steps: designing an amplification primer according to a conserved sequence SEQ ID NO:1 in the genome DNA sequence of the Salmonella typhimurium; extracting a sample DNA and amplifying by a PCR method; and detecting an amplified product by gel electrophoresis and judging whether the sample contains the Salmonella typhimurium or not; the judgment comprises the following concrete steps: if a corresponding single-amplification strip occurs in an electrophoresis result, showing that the sample contains the Salmonella typhimurium; if the corresponding amplification strip does not occur, showing that the sample does not contain the Salmonella typhimurium; the invention also relates to the nucleic acid of which the base sequence is shown as SEQ ID NO:1; and the invention also relates to the primer pair, in particular to the nucleic acid of which the base sequence is shown as SEQ ID NO:2 and SEQ ID NO:3. By adopting the detection method to detect the Salmonella typhimurium, the detection time is short, the cost is low, the detection result is specific, and the result judgment is simple.
Description
Technical field
The PCR detection method and wherein nucleic acid and the primer that the present invention relates to a kind of food safety inspection technology field are right, specifically are that a kind of Salmonella typhimurium PCR detection method nucleic acid and the primer that reach wherein are right.
Background technology
Salmonella typhimurium (Salmonella Typhimurium) is isolated on one's body from mouse by Loffler in 1892, is one of modal Salmonella infection serotype.Salmonella typhimurium belongs to the B crowd of salmonella, and its biological property is similar with Corynebacterium diphtheriae, is Gram-negative bacteria, does not form the brood cell, no pod membrane, but amphitrichous.This bacterium has formed three mutation during evolution, i.e. Copenhagen mutation, guest's Si mutation and the mutation of O type.The Salmonella typhimurium resistibility of environment to external world is stronger, can breed rapidly under the normal temperature, in low temperature, exsiccant environment, can survive, but thermo-labile, but the high temperature deactivation is responsive to acid, ultraviolet ray and various chemostefilant.Salmonella typhimurium is wider in distributed in nature, contains this bacterium in the enteron aisle of many poultry, domestic animal, muroid and wild birds.These animals become main contagium with the crowd of carrying disease germs, and fly, flea are its communication medias.Clinical, Salmonella typhimurium infection pilosity is born in the infant, and main illness is for generating heat, feel sick, vomit and diarrhoea.The detection of Salmonella typhimurium mainly is to rely on traditional cultured method (can referring to GB GB/T4789.4-2008), mentions in the 56th～57 page of being entitled as in " separation of egression Salmonella typhimurium and mirror " literary composition of delivering of " special product research " the 1st phase in 2007 that as Liu Zhen Hunan these method need cultivated through selective enrichment; And test through biochemical reaction and serology and to identify; Though reliable results, complicated operation is wasted time and energy; Usually take 3～5 days and just can draw detected result; Be unfavorable in time diagnosing the cause of disease, search pathogeny, the spreading of disease controlling.For check Salmonella typhimurium that can be quick, accurate, easy, be that the detection method on basis constantly makes further progress in practice test with molecular biology, become one of detection method that replaces the tool potentiality of traditional detection method.Characteristics such as round pcr is easy and simple to handle, quick, highly sensitive with it, high specificity progressively are applied to after the quarantine of Salmonellas, and DIAGNOSIS OF SALMONELLOSIS IN and preventing and controlling have been played active effect.But it is only few to the PCR method of Salmonella typhimurium serotype detection; The specificity that detects target spot is not strong; Like Lim YH; Hirose K; Izumiya H etc. are in the 151st～155 page of being entitled as described in " Multiplex polymerase chain reactionassay for selective detection of Salmonella enterica serovar typhimurium " (multiple PCR method detects the selectivity of Salmonella typhimurium) literary composition of delivering of " Japanese Journal of Infectious Diseases " (Japanese transmissible disease magazine) 2003 the 56th the 4th phases of volume; According to the serotype O4:Hi:1 of Salmonella typhimurium, 2 and adopt the antigenic flagellin gene of corresponding encoded H? IC with? JB and O antigen gene rfbJ form multiplex PCR and detect Salmonella typhimurium, have increased detection of complex property; Interference between the primer influences the raising of detection sensitivity easily, and the bacterial strain that serotype is close can bring difficulty to the judgement of net result.
