CN101748214B - Pullorum-typhoid salmonella PCR detection method, nucleic acid and primer therein - Google Patents
Pullorum-typhoid salmonella PCR detection method, nucleic acid and primer therein Download PDFInfo
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- CN101748214B CN101748214B CN2009103112455A CN200910311245A CN101748214B CN 101748214 B CN101748214 B CN 101748214B CN 2009103112455 A CN2009103112455 A CN 2009103112455A CN 200910311245 A CN200910311245 A CN 200910311245A CN 101748214 B CN101748214 B CN 101748214B
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Abstract
Disclosed are pullorum-typhoid salmonella PCR detection method, nucleic acid and primer therein in food safety inspection technical field; the PCR detection method includes the following procedures: amplification primer is designed according to sequence SEQ ID NO:1; DNA of sample is extracted and the PCR method is carried out for amplification; the amplification product is detected by gel electrophoresis for judging whether the sample contains pullorum-typhoid salmonella; the judging process concretely includes that: if no corresponding single amplification band occurs, the sample contains nopullorum-typhoid salmonella; the invention also relates to a nucleic acid with base sequence shown in SEQ ID NO:1; the invention also relates to a primer with base sequence shown in SEQ ID NO:2 and SEQ ID NO:3. The detection method of the invention is used for detecting the pullorum-typhoid salmonella, has short detection time, low cost, strong practicality, specific detection results and simple result judgment.
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 white dysentery-salmonella typhi PCR detection method nucleic acid and the primer that reach wherein are right.
Background technology
Salmonellosis of chicken is the bacterial infectious disease of the chicken that causes of some pathogenic salmonella by salmonella, generally is divided into white dysentery, fowl typhoid and three kinds of sick types of typhus fever, and is wherein more with white dysentery and fowl typhoid morbidity, endangers stronger.White dysentery is by chicken infected the causing of white dysentery Salmonellas serotype (Salmonella Gallinarum); It mainly is the infringement chick; Chick morbidity back mortality ratio below 2 weeks can reach 40～70%, and infected chick can be death in 1～3 day when acute death in 4～7 days.The chicken infected fowl typhoid illness that causes of fowl typhoid Salmonellas serotype (Salmonella Pullorum) mainly is the chicken that infect to grow up, and chick also can be infected sometimes, goes out behind the shell to begin morbidity in several days, and mortality ratio is higher.In recent years, these two kinds of pathogenic bacterium are classified as a kind, are referred to as white dysentery-salmonella typhi (SalmonellaGallinarum-Pullorum), mainly are to be present in the organs such as ovary, testis, liver, can be infected to chick by the parent chicken that infects.The effective measures of control salmonellosis of chicken are strict management and elimination plans.Therefore, the parent chicken crowd of evaluation and removing infection is most important.At present; Many reports that are applied to quarantine about the PCR detection method of Salmonellas are arranged; Though actively promoted the control of salmonellosis of chicken; But the research report of target spot that the PCR that only is directed against serotype white dysentery-salmonella typhi detects and primer does not almost have, thereby does not have the relevant report of setting up to the PCR method of white dysentery-salmonella typhi serotype detection yet.
Literature search through to prior art is found; For a long time; China identifies the disease chicken through the antibody that uses slide agglutination test to detect white dysentery-salmonella typhi; As Xu Yaohui, Jiao Xinan, Li Qiuchun etc. described in " Chinese animal doctor science and technology " 2005 the 36th the 8th phases of volume the 661st～664 page of " monoclonal antibody of part provinces and cities white dysentery and fowl typhoid is blocked the ELISA serosurvey " literary composition of delivering; This method susceptibility is limited, unstable result usually occurs according to the antigenic quality difference.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide a kind of white dysentery-salmonella typhi PCR detection method and wherein nucleic acid and primer right.Adopt detection method of the present invention to detect white dysentery-salmonella typhi, 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 white dysentery-salmonella typhi PCR detection method, comprise the steps:
In the step 1, said primer is specially: forward primer SEQ ID NO:2 and reverse primer 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 231bp position, if exist, then contain white dysentery-salmonella typhi in the interpret sample; If corresponding single amplified band do not occur, then do not contain white dysentery-salmonella typhi in the sample.
