CN102399854A - PCR detection method of salmonella and primer pair - Google Patents
PCR detection method of salmonella and primer pair Download PDFInfo
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- CN102399854A CN102399854A CN2010102774121A CN201010277412A CN102399854A CN 102399854 A CN102399854 A CN 102399854A CN 2010102774121 A CN2010102774121 A CN 2010102774121A CN 201010277412 A CN201010277412 A CN 201010277412A CN 102399854 A CN102399854 A CN 102399854A
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Abstract
The invention relates to a PCR detection method of salmonella and a primer pair which belong to the detection technical field. The method comprises the following steps: designing an amplimer according to a conserved sequence in a genome DNA sequence of salmonella, extracting DNA of a sample to be detected, performing PCR amplification; detecting an amplified product by gel electrophoresis, determining whether the sample contains salmonella or not; if the corresponding amplification bands are generated in electrophoresis result, the sample to be detected contains salmonella. The base sequence of a forward primer of the primer pair is shown as a SEQ ID NO:2, a base sequence of a reversed primer is shown as a SEQ ID NO:3. The detection method of the present invention has the advantages of short detection time, specific detection result and simple result determination.
Description
Technical field
The present invention relates to the method in a kind of detection technique field, specifically is that PCR detection method and the primer of a kind of Salmonellas is right.
Background technology
In recent years, food origin disease has become current outstanding, broad range of food safety-problems.Salmonellas (Salmonella) is enterobacteriaceae salmonella member, is the endotrophic Gram-negative enterobacteria of conditionality.Salmonellas is the highest a kind of of sickness rate in the bacterial food poisoning, and nearly all serotype can both be caused a disease.Traditional Salmonellas detection method need be cultivated through selective enrichment, and confirms through biochemical reaction and serology test, though this detection method result is comparatively reliable; But complicated operation; Waste time and energy, needed 3～5 days just can obtain detected result usually, be unfavorable in time diagnosing the cause of disease; Search pathogeny, the spreading of disease controlling.
Summary of the invention
First purpose of the present invention is to provide the PCR detection method of a kind of Salmonellas, comprises the steps:
Preferably, in the step 1, the base sequence of said conserved sequence is shown in SEQ ID NO:1.
Preferably, in the step 2, said primer is specially: the base sequence of forward primer is shown in SEQ ID NO:2, and the base sequence of reverse primer is shown in SEQ ID NO:3.
Preferably, in the step 2, in the said pcr amplification, 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, 5uM primer be to 1 μ L, Taq enzyme 1U, and template solution is got 2～5 μ L, last moisturizing to 25 μ L.
Preferably, in the step 2, the reaction conditions in the said pcr amplification is: 94 ℃ of 5min, and 94 ℃ of 30min, 55 ℃ of 30min, 72 ℃ of 30min, 30 circulations, 72 ℃ are extended 10min eventually.
Preferably, in the step 2, said amplified band is the 516bp band.
Second purpose of the present invention is to provide a kind of primer right, and the base sequence of primer centering forward primer is shown in SEQ ID NO:2, and the base sequence of reverse primer is shown in SEQ ID NO:3.
The present invention has following beneficial effect: adopt detection method of the present invention to detect Salmonellas, can shorten to detection time in 10 hours, owing to need not to adopt the antiserum(antisera) that must use in the traditional method, 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 sample detection electrophoresis result figure among the embodiment 1;
Fig. 2 is that the Salmonellas of different serotypes among the embodiment 2 detects electrophoresis result figure;
Fig. 3 is that Salmonellas detects electrophoresis result figure with other non-Salmonellass among the embodiment 2;
Fig. 4 is the detection electrophoresis result figure after the different concns DNA cloning among the embodiment 3;
Fig. 5 is a mixed culture pcr amplification electrophorogram among the embodiment 4;
Fig. 6 is that the Salmonellas culture is cultivated back pcr amplification electrophorogram among the embodiment 4.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for the present invention 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.
Instrument that relates in following examples and reagent are:
Tris-EDTA (TE) damping fluid, 10 * buffer, MgCl
2, dNTP, Taq enzyme all purchase the ltd in JaRa biotechnology Shanghai;
Primer, sepharose, PTC-200PCR gene-amplificative instrament are purchased the M.J.Research company in the U.S.;
5415D desk centrifuge: Eppendorf company; Electrophoresis apparatus: Liuyi Instruments Plant, Beijing; Electrophoresis chamber, TAT gel imaging system: BIO-RAD company;
The various bacterial strains that relate in following examples all can obtain through disclosed commercially available channel.