Literature search through to prior art is found, not invention and Salmonella typhimurium serotype PCR detection method of the present invention, nucleic acid and the relevant report of primer as yet.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide a kind of Salmonella typhimurium PCR detection method and wherein nucleic acid and primer right.Adopt detection method of the present invention to detect Salmonella typhimurium, detection time is short, and cost is low, has practicality more, and detected result is special, and the result judges simply.
The present invention realizes through following technical scheme,
The present invention relates to a kind of Salmonella typhimurium PCR detection method, comprise the steps:
In the step 1, said primer is specially: the sequence of forward primer is shown in SEQ ID NO:2, and the sequence of reverse primer is shown in SEQ ID NO:3.
In the step 2, in the said PCR method, the PCR detection architecture 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 dNTP 1.0 μ L of 2.5mmol/L, 5 μ M primers be to 1 μ L, Taq enzyme 1U, and template solution is got 2～5 μ L, last moisturizing to 25 μ L.
In the step 2, in the said PCR method, PCR detection architecture amplification parameter is specially: 94 ℃ of preparatory sex change 5min of elder generation, begin amplification cycles afterwards, and each round-robin program is: 94 ℃ of sex change 30s, 65 ℃ of annealing 30s, 72 ℃ are extended 30s; Totally 35 of circulations; After the loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
In the step 3, said judgement is specially: detect the detected through gel electrophoresis amplified production and whether have single amplified band in the 915bp position, if exist, then contain Salmonella typhimurium in the interpret sample; If no, then do not contain Salmonella typhimurium in the sample.
The invention still further relates to the nucleic acid that relates in a kind of said PCR detection method, this nucleic acid base sequence shown in SEQ IDNO:1.
It is right to the invention still further relates to the primer that relates in a kind of said PCR detection method, and this primer is to being specially base sequence respectively shown in SEQ ID NO:2 and the nucleic acid shown in SEQ ID NO:3.
Compared with prior art, the present invention has following beneficial effect: adopt detection method of the present invention to detect Salmonella typhimurium, detection time is short, owing to need not adopt the antiserum(antisera) that must use in the traditional detection method, has reduced the detection cost; Simultaneously, detection method of the present invention also can be used to detect some antiserum(antisera) can not detected bacterial strain, like the bacterial strain that antigen is not expressed, remedies the defective of immunodetection, has practicality more; Detection target spot of the present invention has single specificity, and detected result is special, and the result judges simply.
Description of drawings
Fig. 1 is PCR detection method specificity evaluation experimental gel electrophoresis figure as a result among the embodiment 1;
Fig. 2 is PCR detection method sensitivity evaluation experimental gel electrophoresis figure as a result among the embodiment 1.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; For example the 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.