The invention still further relates to the nucleic acid that relates in a kind of said PCR detection method, the base sequence of this nucleic acid is shown in SEQ ID NO: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 white dysentery-salmonella typhi, 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 result among the embodiment 1;
Fig. 2 is PCR detection method sensitivity evaluation experimental 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 white dysentery-salmonella typhi serotype PCR detection method
Through bioinformatic analysis, from white dysentery-salmonella typhi genomic dna sequence, find total specific gene SG1046 and SG1047 (SEQ ID NO:1), with it detection target gene as white dysentery-salmonella typhi.
The dna sequence dna of gene SG1046 and SG1047 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 100～500bp; From alternative primer centering select primer to SG5-L/R as primers designed (primer by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthetic), primer sequence is following:
SG5-L：5’-TTATGTTACGGGACGAGTG-3’(SEQ?ID?NO：2)；
SG5-R：5’-CAAGACTCCCTGCCCTAT-3’(SEQ?ID?NO：3)；
The Salmonellas bacterial strain (as shown in table 1) of various serotypes such as Salmonella typhimurium, Salmonella enteritidis and white dysentery-salmonella typhi 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, 60 ℃ 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.
Table 1 specificity is estimated bacterial strain uses therefor and test-results
In the table 1 ,-: PCR result is negative; +: PCR result is positive.
Salmonellas reference culture such as 16 strain Salmonella typhimuriums, Salmonella enteritidis and white dysentery-salmonella typhi and 31 strain strain isolateds (as shown in table 1, bacterial strain shown in the table be those skilled in the art can obtain through disclosed channel) extract genomic dna respectively according to the method for dna profiling preparation.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.
The detected result that has shown reference culture among Fig. 1, its serotype and strain number see also description of drawings.Among the figure: swimming lane 1～16:AS1.1190, CMCC 50004, and CMCC 50017, and CMCC 50335; AS1.1174, ATCC 14028, ATCC13076, ATCC 7001; ATCC 15611, and ATCC 9150, and ATCC 9270, and NCTC 4840; ATCC 12002, ATCC51812, and NCTC 6017, and CMCC 50770; Swimming lane 17:ddH
2O; Swimming lane M:200bp molecular weight standard.As can beappreciated from fig. 1 except the reference culture CMCC50770 of white dysentery-salmonella typhi, all the other Salmonellass all do not have the specific amplified band, explain that primer is fine to the specificity of SG5-L/R.Table 1 is the PCR qualification result of 16 strain reference cultures and 31 strain isolated strains, from table 1, can know, except the 1 strain reference culture CMCC50770 and 3 strain strain isolateds of white dysentery-salmonella typhi, all the other Salmonellass all do not have the specific amplified band.
Through measuring, the concentration of the total dna solution of white dysentery-salmonella typhi is 68.6 μ g/mL, makes 10 times of gradient dilutions with sterilized water, dilutes 10 gradients altogether, and each gradient is got 5 μ L respectively and added PCR reaction system, detected through gel electrophoresis amplification; The result is as shown in Figure 2, and among the figure: swimming lane 1～10:DNA template concentrations is respectively 34.3ng, 3.43ng, 343pg, 34.3pg, 3.43pg, 343fg, 34.3fg, 3.43fg, 0.343fg, 0.034fg/ reaction; Swimming lane M:200 bp molecular weight standard.Can know that by Fig. 2 can see band clearly at the 7th swimming lane, institute's corresponding DNA concentration is 34.3fg/PCR, and can't see amplified band after the eight lanes.Therefore, judge that the PCR detection sensitivity is 34.3fg/PCR, have higher sensitivity.
