CN101748214A - Pullorum-typhoid salmonella PCR detection method, nucleic acid and primer therein - Google Patents

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 no pullorum-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

Pullorum-PCR detection method of Salmonella typhi and its nucleic acid and primer pair

technical field

The invention relates to a PCR detection method in the technical field of food safety inspection and a nucleic acid and a primer pair therein, in particular to a PCR detection method for pullorum-typhi Salmonella and the nucleic acid and a primer pair therein.

Background technique

Chicken salmonellosis is a bacterial infectious disease of chickens caused by certain pathogenic Salmonella of the genus Salmonella. It is generally divided into three types of pullorum, typhoid and paratyphoid. Among them, pullorum and typhoid are more common. The danger is strong. Pullorum is caused by Salmonella Gallinarum serotype (Salmonella Gallinarum) infected chickens, mainly against chicks, the mortality rate of chicks less than 2 weeks after the onset can reach 40-70%, and infected chicks die within 4-7 days , In acute cases, death can occur within 1 to 3 days. Salmonella pullorum serotype (Salmonella Pullorum) infection of chickens can cause typhoid fever in chickens, mainly infecting adult chickens, sometimes chicks are also infected, and the disease begins a few days after hatching, with a high mortality rate. In recent years, these two pathogenic bacteria have been classified into one species, collectively referred to as Salmonella Gallinarum-Pullorum (Salmonella Gallinarum-Pullorum), which mainly exist in organs such as ovaries, testes, and liver, and can be transmitted from infected parent chickens to chicks . The most effective measures to control chicken salmonellosis are strict management and eradication programmes. Therefore, identification and removal of infected parent flocks is critical. At present, there are many reports on the application of PCR detection methods for Salmonella in quarantine. Although the prevention and control of chicken salmonellosis has been actively promoted, there are almost no research reports on the targets and primers for PCR detection of serotype pullorum-typhi Salmonella. There is therefore no report on the establishment of a PCR method for the detection of pullorum-typhi Salmonella serotypes.

Through the literature search of prior art, it is found that for a long time, my country has mainly used the slide agglutination test to detect the antibody of pullorum-typhoid salmonella to identify sick chickens, just as Xu Yaohui, Jiao Xin'an, Li Qiuchun, etc. in "Chinese Veterinary Science and Technology" 2005 As stated in the article "Serological Survey of Monoclonal Antibody Blocking ELISA on Pullorum and Chicken Typhoid in Some Provinces and Cities" published on pages 661-664 of Volume 36, Issue 8, 2010, the sensitivity of this method is limited, and different antigens often appear depending on the quality of the antigen. Stable results.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a PCR detection method for pullorum-typhi Salmonella and nucleic acid and primer pairs therein. Using the detection method of the invention to detect pullorum-typhi Salmonella has short detection time, low cost, more practicality, specific detection results and simple result judgment.

The present invention is achieved through the following technical solutions,

The present invention relates to a kind of PCR detection method of pullorum-typhi Salmonella, comprising the following steps:

Step 1, designing amplification primers according to the conserved sequence SEQ ID NO: 1 in the genomic DNA sequence of Salmonella pullorum-typhi;

Step 2, extract sample DNA and amplify by PCR method;

Step 3: Gel electrophoresis detects the amplified product, and judges whether the sample contains Salmonella pullorum-typhi; the judgment is specifically: if a corresponding single amplification band appears in the electrophoresis result, it means that the sample contains Salmonella pullorum-typhi; If there is no corresponding single amplified band, the sample does not contain Salmonella pullorum-typhi.

In step 1, the primers are specifically: forward primer SEQ ID NO: 2 and reverse primer SEQ ID NO: 3.

In step 2, in the PCR method, the PCR detection system is specifically: 25 μL reaction system is specifically, 10×PCR reaction buffer 2.5 μL, 25 mmol/L Mg ²⁺ 2.0 μL, 2.5 mmol/L dNTP 1.0 μL, 1 μL of 5 μM primer pair, 1 U of Taq enzyme, 2-5 μL of template solution, and finally add water to 25 μL.

In step 2, in the PCR method, the amplification parameters of the PCR detection system are as follows: first, pre-denature at 94°C for 5 minutes, and then start the amplification cycle. Extend at ℃ for 30s; cycle 35 times in total; after the cycle ends, extend at 72°C for 10 minutes, cool down to 12°C, and end.

In step 3, the judgment is specifically: detecting whether there is a single amplification band in the 231bp position of the amplification product by gel electrophoresis, if it exists, it means that the sample contains Salmonella pullorum-typhi; if there is no corresponding single amplification band If the band is increased, the sample does not contain Salmonella pullorum-typhi.

The present invention also relates to a nucleic acid involved in the PCR detection method, the base sequence of which is shown in SEQ ID NO:1.

