CN101748215B - Detection method for infant salmonella PCR and nucleic acid and primer pair thereinto - Google Patents

Abstract

The invention relates to a detection method for infant salmonella PCR and nucleic acid and primer pair therein, belonging to the technical field of food safety detection; the detection method comprises the following steps: designing an amplimer according to the conserved sequence SEQ ID NO:1 in the genome DNA sequence of the infant salmonella; extracting sample DNA, amplifying by PCR method; detecting the amplified product through gel electrophoresis, and judging whether the sample contains the infant salmonella or not; and the judgment is particular as follows: if corresponding single amplification belt does not appear, the sample does not contain the infant salmonella. The invention also relates to a nucleic acid, the base sequence of which is showed by SEQ ID NO:1; the invention also relates to a pair of primer, particularly the base sequence of which is showed by the nucleic acid SEQ ID NO: 2, and SEQ ID NO: 3. When detecting the infant salmonella by adopting the invention, the detection time is short, the cost is low, the detection result is distinctive and the result is judged easily.

Description

Salmonella infantis PCR detection method and wherein nucleic acid and primer are right in the food

Technical field

The 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, and nucleic acid and primer that salmonella infantis PCR detection method reaches wherein are right.

Background technology

Salmonella infantis (Salmonella Infantis) is one of main Salmonellas serotype that causes newborn infant and infant infection, is to be found first by Wheele and Borman in nineteen forty-three.The main contagium of salmonella infantis is animal (bird is main), food and the crowd etc. that carries disease germs; Can pass through approach such as food, environment, trans-oral infects healthy human body, in its enteron aisle breeding; Invasion and attack intestines wall mucous membrane causes diseases such as enteritis, dysentery, septicemia.Salmonella infantis infects does not have strict seasonal, and the four seasons all can fall ill, and it has very strong resistibility in contaminate environment, can survive several months, thereby can cause newborn infant and infant's repeated infection, is difficult for eliminating.The detection of salmonella infantis mainly is the traditional cultured method (can referring to GB GB/T 4789.4-2008) through GB regulation.As Zou Jingbo, Cheng Yue, Zheng Xianqi the 62nd～63 page of " laboratory medicine is with clinical " 2008 the 5th the 1st phase of volume deliver be entitled as " causing the lab analysis of food poisoning by Salmonella infantis " literary composition; Mention following content in the literary composition: this method needs to cultivate through selective enrichment, and combines tests such as biochemical reaction, serology test to identify, though reliable results; But complicated operation; Detection time is long, takes 3～5 days usually and just can identify the result, is unfavorable in time diagnosing the cause of disease; Search pathogeny, the spreading of disease controlling.For check salmonella infantis that can be quick, accurate, easy; With molecular biology is that basic detection authentication method constantly makes further progress in practice test; Become one of detection method that replaces the tool potentiality of traditional detection method; Wherein, PCR with its sensitivity, special, easy, characteristics become one of important detection technique of setting up in the molecular biology level fast.

At present; The target gene that is used for salmonella infantis serotype PCR detection abroad mainly comes own coding Salmonellas H antigenic flagellin gene flic C and fl jB; Like Kardos G; Farkas T, Antal M etc. are in the 421st～425 page of being entitled as described in " NovelPCR assay for identification of Salmonella enterica serovar Infantis " (identifying the new PCR method of salmonella infantis) literary composition of delivering of " Letters in AppliedMicrobiology " (applied microbiology wall bulletin) 2007 the 45th the 4th phases of volume.Yet; Because the complicacy of antigenic polymorphum of Salmonellas and encoding sox thereof; It is right to be difficult for finding suitable target gene and to detect primer; And the serological type strain of similar antigenic structure is arranged for some and salmonella infantis, and need to combine other relevant genomic constitution multiplex PCRs could judge, increased the complicacy of detection.