The detection of the doubtful bacterial strain of Salmonellas
Utilize the PCR detection architecture of the white dysentery-salmonella typhi of setting up; Detected 44 strains doubtful bacterial strain of isolating Salmonellas from chicken manure; Sample collecting is 5 chicken houses in the outskirts of a town, Shanghai City, and the processing of chicken manure sample and the separation of doubtful bacterial strain are referring to GB GB/T 4789.4-2008.Therefrom detect the doubtful bacterial strain of the 11 strains result that is positive; The doubtful bacterial strain of this 11 strain identifies it is O9:H-:-through salmonella diagnostic serum (Lanzhou Institute of Biological Products), determines that it is white dysentery-salmonella typhi (the serum authentication step sees also product description and GB GB4789.4-1994).Other doubtful bacterial strains are not white dysentery-salmonella typhis through identifying.Prove that through present embodiment the serotype PCR detection method of white dysentery-salmonella typhi Salmonellas has extreme high reliability.
Sequence table
< 110>Shanghai Communications University
< 120>white dysentery-salmonella typhi PCR detection method and wherein nucleic acid and primer are right
<160>3
<170>PatentIn?version?3.3
<210>1
<211>1460
<212>DNA
< 213>white dysentery-salmonella typhi
<400>1
gtggtgttcc?cggcagggct?ggtgaggcgg?ctggacagcc?tggaggcaga?gctgcgggcg 60
gggagagtga?gcagtgagag?ccgtgcatgg?ctggcgcagt?gcggactgac?ggcggagcag 120
atggcggggc?aactggaaga?gcgcgctgaa?ccggaaagga?aaatccatct?ttaccactgc 180
gaccaccgtg?gtctgccggt?ggcgctgata?aacagcgagg?gggcgcggga?atggagcgcg 240
gagtatgatg?tgtggggaaa?ccggcagaag?gaggagaacc?cgcaacagct?tgagcagtta 300
ctgagactgc?cgggccagca?gtatgatgag?gaaacggggc?tgtattataa?ccgttaccgt 360
tactataatc?cggaacaggg?aaggtacatc?acgcaggacc?cgattgggtt?gcgggggggg 420
atggaacctg?tatgcgtatc?cgctgaaccc?ggtgagcggg?actgacccgc?tggggctggt 480
tgtggatgcg?attccctggg?gagcggcgac?acccacggcg?gtgctggatg?cggcactggc 540
cggatttgaa?gcggatgtgg?ccgttccgga?gccaacagat?ggcgcctggc?cgaagtgggc 600
tgggtgggca?gtcgttattg?ctgctgcggc?tgcttttaca?tacctgacct?gtgcgtccgg 660
tgatgattca?acagaggatt?cagaggacag?tgatgctggc?tatggtgaga?agggggactc 720
cacggattca?gggcatggag?tatctggtgc?ggaagacaat?gagagtggca?gcgattcgga 780
tgccacgact?gttgacgatt?taattgcaac?gtcatctaaa?ggtgagcaga?ctacagggag 840
gtcccggctt?tatgaacggc?ctggggggat?tgatgaggct?aacaaggatt?ttgataactt 900
atcccctagc?gatgttaagg?acatcgacaa?taattatgtt?acgggacgag?tgggtacttt 960
gccggatggt?cgcacagtga?ttgtgcgtga?tggcagttct?gatggtggac?ctacacttga 1020
ggtccaatcc?gggaaaaata?aaattaaatt?taggtatgat?tagtgaggaa?tatatgattg 1080
aaaaattggt?tgatattacc?ccccaaaata?tatctttaaa?aggtagtcag?ataattgatt 1140
ttcattatag?ggcagggagt?cttgagatta?tagttactct?tgatggagtg?aattcggatt 1200
ttcgattttt?ttttgattgg?actcattcat?ttcgtgtcac?tgatgaaggt?gatctgttga 1260
aaatgttggg?tgagcaaaaa?ggaaaaatgc?gagtaggtat?ttataaggtt?gaggactcat 1320
cttatctgga?atggtttaat?gaccagagtt?ttaatataca?tgaaaaagag?aaaattattc 1380
attatttgat?tgtgacagta?aatgatatca?ttgatgtttt?gtcctcagag?tctccagtga 1440
tatctaactg?ttctaaataa 1460
<210>2
<211>19
<212>DNA
< 213>artificial sequence
<400>2
ttatgttacg?ggacgagtg 19
<210>3
<211>18
<212>DNA
< 213>artificial sequence
<400>3
caagactccc?tgccctat 18
Claims (4)
1. non-diagnostic purpose white dysentery-salmonella typhi PCR detection method is characterized in that, comprises the steps:
