CN109762914A - HRM serotype method, target gene, specificity amplification primer and the kit of salmonella food-borne pathogens - Google Patents
HRM serotype method, target gene, specificity amplification primer and the kit of salmonella food-borne pathogens Download PDFInfo
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- CN109762914A CN109762914A CN201910078263.7A CN201910078263A CN109762914A CN 109762914 A CN109762914 A CN 109762914A CN 201910078263 A CN201910078263 A CN 201910078263A CN 109762914 A CN109762914 A CN 109762914A
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Abstract
The present invention relates to technical field of food safety detection more particularly to HRM serotype method, target gene, specificity amplification primer and the kits of salmonella food-borne pathogens.The HRM serotype method of salmonella food-borne pathogens, it is characterized in that, method includes the following steps: step 1, according to the ykgD gene order of salmonella, ykgD gene order designs specificity amplification primer pair as shown in SEQ 1, at the both ends of its saltation zone；Step 2 extracts sample DNA, is expanded using specificity amplification primer obtained by step 1 to using HRM method；Step 3, by whether containing salmonella in fluorescence curve judgement sample；Above-mentioned method is not related to treatment and the diagnostic method of disease.When carrying out serotype to Salmonella strains using classifying method of the invention, detection speed is fast, and as a result accurately and reliably, easy to operate, testing cost is low.
Description
Technical field
The present invention relates to technical field of food safety detection more particularly to the HRM of salmonella food-borne pathogens
Serotype method, target gene, specificity amplification primer and kit.
Background technique
Salmonella (Salmonella) it is food-borne pathogens mostly important in enterobacteriaceae, caused by salmonella
Poisoning case occupy the first place poisoned by food all over the world.The bacterium is the main pathogenic bacteria for causing food poisoning and typhoid fever,
It is often propagated with the food of pollution and water source, zoonosis can be caused.The plant type of salmonella is various, and the whole world has been at present
The serotype isolated has 2, and more than 500 kinds, 46 serum groups, in China, there are about 300 kinds of serotypes, adheres to 37 serum groups separately.No
Cross serotype related with human diseases and focus primarily upon A~E groups, wherein the pathogenic salmonella of A~D group accounts for 70%, especially with
Salmonella typhimurium and Bacterium enteritidis are most commonly seen.The division of Salmonella serogroup has the classification of bacterial strain with identification
Important meaning, for monitor with control pathogenic bacteria propagation also play an important role.
What the difference that the basic principle of HRM technology is mainly based upon nucleic acid molecules physical property developed.Different nucleic acid
Molecule GC content, GC distribution etc. is different, therefore double-stranded DNA molecule has oneself melting curve in heat denatured
Shape and position.The principle of HRM technology is exactly to distinguish sample according to the difference of nucleic acid molecules melting temperature and melting shape.
The fluorescent dye LC Green of saturation is introduced in PCR reaction, fluorescent dye is embedded in DNA double during PCR amplification
In chain.When being embedded with the DNA molecular unwinding of dyestuff, instrument is acquired fluorescence signal, draws out the melting of DNA molecular
Curve.The melting curve of DNA molecular and the G/C content of DNA molecular, GC distribution, the properties such as fragment length are related, different samples
Since the difference of base active force shows different melting curve shape and melting temperature (Tm value).
It is influenced due to receiving host and environment, some gene orders of the genome of salmonella different serotypes exist
Sequence difference, and these genes can be used for HRM parting.For example, foreign study person Zeinzinger et al. is according to salmonella
'sfljB、gyrBWithycfQGene establishes a set of HRM serotype method, and this method is published in " Applied and
Environmental Microbiology " magazine 3352-3360 pages of 09 phase of volume 78 in 2012, entitled " One-step
triplex high-resolution melting analysis for rapid identification and
simultaneous subtyping of frequently isolated Salmonella serovars".But this
Three genes that method usesfljB、gyrBWithycfQBe not the distinctive gene of salmonella, in other bacteriums there is also
The similar gene of DNA sequence dna.When therefore, using the method parting, needs first to determine that the bacterial strain of parting is salmonella, otherwise can
Other bacteriums are mistakenly considered to the serotype of a certain salmonella.Moreover, being not only easy by primer two using multiple PCR method
The interference of aggressiveness also will increase the probability of the mispairing of primer and cause parting mistake.