Literature search through to prior art is found, not invention and salmonella infantis 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 infantis PCR detection method and wherein nucleic acid and primer right.Adopt detection method of the present invention to detect salmonella infantis, 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 infantis PCR detection method, comprise the steps:

Step 1 is according to the conserved sequence SEQ ID NO:1 design of amplification primers in the genomic dna sequence of salmonella infantis;

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 infantis; Said judgement is specially: if corresponding single amplified band appears in electrophoresis result, then contain salmonella infantis in the interpret sample; If corresponding single amplified band do not occur, then do not contain salmonella infantis in the sample.

In the step 1, said primer is specially: the sequence of forward primer is shown in SEQ ID NO:2, and reverse primer is shown in SEQ IDNO: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 197bp position, if exist, then contain salmonella infantis in the interpret sample; If corresponding single amplified band do not occur, then do not contain salmonella infantis 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 salmonella infantis, 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.

Embodiment 1

The foundation of salmonella infantis serotype PCR detection method

Step 1 is according to the conserved sequence design of amplification primers in the genomic dna sequence of salmonella infantis

From the salmonella infantis genomic dna sequence, find special restriction endonuclease gene, with it detection target gene as salmonella infantis, 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 150～300bp; Select primer from alternative primer centering, primer sequence is (primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) as follows:

SIN5-L：5’-AGCCAACGCCACCTACTACT-3’(SEQ?ID?NO：2)；

SIN5-R：5’-TGAACACCATATCCATCCACAT-3’(SEQ?ID?NO：3)；

Step 2, the dna profiling preparation

The Salmonellas bacterial strain (as shown in table 1) of various serotypes such as Salmonella typhimurium, Salmonella enteritidis and salmonella infantis 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;

Afterwards, 3, the centrifugal 10min of 000r/min gets supernatant, 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.

Step 3, PCR detection method specificity evaluation experimental

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 infantis and 29 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 in the PCR reaction system as the PCR reaction template, carries out amplified reaction by aforementioned PCR detection method.

Fig. 1 is the detected result of Salmonellas reference culture, and its serotype and strain number see also description of drawings and table 1.Among the figure: swimming lane 1～15:Salmonella Typhimurium AS1.1174, Salmonella Choleraesuis AS1.1190, Salmonella Gallinarum CMCC50770; Salmonella Enteritidis ATCC13076, SalmonellaEnteritidis CMCC50335, Salmonella Paratyphi A ATCC9150; Salmonella Paratyphi BCMCC50004, Salmonella Vellore ATCC15611, Salmonella Anatum ATCC9270; SalmonellaPoona NCTC4840; Salmonella Tallahassee ATCC12002, Salmonella HirschfeldiiCMCC50017, Salmonella Abony NCTC6017; Salmonella Choleraesuis ATCC 7001, Salmonella Typhimurium ATCC 51812; Swimming lane 16:ddH ₂O; Swimming lane 17:Salmonella InfantisATCC 51741; Swimming lane M:200bp molecular weight standard.Can know that from Fig. 1 except Salmonella typhimurium reference culture ATCC51744, all the other serotypes all do not have 197bp specific amplified band.Table 1 is the PCR qualification result of 16 strain reference cultures and 29 strain isolated strains, from table 1, can know, except 1 strain salmonella infantis reference culture ATCC51744 and 2 strain strain isolateds, all the other Salmonellass all do not have the specific amplified band.

Table 1 specificity is estimated bacterial strain uses therefor and test-results

In the table 1 :-: PCR result is negative; +: PCR result is positive.

Step 5, the sensitivity evaluation test

Through measuring, the concentration of the total dna solution of salmonella infantis is 599.96 μ g/mL, makes 10 times of gradient dilutions with sterilized water; Dilute 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 299.83ng, 29.983ng, 2.998ng, 299.83pg, 29.983pg, 2.998pg, 299.83fg, 29.983fg, 2.998fg, 0.29983fg/ reaction; Swimming lane M:200bp molecular weight standard.Can see band clearly at eight lanes, institute's corresponding DNA concentration is 29.983fg/PCR, and can't see amplified band after the 9th swimming lane.Therefore, judge that the PCR detection sensitivity is 29.983fg/PCR, have higher sensitivity.