Step 1 is according to the conserved sequence SEQID NO:1 design of amplification primers in the genomic dna sequence of white dysentery-salmonella typhi;
Step 2 is extracted sample DNA, the amplification of PCR method;
Step 3, the detected through gel electrophoresis amplified production, whether judgement sample contains white dysentery-salmonella typhi; Said judgement is specially: if corresponding single amplified band appears in electrophoresis result, then contain white dysentery-salmonella typhi in the interpret sample; If corresponding single amplified band do not occur, then do not contain white dysentery-salmonella typhi in the sample;
In the step 1, said primer is specially: forward primer SEQ ID NO:2 and reverse primer SEQ IDNO:3;
In the step 3, said judgement is specially: detect the gel electrophoresis amplified production and whether have single amplified band in the 231bp position, if exist, then contain white dysentery-salmonella typhi in the interpret sample; If corresponding single amplified band do not occur, then do not contain white dysentery-salmonella typhi in the sample.
2. non-diagnostic purpose white dysentery according to claim 1-salmonella typhi PCR detection method 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, T aq enzyme 1U, and template solution is got 2-5 μ L, last moisturizing to 25 μ L.
3. non-diagnostic purpose white dysentery according to claim 1-salmonella typhi PCR detection method is characterized in that, in the step 2; In the said PCR method, PCR detection architecture parameter is specially: 94 ℃ of preparatory sex change 5min of elder generation begin amplification cycles afterwards; 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. the primer of non-diagnostic purpose white dysentery as claimed in claim 1-salmonella typhi PCR detection method is right, it is characterized in that, 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.
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| CN104846066A (en) * | 2014-09-23 | 2015-08-19 | 中国农业大学 | PCR detection primers and detection method of Salmonella pullorum |
| CN105087806A (en) * | 2015-09-09 | 2015-11-25 | 江苏省家禽科学研究所 | Primer group, kit and method for identifying salmonella pullorum and salmonella gallinarum |
| CN106086209B (en) * | 2016-08-03 | 2019-06-18 | 扬州大学 | A kind of PCR detection kit of Rapid identification white diarrhea and Salmonella gallinarum |
| CN106834500B (en) * | 2017-03-03 | 2020-07-14 | 中国农业科学院哈尔滨兽医研究所 | Specific primer for detecting salmonella pullorum, kit containing primer and application of kit |
| CN110714089B (en) * | 2019-10-29 | 2022-06-14 | 扬州大学 | PCR primer of specific targeting flagellum hook gene flgE and application thereof in rapid detection of flagella and flagellum-free salmonella |
| CN110699470A (en) * | 2019-11-14 | 2020-01-17 | 四川大学 | Dual PCR primer, kit, application and method for detecting salmonella and identifying pullorum disease/gallinarum serotype |
| CN112725477A (en) * | 2021-01-07 | 2021-04-30 | 四川大学 | Primer and probe composition of pullorum disease/salmonella gallinarum TaqMan probe fluorescent quantitative PCR detection method |
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| CN101045943A (en) * | 2007-04-26 | 2007-10-03 | 上海交通大学 | Process of PCR detecting salmonella with added amplifying internal standard |
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