PCR detection method of salmonella and primer pair

A Salmonella and detection method technology, applied in the detection field, can solve problems such as unfavorable diagnosis of etiology, time-consuming and laborious, complicated operation, etc., and achieve the effects of specific detection results, simple result judgment, and reduction of detection costs.

CN102399854AInactive Publication Date: 2012-04-04SHANGHAI HAIDI GARDENING
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2012-04-04
Estimated Expiration
Not applicable · inactive patent

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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.
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Description

technical field

[0001] The invention relates to a method in the technical field of detection, in particular to a PCR detection method for Salmonella and a pair of primers. Background technique

[0002] In recent years, foodborne diseases have become a prominent and widespread food safety problem. Salmonella (Salmonella), a member of the Enterobacteriaceae Salmonella genus, is a conditional intracellular parasitic Gram-negative enterobacteriaceae. Salmonella has the highest incidence of bacterial food poisoning, and almost all serotypes can cause disease. The traditional detection method of Salmonella requires selective enrichment culture, and is determined by biochemical reaction and serological test. Although the result of this detection method is relatively reliable, the operation is complicated, time-consuming and laborious, and usually takes 3 to 5 days to obtain the detection result , It is not conducive to timely diagnosis of the cause of disease, to find the source ...

Examples

Embodiment 1

[0027] 1. Select the conserved sequence in the Salmonella genomic DNA sequence and design primers

[0028] The base sequence selected from the genome DNA sequence of Salmonella, such as the sequence shown in SEQ ID NO: 1, is a conserved sequence. Then use the Primer Premier 5.0 software to design primers, and the primer sequences are as follows:

[0029] The base sequence of the forward primer is: gagttatga accaacagct (SEQ ID NO: 2);

[0030] The base sequence of the reverse primer is: gccgctaaac tacacgatga (SEQ ID NO: 3);

[0031] Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0032] 2. Extract the DNA of the sample to be tested and amplify it by PCR;

[0033] Refer to the "People's Republic of China Import and Export Commodity Inspection Industry Standard Export Food Salmonella" (SN 0170-92) for enrichment culture of oysters to be inspected.

[0034] Inoculate each bacterial culture into 30mL lysing broth (LB) liquid medium, sha...

Embodiment 2

[0038] Embodiment 2, broad spectrum and specificity test

[0039] Different serotypes of Salmonella and other non-Salmonella standard strains were purely cultured, and the DNA template was extracted according to the method in Example 1, PCR amplification was performed, and the amplified products were detected by electrophoresis. see results figure 2 and image 3 , figure 2 The strains represented by each lane are: 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; image 3 The strains represented by each lane are: 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 results showed that the 8 different serotypes of Salmonella could amplify the bands, but none of the non-Salmonella could amplify the bands. It can be seen that the detection method in Example 1 has ...

Embodiment 3

[0040] Embodiment 3, sensitivity test

[0041] Take 1 mL of Salmonella culture to extract the DNA template and measure the concentration, and then dilute it to different concentrations in a 10-fold gradient. Take 1 μL of each concentration for PCR amplification, and detect the amplified product. The detection method refers to Example 1. The liquid culture of Salmonella was diluted 10 times, and the DNA template was extracted for amplification detection, and a plate colony counting was performed at the same time. The result is as Figure 4 as shown, Figure 4 Middle, 1, undiluted template; 2, 10 times diluted; 3, 10 2 Double dilution; 4, 10 3 Double dilution; 5, 10 4 1-fold dilution; 6, 10 5 double dilution. It can be seen from the figure that diluting the concentration of the extracted DNA template to 10 4 When multiple times, the target DNA band can still be clearly observed. The DNA template was detected by ultraviolet light, and the concentration of the stock soluti...