The foundation of Salmonella typhimurium serotype PCR detection method
From the Salmonella typhimurium genomic dna sequence, find special STM4495 gene, with it detection target gene as Salmonella typhimurium, gene order is shown in SEQ ID NO:1;
The dna sequence dna of restriction endonuclease gene is input among the primer-design software Primer Premier 5.0 designs primer; It is 40～60% that the GC% scope is set; The product magnitude range is 100～1000bp; Select primer from alternative primer centering, primer sequence is (primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) as follows:
STM4-L：5’-GGTGGCAAGGGAATGAA-3’(SEQ?ID?NO：2)；
STM4-R：5’-CGCAGCGTAAAGCAACT-3’(SEQ?ID?NO：3)；
The Salmonellas bacterial strain (as shown in table 1) of various serotypes such as Salmonella typhimurium, Salmonella enteritidis and Salmonella choleraesuls is connect bacterium respectively to the LB liquid nutrient medium of 50mL, 37 ℃ increase bacterium 8h after, get 1mL bacterium liquid, put into the 1.5mL centrifuge tube;
3, the centrifugal 10min of 000r/min gets supernatant afterwards, and again 12, the centrifugal 5min of 000r/min collects thalline.With the aseptic double-distilled water thalline that suspends again, add the aseptic ultrapure water of 100 μ L behind the centrifuge washing, in boiling water bath, boil 15min, take out immediately, place 30min at-20 ℃.37 ℃ thaw after, 12, the centrifugal 5min of 000r/min, get supernatant place-20 ℃ subsequent use.
The PCR detection method is following: add 16.1 μ L sterilized waters earlier in reaction tubes, add 10 * PCR reaction buffer, 2.5 μ L more successively, the Mg of 25mmol/L
2+2.0 μ L, the dNTP 1.0 μ L of 2.5mmol/L, 5 μ M primers, 1.0 μ L, 2.5U/ μ L Taq enzyme 0.4 μ L adds template solution 2 μ L at last, and is the negative control of template as reaction with the sterilized water.Then with reaction tubes centrifugal after, put into PCR reaction appearance, carry out according to following PCR loop parameter: at 94 ℃ of preparatory sex change 5min; Then do 35 circulations, each round-robin program comprises 94 ℃ of sex change 30s, 65 ℃ of annealing temperatures; Annealing time is 30s, extends 30s at 72 ℃ then, extends 10min at 72 ℃ after the loop ends; Be cooled to 12 ℃ at last, finish all operations program.
It is (as shown in table 1 to get Salmonellas reference cultures such as 16 strain Salmonella typhimuriums, Salmonella enteritidis and Salmonella choleraesuls and 39 strain isolated strains; Bacterial strain shown in the table is that those skilled in the art can obtain through disclosed channel); According to the dna profiling process for extracting, extract genomic dna respectively.The dna solution of every strain bacterial strain is all got 2 μ L and is added to as the PCR reaction template and carry out amplified reaction in the PCR reaction system.
Shown in Figure 1 is the detected result of reference culture, and its serotype and strain number see also accompanying drawing and subordinate list explanation.Among the figure: swimming lane 1～16:AS1.1174, ATCC14028, ATCC13311, ATCC51812, CMCC50335, CMCC50770; AS1.1190, ATCC9150, CMCC50004, ATCC13076, ATCC15611; ATCC9270, NCTC4840, ATCC12002, CMCC50017, NCTC6017; Swimming lane 17:ddH2O; Swimming lane M:200bp molecular weight standard.From Fig. 1, can know, except Salmonella typhimurium reference culture AS1.1174, ATCC14028, ATCC13311, outside the ATCC51812, all the other serotypes all do not have 915bp specific amplified band.Table 1 is the PCR qualification result of 16 strain reference cultures and 39 strain isolated strains, from table 1, can know, except the reference culture AS1.1174 of Salmonella typhimurium; ATCC14028; ATCC13311, outside ATCC51812 and the strain isolated, all the other Salmonellass all do not have the specific amplified band.
In the table 1 ,-: PCR result is negative; +: PCR result is positive.
Table 1 specificity is estimated bacterial strain uses therefor and test-results
Through measuring, the concentration of the total dna solution of Salmonella typhimurium is 65.7 μ g/mL, makes 10 times of gradient dilutions with sterilized water, dilutes 10 gradients altogether; Each gradient is got 5 μ L respectively and is added the PCR reaction system, and the detected through gel electrophoresis amplified production is observed the gel electrophoresis result in the gel imaging appearance, as shown in Figure 2; Among the figure: swimming lane 1～10:DNA template concentrations is respectively 32.85ng, 3.285ng, 328.5pg, 32.85pg; 3.285pg, 328.5fg, 32.85fg; 3.285fg, 0.3285fg, 0.033fg/PCR; Swimming lane M:200bp molecular weight standard.Can know that by Fig. 2 can see band clearly at the 7th swimming lane, institute's corresponding DNA concentration is 32.85fg/PCR, and can't see amplified band after the eight lanes.Therefore, judge that the PCR detection sensitivity is 32.85fg/PCR, have higher sensitivity.