Summary of the invention
For overcome the deficiencies in the prior art, the first purpose of this invention is to provide salmonella food-borne pathogens
HRM serotype method, second object of the present invention is to provide the HRM serotype of salmonella food-borne pathogens
Target gene, third object of the present invention are to provide the HRM serotype specificity of salmonella food-borne pathogens
Amplimer, fourth object of the present invention are to provide the HRM serotype kit of salmonella food-borne pathogens.
Serotype is carried out to salmonella using classifying method of the invention, the parting time can be shortened, at low cost, result judgement letter
It is single, quick, accurate.
In order to realize first above-mentioned purpose, the present invention is achieved by the following technical solutions:
The HRM serotype method of salmonella food-borne pathogens, which is characterized in that method includes the following steps:
Step 1, according to the ykgD gene order of salmonella, ykgD gene order is as shown in SEQ 1, the two of its saltation zone
End design specificity amplification primer pair；
Step 2 extracts sample DNA, is expanded using specificity amplification primer obtained by step 1 to using HRM method；
Step 3, by whether containing salmonella in fluorescence curve judgement sample；
Above-mentioned method is not related to treatment and the diagnostic method of disease.
In step 1, specificity amplification primer is made of upstream primer and downstream primer, and the base sequence of upstream primer is such as
Shown in SEQ 8, the base sequence of downstream primer is as shown in SEQ 9.
In step 2, in the HRM method, HRM serotype system (20 μ L) specifically:
Take the MgCl of the 2*buffer and 2.4 μ L of 10 μ L2, upstream and downstream primer respectively takes 1 μ L, and DNA profiling takes 2 μ L, finally plus H2O
For 3.6 μ L.
In step 2, in the HRM method, HRM serotype system Amplification specifically:
94 DEG C of initial denaturation 5min start amplification cycles, the program of each circulation later are as follows: 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s,
72 DEG C of extension 30s；Circulation totally 35；After circulation terminates, 72 DEG C of extension 10min are cooled to 10 DEG C, terminate.
In order to realize second above-mentioned purpose, the present invention is achieved by the following technical solutions:
The HRM serotype target gene of salmonella food-borne pathogens, the base sequence of the target gene such as 1 institute of SEQ
Show.
In order to realize above-mentioned third purpose, the present invention is achieved by the following technical solutions:
The HRM serotype specificity amplification primer of salmonella food-borne pathogens, the specificity amplification primer is by upstream
Primer and downstream primer are constituted, and the base sequence of upstream primer is as shown in SEQ 8, the base sequence of downstream primer such as 9 institute of SEQ
Show.
In order to realize the 4th above-mentioned purpose, the present invention is achieved by the following technical solutions:
The HRM serotype kit of salmonella food-borne pathogens, the kit include DNA profiling, specific amplification
Primer, 2*buffer and MgCl2, the specific amplification primers are made of upstream primer and downstream primer, the alkali of upstream primer
Basic sequence is as shown in SEQ 8, and the base sequence of downstream primer is as shown in SEQ 9.
Compared with prior art, the present invention is with following the utility model has the advantages that using classifying method of the invention to Salmonella
When bacteria strain carries out serotype, detection speed is fast, and as a result accurately and reliably, easy to operate, testing cost is low；Detection of the invention
Method can be used for the detection of food samples；Target dna sequence of the invention is voluntarily by comparing genomics, biological information
It learns and screens and by there is single specificity, testing result is reliable, and result judgement is simple obtained from biometric authentication.
Detailed description of the invention
Fig. 1 is the genotyping result of 7 plants of salmonella reference cultures of bcfC primer pair.
Fig. 2 is the genotyping result of 7 plants of salmonella reference cultures of ykgC primer pair.
Fig. 3 is the genotyping result of 7 plants of salmonella reference cultures of ykgD primer pair.