The present invention also relates to a pair of primers involved in the PCR detection method. The primer pair is specifically a nucleic acid whose base sequences are respectively shown in SEQ ID NO: 2 and SEQ ID NO: 3.

Compared with the prior art, the present invention has the following beneficial effects: the detection time of Salmonella pullorum-typhi is detected by the detection method of the present invention, and the detection cost is reduced because the antiserum that must be used in the traditional detection method is not used; At the same time, the detection method of the present invention can also be used to detect some strains that cannot be detected by antiserum, such as strains that do not express antigens, to make up for the defects of immune detection and to be more practical; the detection target of the present invention has a single specificity , the detection result is specific, and the result determination is simple.

Description of drawings

Fig. 1 is PCR detection method specificity evaluation experiment result among the embodiment 1;

Fig. 2 is the result of the sensitivity evaluation experiment of the PCR detection method in Example 1.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Example 1

Establishment of PCR detection method for pullorum-typhi Salmonella serotypes

Through bioinformatics analysis, the common specific genes SG1046 and SG1047 (SEQ ID NO: 1) were found from the genome DNA sequence of pullorum pullorum-typhi Salmonella, which were used as the detection target genes of pullorum pullorum-typhi salmonella.

Input the DNA sequences of genes SG1046 and SG1047 into the primer design software Primer Premier 5.0 to design primers, set the GC% range to 40-60%, and the product size range to 100-500bp, and select the primer pair SG5- L/R is used as the identification primer (the primer was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.), and the primer sequence is as follows:

SG5-L: 5'-TTATGTTACGGGACGAGTG-3' (SEQ ID NO: 2);

SG5-R: 5'-CAAGACTCCCTGCCCTAT-3' (SEQ ID NO: 3);

Step 2, DNA template preparation

Inoculate various serotypes of Salmonella strains (as shown in Table 1) such as Salmonella typhimurium, Salmonella enteritidis, and Salmonella pullorum-typhi into 50 mL of LB liquid medium, and after enriching at 37°C for 8 hours, take 1 mL Bacterial solution, put into a 1.5mL centrifuge tube;

Then centrifuge at 3,000r/min for 10min, take the supernatant, and then centrifuge at 12,000r/min for 5min to collect the bacteria. Resuspend the bacteria with sterile double distilled water, centrifuge and wash, add 100 μL sterile ultrapure water, boil in a boiling water bath for 15 minutes, take it out immediately, and place it at -20°C for 30 minutes. After thawing at 37°C, centrifuge at 12,000r/min for 5min, and take the supernatant and store it at -20°C for later use.

Step 3, PCR detection method specificity evaluation experiment

The PCR detection method is as follows: first add 16.1 μL sterile water to the reaction tube, then add 2.5 μL of 10×PCR reaction buffer, 2.0 μL of 25 mmol/L Mg ²⁺ , 1.0 μL of 2.5 mmol/L dNTP, and 5 μM primers 1.0 μL, 2.5U/μL Taq enzyme 0.4 μL, finally add 2 μL of template solution, and use sterile water as the template as the negative control of the reaction. Then centrifuge the reaction tube, put it into the PCR reactor, and proceed according to the following PCR cycle parameters: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles, each cycle program includes denaturation at 94°C for 30 seconds, annealing temperature at 60°C, The annealing time is 30s, and then extended at 72°C for 30s, after the end of the cycle, extended at 72°C for 10min, and finally cooled to 12°C to end all operating procedures.

Table 1 The strains and test results used for specificity evaluation

In Table 1, -: PCR result is negative; +: PCR result is positive.

16 Salmonella standard strains such as Salmonella typhimurium, Salmonella enteritidis and Salmonella pullorum-typhi and 31 isolated strains (as shown in Table 1, the strains shown in the table are those skilled in the art can obtain through open channels) Genomic DNA was extracted separately according to the method of DNA template preparation. 2 μL of the DNA solution of each strain was taken as a PCR reaction template and added to the PCR reaction system for amplification reaction.

Figure 1 shows the detection results of the standard strains, please refer to the accompanying drawings for their serotypes and strain numbers. In the picture: lanes 1 to 16: AS1.1190, CMCC 50004, CMCC 50017, CMCC 50335, AS1.1174, ATCC 14028, ATCC13076, ATCC 7001, ATCC 15611, ATCC 9150, ATCC 9270, NCTC 4840, ATCC 12002, ATCC51812 NCTC 6017, CMCC 50770; Lane 17: ddH ₂ O; Lane M: 200bp molecular weight standard. It can be seen from Figure 1 that except for the standard strain CMCC50770 of Salmonella pullorum-typhi, other Salmonella have no specific amplification bands, indicating that the primers have good specificity to SG5-L/R. Table 1 shows the PCR identification results of 16 standard strains and 31 isolated strains. It can be seen from Table 1 that, except for 1 standard strain CMCC50770 and 3 isolates of Salmonella pullorum-typhi, all other Salmonella have no specific amplification bands. bring.