The detection of the doubtful bacterial strain of Salmonella typhimurium
Utilize the PCR detection method of the Salmonella typhimurium of embodiment 1 foundation; Detected 61 strains doubtful bacterial strain of isolating Salmonellas from food samples; Food samples is collected in the 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/T 4789.4-2008.
Therefrom detect the doubtful bacterial strain of the 9 strains result that is positive; The doubtful bacterial strain of this 9 strain identifies it is O4:Hi:5 through salmonella diagnostic serum (Lanzhou Institute of Biological Products); Determine that it is Salmonella typhimurium (the serum authentication step sees also product description and GB GB/T 4789.4-2008), other doubtful bacterial strains are not Salmonella typhimuriums through identifying.Prove that through present embodiment the PCR detection method of Salmonella typhimurium has extreme high reliability.
Sequence table
< 110>Shanghai Communications University
< 120>Salmonella typhimurium PCR detection method and wherein nucleic acid and primer are right
<160>3
<170>PatentIn?version?3.3
<210>1
<211>3678
<212>DNA
< 213>Salmonella typhimurium
<400>1
atgaatacca?ataacatcaa?aaaatacgct?ccacaggccc?gtaaccagtt?ccgcgatgcg 60
gtgatccaaa?agctaaccac?gctggggatt?tccgctgata?aaaaaggcaa?tctgcaaatt 120
gcggatgccg?agctcgtcgg?cgaaaccatg?cgctatggtc?agttcgacta?ccccaaatcc 180
accctcaccc?gccgcgatcg?tctggtaaaa?cgcgcccgcg?agcagggctt?tgacgtgctg 240
gttgagcact?gtgcctacac?ctggttcaac?cgcctgtgcg?ccattcgtta?tatggaaatc 300
cacggttatc?ttgaccacgg?cttccacatg?ctctcgcacc?cggataaccc?gacaggcttt 360
gaagtgctgg?accacgtacc?ggaagtcgcg?gaagcattac?taccagagaa?aaaggcgcag 420
ctggtcgaga?tgaagctttc?cggcaaccag?gacgaagcca?tctaccgtga?actgctgctg 480
gcccagtgcc?acgccctgca?ccgcgcgatg?ccgttcctgt?ttgaagctgt?ggacgatgaa 540
gctgaactac?tgctgccgga?taacctgacc?cgcaccgact?ccattctacg?cggtctggtg 600
gacggtattc?cggaagaaga?ctggcaagaa?gttgaggtta?tcggctggct?gtatcagttc 660
tatatctctg?agaaaaaaga?tgcggttatc?ggtaaggtgg?tgaagagcga?agatattcct 720
gccgccaccc?agctgtttac?cccaaactgg?attgtgcagt?atctggtaca?gaactccgtc 780
ggccgccagt?ggttgcagac?ctacccggat?tcgccgctga?aaggcaaaat?ggactactac 840
attgagccag?ccgaacagac?gccagaagtg?caggcgcagc?tggccgccat?tacacctgcc 900
agtattgaac?cggaaagcat?caaagtactc?gacccggcct?gcggctccgg?gcatattctg 960
attgaagtct?ataatgtgct?gaaaaatatc?tatgaagagc?gcggctatcg?cgcccgcgat 1020
attccacagc?taattctgga?aaataatatt?tttggtctcg?acattgacga?ccgtgctgcc 1080
cagctttccg?gctttgcctt?attaatgatg?gcccgtcagg?atgaccgccg?gatattcacc 1140
cgcgacgtgc?gtctgaatat?tgtctccctg?caggagagcc?tgcatctgga?tattgctaag 1200
ctgtggcagc?agctgaactt?ccaccagcag?aaccagactg?gtagcatggg?ggatatgttt 1260