Fig. 4 is the genotyping result of salmonella typhimurium ATCC14028 in food.
Specific embodiment
Embodiment 1:
Step 1, according to salmonellabcfC、ykgC、ykgDGene order design primer
By comparing genome and bioinformatic analysis, distinctive gene sequence is found in salmonella gene group DNA sequence dna
ColumnbcfC、ykgCWithykgD, using it as the parting target spot of salmonella (as shown in SEQ 1, SEQ 2 and SEQ 3).
The nucleic acid base sequence of these genes all having a certain difference property in different Salmonella serogroups；
This DNA nucleic acid sequence is input to design primer in primer-design software Primer Premier 5.0, upstream and downstream primer
Sequence is located at the both ends in gene mutation area, and sequence is following (primer is by Dalian treasured bioengineering Co., Ltd):
bcfCUpstream primer: 5 '-GCAGAATGCGGGCCTTAG -3 ', (SEQ 4)
bcfCDownstream primer: 5 '-CGCGCGTCCGTATGAAT -3 ', (SEQ 5)
ykgCUpstream primer: 5 '-GAACAGTCCGCCAGTATG -3 ', (SEQ 6)
ykgCDownstream primer: 5 '-GCAGGTAATCATCCACGAT -3 ', (SEQ 7)
ykgDUpstream primer: 5 '-GATTCGCTCGCATAACCTACC -3 ', (SEQ 8)
ykgDDownstream primer: 5 '-GTGATTCGCTGGCATACCGT -3 ', (SEQ 9).
Step 2, the system and response parameter of HRM serotype method
HRM reaction system (20 μ L) are as follows: take the MgCl of the 2*buffer and 2.4 μ L of 10 μ L2, upstream and downstream primer respectively takes 1 μ L, DNA
Template takes 2 μ L, finally plus H2O is 3.6 μ L.
Then it after reaction tube being centrifuged, is put into PCR instrument, is carried out according to following parameter:
94 DEG C of initial denaturation 5min start amplification cycles, the program of each circulation later are as follows: 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s,
72 DEG C of extension 30s；Circulation totally 35；After circulation terminates, 72 DEG C of extension 10min are cooled to 10 DEG C, terminate.
Step 3, parting
Salmonella typhimurtum ATCC14028, Bacterium enteritidis ATCC13076, Vylor salmonella ATCC15611, A type
Bacillus paratyphosus CMCC50093, Salmonella meleagridis CICC21511, salmonella dublin CICC21497, love Dare fort are husky
The DNA solution of door Salmonella CICC21487 takes 2 μ L to be added in HRM system as reaction template and carries out amplification reaction.It extracts
Process is as follows: above 7 plants of reference cultures being respectively connected in the LB liquid medium of 50 mL, after 37 DEG C of 8 h of increasing bacterium, are taken
1mL bacterium solution is put into 1.5 mL centrifuge tubes；10 min are centrifuged in 3,000 r/min later, take supernatant, then 12,000
R/min is centrifuged 5 min, collects thallus.It is sterile ultrapure that 100 μ L are added with aseptic double-distilled water again suspension thalline, after centrifuge washing
Water boils 15 min in boiling water bath, takes out immediately, in -20 DEG C of 30 min of placement.37 DEG C thaw after, 12,000 r/min from
5 min of the heart takes -20 DEG C of supernatant placement spare.
As shown in Figure 1,bcfCIn the genotyping result of 7 plants of salmonella reference cultures of primer pair of gene, HRM curve will
Bacterial strain, which is divided into, is roughly divided into two types, and wherein the HRM curve of A6, B6, C6, D6, E6, F6 type strain is more similar, therefore
It is difficult to distinguish 7 kinds of common serological type strains by these curves.The number information of bacterial strain uses therefor refers to attached drawing and says
It is bright.
In Fig. 1 the template of HRM curve be respectively as follows: Salmonella typhimurtum ATCC14028, Bacterium enteritidis ATCC13076,
Vylor salmonella ATCC15611, paratyphosus A bacillus CMCC50093, Salmonella meleagridis CICC21511, Dublin
Salmonella CICC21497 and love Dare fort salmonella CICC21487.