Embodiment 2

The detection of the doubtful bacterial strain of Salmonellas

Utilize the salmonella infantis serotype PCR detection method of embodiment 1 that 53 strains doubtful bacterial strain of isolating Salmonellas from food samples is detected; 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 2 strains result that is positive; The doubtful bacterial strain of this 2 strain identifies it is O7:Hr:5 through salmonella diagnostic serum (Lanzhou Institute of Biological Products); Determine that it is salmonella infantis (the serum authentication step sees also product description and GB GB/T 4789.4-2008), other doubtful bacterial strains are not salmonella infantises through identifying.Prove that through present embodiment the PCR detection method of salmonella infantis has extreme high reliability.

Sequence table

< 110>Shanghai Communications University

< 120>salmonella infantis PCR detection method and wherein nucleic acid and primer are right in the food

<160>3

<170>PatentIn?version?3.3

<210>1

<211>692

<212>DNA

< 213>salmonella infantis

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atgtcagagg?cagtgttttt?cgtcgagaat?gctgaagagt?tggcaaaaca?gaaaatggat 60

aatattaatc?ctgaactttc?tgaaaaattc?cagcttttaa?ttaaatttct?ttcaagattt 120

cctgaaagtt?gttcaaatcc?gcgctcgaaa?caagttagaa?aaaactttgg?taaagcagaa 180

catatagaat?atttagctca?aaatttcaat?gaaagtcggc?ttccgaaaaa?gccaacgcca 240

cctactacta?tccctgatga?ggttgtcagt?ttagtcctta?atgtaagctt?tgatataccg 300

caagaaaatc?tcaatagaat?taaagaagaa?catcgactct?ctatggcttc?tgaaaacatt 360

gttggagatc?ttctggaaag?atatcttgct?gaaaaattag?agccatgtgg?atggatatgg 420

tgttcaggaa?caagtgtaaa?agcagtagat?tttattcatt?atgacaatga?aaaagatgag 480

tggggtcttc?tacaagtaaa?aaatagagat?aatactgaga?actcttcaag?tagcaaaatt 540

cgcgacaata?cgccaattaa?aaaatggttt?agaacattct?cacaaagaga?tgctacaaat 600

tgggaaaatt?tccctgacga?agtttcatcc?aaagacctca?atgaagatga?ttttagagct 660

tttgttgaaa?gctacttacg?aaaaattaaa?ta 692

<210>2

<211>20

<212>DNA

< 213>artificial sequence

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agccaacgcc?acctactact 20

<210>3

<211>22

<212>DNA

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tgaacaccat?atccatccac?at 22

Claims (5)

1. a method that detects salmonella infantis PCR in the food is characterized in that, comprises the steps:

Step 1 is according to the conserved sequence SEQ ID NO:1 design of amplification primers in the genomic dna sequence of salmonella infantis;

Said primer is specially: the sequence of forward primer shown in SEQ ID NO:2 with the sequence of reverse primer 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 infantis; Said judgement is specially: if corresponding single amplified band appears in electrophoresis result, then contain salmonella infantis in the interpret sample; If corresponding single amplified band do not occur, then do not contain salmonella infantis in the sample.

2. 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 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.

3. 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; Each round-robin program is: 94 ℃ of sex change 30s, and 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. method according to claim 1 is characterized in that, in the step 3, said judgement is specially: detect the detected through gel electrophoresis amplified production and whether have single amplified band in the 197bp position, if exist, then contain salmonella infantis in the interpret sample; If corresponding single amplified band do not occur, then do not contain salmonella infantis in the sample.

One kind right according to the primer in the said method of claim 1, it is characterized in that this primer is to being specially like base sequence respectively shown in SEQ ID NO:2 and the nucleic acid shown in SEQ ID NO:3.