gccgaaaata?cagcattagc?ccataccgac?agcgcagaat?atcagctgct?gatgcgcacg 1320
ctgaagcgct?ttgtgaacgc?caaaacgctg?ggctcgctga?tccaggtgcc?acaggaagaa 1380
gaggcggaac?tgaaggcgtt?tctcgaagcg?ctatatcgca?tggagcagga?aggcgatttc 1440
cagcagaagg?cagcggcgaa?agcgtttatt?ccgtatattc?agcaggcgtg?gatcctggcg 1500
cagcggtatg?atgcggtagt?ggcgaatccg?ccgtatatgg?gtggcaaggg?aatgaatagt 1560
gagctgaaag?agtttgccaa?aaataacttc?ccggatagta?aagctgattt?gtttgcaatg 1620
tttatgcaga?atgcattttc?tttgcttaaa?gaaaatgggt?ttaatgctca?agtcaatatg 1680
caatcatgga?tgtttttgtc?aagttatgaa?gcactacgta?actggttatt?ggacaataaa 1740
acatttatta?cgatggcaca?tttgggagct?cgggcttttg?ggcaaatttc?tggagaggtt 1800
gtacagacaa?ctgcctgggt?gattaaaaac?caacactccg?aacgttacca?acctgtattt 1860
tttagactta?tagatggtag?ggaagaagta?aagaaaagcg?atctacttct?aaggaaaaat 1920
atatttgata?aatttacaca?gcatgatttt?aaaaacatac?caggaatgcc?aatagcatat 1980
tggatagact?taccgagtct?attatctttt?cgccaccata?aaaaacttgg?agaaaaaata 2040
gcattaaaag?caggcatgtc?caccggtgac?aatattaaat?ttcaaagata?ttggtacgag 2100
gtttcaataa?aaaaaaccct?tatcacaaat?aaagaatcaa?atacaaaaat?cgacattcat 2160
aatatcaaat?ggtttccttg?tagtagtgga?ggtgaatatc?gaaagtggta?tggtaataac 2220
gaaatagttg?taaattggga?aaataatggt?tacgaaatac?gaaattttaa?atttgagaat 2280
ggcaaaactc?gctctgccgt?aagaaatgat?gagtattact?ttagagaagg?cataacatgg 2340
tcaaaaataa?gccaaggtaa?tttttgcgtg?agatatcgtc?caaaagggtt?tgtttttgat 2400
gatacaggcc?gttgcggctt?ttcaaataac?aaaaatgagt?tgctttacgc?tgcgggatta 2460
atgtgcactc?cggttgtaaa?ccattattta?tcaatactag?cccccacact?tagctttact 2520
agtggtgaat?tagcctcagt?accatatcca?gaaattgaag?atgaaattat?cgaattagtc 2580
accaatgcta?ttgaaatagc?taaaaatgac?tgggactctc?aagagcaatc?atgggattat 2640
gtttgttcac?cattgcttga?acacaattca?actcaattgc?tacggaatat?ctacaaacaa 2700
aaaatcaata?caaatatcaa?gttagtcgaa?acacttctcc?taatagaaaa?tactataaat 2760
aatattttta?tagataaatt?acaacttgac?aaaactataa?ttaaagctgt?attgcaaagt 2820
gaaataactc?tactatgtaa?tccaaactat?cgatataaaa?atattcaaga?tcataccgat 2880
ctaaccaata?agtattacac?tgatatcact?atagatatct?taagctacat?catcggctgc 2940
atgatgggcc?gctactccct?cgatcgcgaa?ggactggtct?atgcccatga?aggcaataaa 3000
ggctttgccg?aactggtcgc?tgaagacgcg?tacaaaacct?tcccggctga?caatgacggt 3060
atcctgccgc?tgatggatga?cgagtggttt?gacgatgacg?ttacctctcg?cgtcaaagag 3120
tttgttcgca?ccgtttgggg?cgaagaacac?ttgcaggaaa?atctcgaatt?tatcgccgaa 3180
agcctctgtt?tatacgcgat?aaaaccaaaa?aaaggcgaat?ctgcgctgga?taccatccgc 3240
cgctatcttt?ccactcagtt?ctggaaagat?catatgaaga?tgtataaaaa?gcgtccgata 3300
tactggctgt?tcagctccgg?taaagagaaa?gcgtttgagt?gcctggtcta?tctgcatcgc 3360
tacaacgatg?cgacgctggc?gagaatgcgt?accgaatatg?tagtgccgct?gctggcgcgc 3420
tatcaggcca?atatcgatcg?cctgaacgaa?caggtcgatg?gagcctcagg?cggcgaagcc 3480
acccgcctga?aacgtgagcg?tgatagcctg?agcaaaaaat?tcaacgaact?gcgcagcttc 3540