As shown in Fig. 2,ykgCIn the genotyping result of 7 plants of salmonella reference cultures of primer pair of gene, HRM curve will
Bacterial strain is divided into three types, wherein has the HRM type of two plants of bacterium much like in A7, B7, D7, G7, therefore also not by these curves
This 7 kinds of common serological type strains can clearly be distinguished.The number information of bacterial strain uses therefor refers to Detailed description of the invention.
In Fig. 2 the template of HRM curve be respectively as follows: Salmonella typhimurtum ATCC14028, Bacterium enteritidis ATCC13076,
Vylor salmonella ATCC15611, paratyphosus A bacillus CMCC50093, Salmonella meleagridis CICC21511, Dublin
Salmonella CICC21497 and love Dare fort salmonella CICC21487.
As shown in figure 3,ykgDIn the genotyping result of 7 plants of salmonella reference cultures of primer pair of gene, every kind of serum
The HRM curve of type bacterial strain is different from, therefore can be distinguished by these curves to 7 kinds of common serological type strains.Institute
Detailed description of the invention is referred to the number information of bacterial strain.
In Fig. 3 the template of HRM curve be respectively as follows: Salmonella typhimurtum ATCC14028, Bacterium enteritidis ATCC13076,
Vylor salmonella ATCC15611, paratyphosus A bacillus CMCC50093, Salmonella meleagridis CICC21511, Dublin
Salmonella CICC21497 and love Dare fort salmonella CICC21487.
Step 4 establishes HRM classifying method
From genotyping result it is found that third to primer (ykgDGene primer) 7 kinds of Salmonella serogroups can be distinguished significantly,
Therefore, may be selected withykgDGene is parting target gene, with following primer:
Upstream primer: 5 '-GATTCGCTCGCATAACCTACC -3 ', (SEQ 8)
Downstream primer: 5 '-GTGATTCGCTGGCATACCGT -3 ', (SEQ 9)
The HRM classifying method of foundation can Salmonella typhimurtum, Bacterium enteritidis, Vylor's Salmonella to salmonella
Bacterium, paratyphosus A bacillus, Salmonella meleagridis, salmonella dublin and love Dare fort salmonella carry out parting.
Embodiment 2:
10 parts of milk samples are taken, salmonella typhimurium ATCC14028 is connect into bacterium into wherein 3 parts of samples, utilizes embodiment 1
Detection method carries out detection and parting to food samples.The results show that HRM serotype method not only can be with accurate detection
Salmonella typhimurium out, and it is consistent with the result figure of embodiment 1 (as shown in Figure 4) to strain typing result.
The above are the descriptions to the embodiment of the present invention to keep this field special by the foregoing description of the disclosed embodiments
Industry technical staff can be realized or using the present invention.Various modifications to these embodiments carry out those skilled in the art
Saying will be apparent.The general principles defined herein can be the case where not departing from the spirit or scope of the present invention
Under, it realizes in other embodiments.Therefore, the present invention is not intended to be limited to these implementation columns shown in this article, but to accord with
Close the widest scope consistent with principles disclosed herein and novel point.