1, selects the conserved sequence in the salmonella gene group dna sequence dna and design primer
From selecting base sequence sequence shown in SEQ ID NO:1 the salmonella gene group dna sequence dna is conserved sequence.Utilize Primer Premier 5.0 software design primers afterwards, primer sequence is following:
The base sequence of forward primer is: gagttactga accaacagct (SEQ ID NO:2);
The base sequence of reverse primer is: gccgctaaac tacacgatga (SEQ ID NO:3);
Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
2, extract the DNA of sample to be checked, pcr amplification;
With reference to " the People's Republic of China's import-export commodity inspection industry standard export food salmonella " (SN 0170-92) oyster to be checked being increased bacterium cultivates.
Each bacterium culture is inoculated in respectively in 30mL bacteriolyze meat soup (LB) liquid nutrient medium, and 37 ℃ of shaking culture 8h get the 1.0mL inoculum, the centrifugal 1min of 12000r/min; Get deposition, repeat to wash twice with 100 μ L TE solution (pH8.0), the centrifugal 1min of 12000r/min gets deposition; Add 100 μ L TE solution, suspend, 98 ℃ of water-bath 10min, ice bath is placed 5min; The centrifugal 1min of 12000r/min gets supernatant, gets the DNA of sample.
PCR reaction system (25 μ L): 10 * PCR damping fluid, 2.5 μ L, dNTP 0.5 μ L, primer (10nmol/ μ L) 1 μ L, template DNA is 1 μ L, Taq enzyme (5U/ μ L) 0.5 μ L uses ddH
2It is 25 μ L that O replenishes volume.PCR reaction conditions: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30,30 circulations; 72 ℃ are extended 10min eventually.
3, electrophoresis detection amplified production
Add 3 μ L, 10 * loading buffer to pcr amplification product, draw 8 μ L, MarkerDL2000 does reference, with sepharose electrophoresis 40min under the 100V condition of concentration 1%, utilizes gel imaging system to observe electrophoresis result.The result is as shown in Figure 1, and is as shown in the figure, all amplifies the specific band of 516bp in all repetitions, contains Salmonellas in the interpret sample.The detection method of present embodiment can be accomplished in 10 hours, had greatly shortened detection time.
The Salmonellas of different serotypes is carried out pure culture with other non-Salmonellas reference cultures, extract dna profiling, carry out pcr amplification and electrophoresis detection amplified production with reference to the method for embodiment 1.The result sees Fig. 2 and Fig. 3, and the bacterial strain of each swimming lane representative is among Fig. 2: 1, S.indiana; 2, S.enterilids; 3, S.derby; 4, S.typhimurium; 5, S.senftenberg; 6, S.kentucky; 7, S.cholerae suis; 8, S.hartford; The bacterial strain of each swimming lane representative is among Fig. 3: 1, S.hartford; 2, E.coli; 3, S.Indiana; 4, Shigella; 5, S.cholerae suis; 6, Staphyloccocus; 7, S.typhimurium; 8, Citrobacter; 9, S.enteritids; 10, Enterobacter cloacae; 11, S.derby.The result shows that the Salmonellas of 8 kinds of different serotypes all can amplify band, but not Salmonellas all can not amplify band, and the detection method specificity of visible embodiment 1 is stronger.
Getting 1mL Salmonellas culture and extract dna profiling and measure concentration, is that gradient dilution becomes different concentration with it with 10 times, and each concentration is got 1 μ L respectively and is used for pcr amplification, and amplified production is detected, and detection method is with reference to embodiment 1.The Salmonellas liquid culture is extracted dna profiling after with 10 times of gradient dilutions carry out augmentation detection, make the flat-plate bacterial colony meter simultaneously.The result is as shown in Figure 4, among Fig. 4, and 1, undiluted template; 2,10 times of dilutions; 3,10
2Doubly dilution; 4,10
3Doubly dilution; 5,10
4Doubly dilution; 6,10
5Doubly dilution.Can know by figure, with the concentration dilution to 10 of the dna profiling that extracts
4Times the time, still can clearly observe the target dna band.Dna profiling is through ultraviolet determination, and original liquid concentration is approximately 450ng/ μ L, after dilution, can detect the dna profiling of 0.045ng/ μ L.When the Salmonellas liquid culture is diluted to concentration is 10
3During cfu/ml, extract DNA and carry out the pcr amplification rear electrophoresis, still can detect band, the pcr amplification detection architecture in this illustrative embodiment 1 has quite high sensitivity.
Method with reference to embodiment 1 detects as follows:
1, infects a sterile sampling simultaneously with Salmonellas reference culture and other non-Salmonellas bacterial strain pure growths.To infect the back article and cultivate, and extract dna profiling and carry out pcr amplification, electrophoresis inspection product.