gacgatcgcc?tgcgccacta?tgctgatatg?agaatcagta?ttgatctcga?cgacggcgtt 3600
aaggttaact?acggtaagtt?tggcgatctg?ctggcggatg?tgaaagccat?taccggcaat 3660
gcaccggaga?ttatttaa 3678
<210>2
<211>17
<212>DNA
< 213>artificial sequence
<400>2
ggtggcaagg?gaatgaa
<210>3
<211>17
<212>DNA
< 213>artificial sequence
<400>3
cgcagcgtaa?agcaact
Claims (4)
1. a non-diagnostic purpose Salmonella typhimurium PCR detection method is characterized in that, comprises the steps:
Step 1 is according to the conserved sequence SEQ IDNO:1 design of amplification primers in the genomic dna sequence of Salmonella typhimurium;
Said primer is specially: the sequence of forward primer is shown in SEQ ID NO:2, and the sequence of reverse primer is shown in SEQ ID NO:3;
Step 2 is extracted sample DNA, the amplification of PCR method;
Step 3, the detected through gel electrophoresis amplified production, whether judgement sample contains Salmonella typhimurium;
Said judgement is specially: detect the detected through gel electrophoresis amplified production and whether have single amplified band in the 915bp position, if exist, then contain Salmonella typhimurium in the interpret sample; If no, then do not contain Salmonella typhimurium in the sample.
2. non-diagnostic purpose Salmonella typhimurium PCR detection method according to claim 1 is characterized in that, in the step 2; In the said PCR method; The PCR detection architecture 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 dNTP1.0 μ L of 2.5mmol/L, 5 μ M primers be to 1 μ L, Taq enzyme 1U, and template solution is got 2～5 μ L, last moisturizing to 25 μ L.
3. non-diagnostic purpose Salmonella typhimurium PCR detection method according to claim 1 is characterized in that, in the step 2; In the said PCR method; PCR detection architecture amplification parameter is specially: 94 ℃ of preparatory sex change 5min of elder generation, begin amplification cycles afterwards, and each round-robin program is: 94 ℃ of sex change 30s; 65 ℃ of annealing 30s, 72 ℃ are extended 30s; Totally 35 of circulations; After the loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
4. a primer that is used for the described Salmonella typhimurium PCR of claim 1 detection method is right, it is characterized in that, this primer is to being specially the base sequence nucleic acid shown in SEQ ID NO:2 and shown in SEQ IDNO:3 respectively.
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| US6251607B1 (en) * | 1999-12-09 | 2001-06-26 | National Science Council Of Republic Of China | PCR primers for the rapid and specific detection of Salmonella typhimurium |
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