Sequence table
<110>Zhejiang Entry-Exit Inspection and Quarantine Bureau
Xibei Univ. of Agricultural & Forest Science & Technology
<120>HRM serotype method, target gene, specificity amplification primer and the reagent of salmonella food-borne pathogens
Box
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2622
<212> DNA
<213>salmonella (Salmonella)
<400> 1
atgcgtaatc agcttttcat gacgcgatat tactccagcg tagctaaacc cgtattaact 60
ccgctggcgt tggctatcgc actggcgcct gcgcctgggt gggcggaaaa ctatttcaac 120
ccggcatttc tgtctgacga cccgtctgcg gtggccgact tatcgacctt ttcccgtaat 180
gcccaggcgg cgggaatgta tcgcgttgac gtttacctga acaatacgtt tctcgcgacc 240
agagacattg ccttccaggc ggtgaagacg acgggaaaaa gcgcgcccac cgatgacagc 300
ggattacgcg cctgcctgac gcctgaaatg cttaaaaata tgggggtaaa caccggggca 360
tttccactgt tggcgaaggc ggcggcggga agttgtccgg atctcgccag tgcgataccg 420
gccgcccgga cccgctttga ttttgcgcag caacgtctcg acattagcat cccgcaggcg 480
gcgatggttg ccagcgccag aggctatatc ccaccgcaat actgggatga aggtattaac 540
gcgttgctat tgaattacac ctttaccggc gcgaatagtc aggatcggag cccaggcggc 600
agtgcggaga acagctattt tcttggattg aatagcggcc ttaatctggg ggcctggcgg 660
ttacgcgact actccacatg gaacgcgaat agcggcgatc agaatagcga cagcgactgg 720
cagcacatca gtacttatct ggaacgtgat gtggtctttt tgcagggaga actgacggca 780
ggcgatagtt ataccccctc cgcattattc gatagccttc cttttcgcgg gctacaactg 840
gcgtctgacg acaatatgtt gccagacagc atgaagggct tcgcgccgac cattcacggc 900
attgccagaa gcaacgtgta agtgaccatt cggcaaaacg gctacatcat caatcagcgc 960
tatgtgccgc ccggggcatt tactattaat gatctctatc ctaccgccgc cagcggcgat 1020
ttgactgtgg aagtcaaaga gtccgacggt tctattaatc gctataacgt gccctattcc 1080
gccgtgccga ttctacaacg agaagggcgg ctgaagtatg cggcgacggt ggcggagtat 1140
cgtagcgata gtagtcaaaa agagaaggtg aaattcagtc aggcgacctt gatatggggg 1200
ttaccgcatg gttttacgct gtatggcgga acacaacttt ccagtcatta tcacgcgctg 1260
gcgatcggca gcggcgcaaa tctgggcgac tggggcgcgg tgtcgctgga tgtcacccag 1320
gctaccagta cgctggcgga taataactcc taccaggggc aatcgctgcg tttcctgtat 1380
gccaaatcgc ttgcacagtc aggaaccaat ttacagctta tgggctatcg ctattcaacc 1440
tcgggctttt acacgttgga tgataccacg tggaaacgga tgagcggcta tgacgatgac 1500
aatcggactg acagcgataa aagcaggccg gaatgggcgg attattacaa tctttattat 1560
accaggaggg gcaaagtaca actcgatatc aatcaacagc taggcgggtt gggatcgctt 1620
tttattaccg gcagtcagca aagttactgg cacactgatg aaaaggattc tttgttgcag 1680
gtgggataca gcgatacgct ggcgggtatt gcatggagcg tttcttacaa caataacaaa 1740
tccgcaggcg atgcggagcg cgatcaaatt ttcgccctga atatctcggt gccgctaagt 1800
caatggctgc aacatgatga tgaggtcacg caccatcaca atgtttacgc tacctttagc 1860
accagtacgg acaaacagca taacgttacg cagaatgcgg gccttagcgg cacactactg 1920
gacgaaaaca atcttagtta caacatacag taaggctatc agaatcacgg tattggcgaa 1980
agcggcgccg ccagcctgga atacgatggg gcgaaaggca acgccaatat tggctataac 2040
gttagcgata acggcgatta ccagcaggtg aattatggcc tgagcggcgg cctggtggcg 2100
cacgcgcatg gagtgacgct aagccagccg ttaggcaata ccaatatttt gattgccgcg 2160
ccgggcgcag ccaatgtcgg cgttgtcgac cagccgggta ttcatacgga cgcgcgtggc 2220
tatgcggtgg tgccgtatgc gacgacatat cgccaaaacc gtatggcgct ggacgttaac 2280
gccatggctg atgatgtcga tattgatgac gcggtgactc gcgttgtgcc gaccgaaggc 2340
gcgctggttc tggcccgctt taaagcgcga gtcggcgtgc gtgccctggt aacgctgaat 2400
cataatggta agcctgtacc ctttggcgca acggtgacgg tgaatgatcg ccatgcggag 2460