2, the mixed culture with Salmonellas reference culture and other non-Salmonellas bacterial strains infects sterile sampling.Cultivate the back and extract dna profiling, its product of pcr amplification and electrophoresis detection.
3, with the LB substratum with the rare 10 ° of cfu/mL of Salmonellas culture, in 37 ℃ of cultivations, every get the 1mLDNA rear electrophoresis that increases at a distance from 2h and detect.
Infect sample with Salmonellas and other single bacterium and mixed bacterium and cultivate back extraction DNA through the short period of time and carry out pcr amplification, the result is as shown in Figure 5, among Fig. 5, and 1, S.enteritids; 2, S.Enteritids and E.coli; 3, S.typhimurium; 4, S.typhimurium and E.coli; 5, E.coli; 6, Shigella; 7, E.coli and Shigella; 8, E.coli and Staphyloccocus; 9, Shigella and Staphyloccocus; 10, Staphyloccocus; Can know by figure, all can amplify target stripe through single bacterium of Salmonellas and the positive sample that infects of mixed bacterium, and the sample of other non-Salmonella infection none can amplify target stripe, the primer among this proof embodiment 1 is to having very strong specificity and practicality.Using bacterial concentration is that 10 ° of cfu/mL Salmonellas liquid inductances dye oyster sample to be checked; Extract DNA behind 37 ℃ of cultivation 6h and carry out pcr amplification; Electrophoresis can detect target stripe, and is as shown in Figure 6, among the figure; The oyster sample of swimming lane 1 and 2 for dying with non-Salmonellas liquid inductance, the oyster sample of swimming lane 3 and 4 for dying with the Salmonellas liquid inductance.
In sum, detect Salmonellas, can shorten to detection time in 10 hours, reduced the detection cost simultaneously with detection method of the present invention; Detection target spot of the present invention has single specificity, and detected result is special, and the result judges simply.
Claims (7)
1. the PCR detection method of a Salmonellas is characterized in that, comprises the steps:
Step 1 is according to the conserved sequence design of amplification primers in the salmonella gene group dna sequence dna;
Step 2 is extracted the DNA of sample to be checked, pcr amplification;
Step 3, whether the detected through gel electrophoresis amplified production contains Salmonellas in the judgement sample; Said being judged as:, then contain Salmonellas in the interpret sample if corresponding amplified band appears in electrophoresis result.
2. the PCR detection method of Salmonellas as claimed in claim 1 is characterized in that, in the step 1, the base sequence of said conserved sequence is shown in SEQ ID NO:1.
3. the PCR detection method of Salmonellas as claimed in claim 2 is characterized in that, in the step 2, said primer is specially: the base sequence of forward primer is shown in SEQ ID NO:2, and the base sequence of reverse primer is shown in SEQ ID NO:3.
4. the PCR detection method of Salmonellas as claimed in claim 3 is characterized in that, in the step 2, in the said pcr amplification, 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, 5uM primer be to 1 μ L, Taq enzyme 1U, and template solution is got 2～5 μ L, last moisturizing to 25 μ L.
5. the PCR detection method of Salmonellas as claimed in claim 4 is characterized in that, in the step 2, the reaction conditions in the said pcr amplification is: 94 ℃ of 5min, and 94 ℃ of 30min, 55 ℃ of 30min, 72 ℃ of 30min, 30 circulations, 72 ℃ are extended 10min eventually.
6. the PCR detection method of Salmonellas as claimed in claim 2 is characterized in that, in the step 2, said amplified band is the 516bp band.
7. a primer is right, it is characterized in that, the base sequence of forward primer is shown in SEQ ID NO:2, and the base sequence of reverse primer is shown in SEQ ID NO:3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898230A (en) * | 2014-04-18 | 2014-07-02 | 北京农学院 | Method for assessing food-borne pathogenic microbe infected leafy vegetables |
| CN106755353A (en) * | 2016-12-05 | 2017-05-31 | 西昌学院 | A kind of salmonella pathogenicity island gene mgtC, sseL dual PCR detection method and purposes |
-
2010
- 2010-09-09 CN CN2010102774121A patent/CN102399854A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898230A (en) * | 2014-04-18 | 2014-07-02 | 北京农学院 | Method for assessing food-borne pathogenic microbe infected leafy vegetables |
| CN103898230B (en) * | 2014-04-18 | 2016-02-17 | 北京农学院 | A kind of food-borne pathogenic microorganism infects the appraisal procedure of leafy vegetable |
| CN106755353A (en) * | 2016-12-05 | 2017-05-31 | 西昌学院 | A kind of salmonella pathogenicity island gene mgtC, sseL dual PCR detection method and purposes |
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Application publication date: 20120404 |