Step five, sensitivity evaluation test

After determination, the concentration of the total DNA solution of pullorum-typhi Salmonella was 68.6 μg/mL, which was diluted 10 times with sterile water, and a total of 10 gradients were diluted. 5 μL of each gradient was added to the PCR reaction system and detected by gel electrophoresis. Amplification results; the results are shown in Figure 2, in the figure: lanes 1 to 10: DNA template concentrations were 34.3ng, 3.43ng, 343pg, 34.3pg, 3.43pg, 343fg, 34.3fg, 3.43fg, 0.343fg, 0.034 fg/reaction; lane M: 200 bp molecular weight standard. It can be seen from Figure 2 that a clear band can be seen in the seventh lane, corresponding to a DNA concentration of 34.3fg/PCR, and no amplified band can be seen after the eighth lane. Therefore, it is determined that the detection sensitivity of PCR is 34.3fg/PCR, which has a high sensitivity.

Example 2

Detection of suspected strains of Salmonella

Using the established PCR detection system for pullorum-typhoid Salmonella, 44 suspected strains of Salmonella isolated from chicken manure were detected. The samples were collected from 5 chicken farms in the suburbs of Shanghai. The treatment of chicken manure samples and the isolation of suspected strains refer to the national standard GB/T 4789.4-2008. From it, 11 suspected strains were detected to be positive results, and these 11 suspected strains were identified as O9:H-:- by the Salmonella diagnostic serum (Lanzhou Institute of Biological Products), and it was determined that it was Pullorum pullorum-Salmonella typhi (serum identification steps please See the product manual and the national standard GB4789.4-1994). None of the other suspected strains was identified as Salmonella pullorum-typhi. This example proves that the PCR detection method for the serotype of Salmonella pullorum-typhi Salmonella has very high reliability.

sequence listing

<110> Shanghai Jiaotong University

<120>PCR detection method of pullorum-typhi Salmonella and its nucleic acid and primer pair

<160>3

<170>PatentIn version 3.3

<210>1

<211>1460

<212>DNA

<213> Pullorum-Typhi Salmonella

<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 accacggcg 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 (7)

1. a pullorum-salmonella typhi serotype PCR detection method, is characterized in that, comprises the steps: Step 1, designing amplification primers according to the conserved sequence SEQ ID NO: 1 in the genomic DNA sequence of Salmonella pullorum-typhi; Step 2, extract sample DNA and amplify by PCR method; Step 3: Gel electrophoresis detects the amplified product, and judges whether the sample contains Salmonella pullorum-typhi; the judgment is specifically: if a corresponding single amplification band appears in the electrophoresis result, it means that the sample contains Salmonella pullorum-typhi; If no corresponding single amplification band appears, the sample does not contain Salmonella pullorum-typhi. 2. pullorum pullorum-salmonella typhi serotype PCR detection method according to claim 1 is characterized in that, in step 1, described primer is specifically: forward primer SEQ ID NO: 2 and reverse primer SEQ ID NO: 3. 3. the pullorum-typhi Salmonella serotype PCR detection method according to claim 1 is characterized in that, in step 2, in the PCR method, the PCR detection system is specifically: 25 μL reaction system is specifically, 10×PCR reaction Buffer 2.5 μL, 25 mmol/L Mg2+2.0 μL, 2.5 mmol/L dNTP 1.0 μL, 5 μM primer pair 1 μL, Taq enzyme 1U, template solution 2-5 μL, and finally add water to 25 μL. 4. the pullorum-typhi Salmonella serotype PCR detection method according to claim 1 is characterized in that, in step 2, in the PCR method, the PCR detection system parameters are specifically: first 94 ° C pre-denaturation for 5 minutes, and then start Amplification cycle, the program of each cycle is: denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 30 s; a total of 35 cycles; after the cycle, extend at 72°C for 10 min, cool to 12°C, and end. 5. the pullorum-typhi Salmonella serotype PCR detection method according to claim 1 is characterized in that, in step 3, the judgment is specifically: whether there is a single amplification in the detection gel electrophoresis detection amplification product at the 231bp position Bands, if present, indicate that the sample contains Salmonella pullorum-typhi; if there is no corresponding single amplification band, the sample does not contain Salmonella pullorum-typhi. 6. a nucleic acid in the pullorum-typhi Salmonella PCR detection method as claimed in claim 1, is characterized in that, the base sequence of this nucleic acid is as shown in SEQ ID NO:1. 7. A primer pair of pullorum-typhi Salmonella PCR detection method as claimed in claim 1, characterized in that, the primer pair is specifically base sequence as shown in SEQ ID NO: 2 and as SEQ ID NO: Nucleic acids indicated in 3.

Images