gcgattgttg acgaggccgg ggaggtctat ctttccgggt tgtcagcgca aggcgttctg 2520
cacgttcgct gggggaacct gccggatcaa cagtgcgtcg cgtcctatca tctctcttcc 2580
tcccgtcaga ttctgagtcg acaatatgcg gagtgtcatt aa 2622
<210> 2
<211> 1326
<212> DNA
<213>salmonella (Salmonella)
<400> 2
atgacacagt atcaggcgct cattattggt tttggcaagg caggaaaaac gttggcggca 60
actctggcga agacgggatg gcgcgtggcg attatcgaac agtccgccag tatgttcggc 120
ggaacctgca tcaacattgg ctgtattcca acgaaaacgc tggtgcatga cgcagaacgt 180
gagggcgatt tttctgtcgc catgcaacgc aaagcggcgg tcgtaaattt tttacgcgac 240
aaaaattttc ataatcttgc cgacctggac aatgtcgatg tgattgaggg aagggcggag 300
tttattgata accatacctt acgggtattt caggcggacg gcgagcgggt gttacggggg 360
gaaaagatct tcattaatac tggcgcagag tcggtgatcc cggctattac gggcttaaca 420
acgacggcag gggtgttcga tagcaccggg ctactcagcc tgagccagcg tccggcgcgg 480
ctggggattt taggcggtgg ttatattggc cttgaatttg cctcaatgtt cgccaacttt 540
ggtacgaagg tcactatctt tgaagcagcg ccgcaattcc tgcctcgtga agatcgggat 600
atcgcgcagg caattacccg tattttacag gaaaagggcg tcgagctgat tttaaacgcc 660
aacgtacagg ccgtatcctc aaaggagggc gcggtacaag ttgaaacgcc ggaaggcgcg 720
catcttgtgg atgccctgct ggtggcgtcg gggcgaaaac cggcgacggc tggcttacag 780
ttgcagaacg ccggcgtggc ggtgaacgaa cgtggcggga tcatcgtgga tgattacctg 840
cgtaccacgg cggacaatat ctgggcgatg ggcgatgtta ccggagggtt gcagtttacc 900
tatatttcac tggatgattt tcgtattgta cgcgacgggt tattgggaga tggcaaacgc 960
agtacccgcg atcgccagaa cgttccatac tccgtcttta tgacgccccc gctctctcgt 1020
gtcgggctga cggaagaaca agcgcgggca agcggcgcaa cggtgcaggt ggtaacatta 1080
ccggtggcgg cgatccctcg cgcccgtgtt atgaacgata cccgtggcgt actaaaagcg 1140
gtagtggacg tgaatacgca gcgtattgta ggggtgtcat tgttatgcgt cgattctcat 1200
gaaatgatta atatcgttaa aaccgttatg gatgcagatt taccttatac ggtgttacgc 1260
gaccagatat ttacccatcc tacaatgagt gagtctttaa acgatctctt ctctctcatt 1320
aagtaa 1326
<210> 3
<211> 855
<212> DNA
<213>salmonella (Salmonella)
<400> 3
ttatcgtccg agcgcgctga cccgtttgcg gtactcgcct ggtgtacaac cgaattcacg 60
gacaaaagct ttgtgaaaag aagattcgct cgcataacct accgactcgg caatcaccat 120
caccggcagc acttcacgcg acagggtctg ggcggcgatt tgcaggcgca gcgttgttaa 180
cacggctaag ggcgttgtag cggacacgtc gcgaaatagc tgggcgaagc tcgcccgcga 240
catgtgtacc cgttgcgcca gcgtttcgac cgtccaggga tgcgccggtg tttccagcat 300
gtgaaggatc acgcggccca gccggggatg cagtaataaa ttgagcacgc ttttcgccgt 360
gccagcctgc gatagccagt cgcgaacggc gagggtaaac agcgtggcgc actgctgact 420
gtataagaca tcagcgccag gtaaatggcg ttctgactcc tgctgtaaca gcaaaatggt 480
ggcgttaatc cagatactgg cggggctatg cggcggcggg gcaagacata acacttccgg 540
caacgcggta aggaaataac gtgacgtcgt gtgcagacgt agactaccgc agacgatatg 600
tgtcggtgat tcgccagact ggcgcaaacg gtgggcggag ttttgcggca gaatcaccac 660
ctttcccggc gtaagcgtca tggcgtcgcc ggtaggcatt tccagttgcg cttcgccctg 720
cgtaacggta tgccagcgaa tcaccgatag ttcgccagcg gcgtgcggca actgccagtc 780
gccgcctaac gggcaattct tgtcgattga gccctgaggg gcgttaagcg tcaatagccg 840
actaagcgca tccat 855
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gcagaatgcg ggccttag 18
<210> 5
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cgcgcgtccg tatgaat 17
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gaacagtccg ccagtatg 18
<210> 7
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gcaggtaatc atccacgat 19
<210> 8
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gattcgctcg cataacctac c 21
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gtgattcgct ggcataccgt 20
Claims (7)
1. the HRM serotype method of salmonella food-borne pathogens, which is characterized in that method includes the following steps:
Step 1, according to salmonellaykgDGene order,ykgDGene order is as shown in SEQ 1, the two of its saltation zone
End design specificity amplification primer pair；
Step 2 extracts sample DNA, is expanded using specificity amplification primer obtained by step 1 to using HRM method；
Step 3, by whether containing salmonella in fluorescence curve judgement sample.
2. the HRM serotype method of salmonella food-borne pathogens according to claim 1, which is characterized in that step
A rapid specificity amplification primer is made of upstream primer and downstream primer, and the base sequence of upstream primer is as shown in SEQ 8, downstream
The base sequence of primer is as shown in SEQ 9.
3. the HRM serotype method of salmonella food-borne pathogens according to claim 1, which is characterized in that step
In rapid two, in the HRM method, HRM serotype system is specific as follows:
20 μ L HRM reaction systems: the MgCl of the 2*buffer and 2.4 μ L of 10 μ L are taken2, upstream and downstream primer respectively takes 1 μ L, DNA profiling
2 μ L are taken, finally plus H2O is 3.6 μ L.
4. the HRM serotype method of salmonella food-borne pathogens according to claim 1 or 2 or 3, feature exist
In, in step 2, in the HRM method, HRM serotype system Amplification specifically:
94 DEG C of initial denaturation 5min start amplification cycles, the program of each circulation later are as follows: 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s,
72 DEG C of extension 30s；Circulation totally 35；After circulation terminates, 72 DEG C of extension 10min are cooled to 10 DEG C, terminate.
5. the HRM serotype target gene of salmonella food-borne pathogens, which is characterized in that the base sequence of the target gene
Column are as shown in SEQ 1.
6. the HRM serotype specificity amplification primer of salmonella food-borne pathogens, which is characterized in that the specificity expands
Increase primer to be made of upstream primer and downstream primer, the base sequence of upstream primer is as shown in SEQ 8, the base sequence of downstream primer
Column are as shown in SEQ 9.
7. the HRM serotype kit of salmonella food-borne pathogens, which is characterized in that the kit includes DNA mould
Plate, specificity amplification primer, 2*buffer and MgCl2, the specific amplification primers are made of upstream primer and downstream primer,
The base sequence of upstream primer is as shown in SEQ 8, and the base sequence of downstream primer is as shown in SEQ 9.
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| CN101115832A (en) * | 2004-11-26 | 2008-01-30 | 协和发酵工业株式会社 | Industrially useful microorganism |
| US20090075333A1 (en) * | 2005-08-20 | 2009-03-19 | Campbell John W | Reduced Genome E. Coli |
| CN101892320A (en) * | 2010-02-04 | 2010-11-24 | 上海交通大学 | Multiple PCR identification method of salmonella serogroup A, B, C1, C2 or D |
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