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Specific primer and multiplex-PCR detection kit for detecting spoilage pathogenic bacteria

A specific and pathogenic technology, applied in microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of time-consuming and laborious, and achieve the effect of simple and convenient operation, low cost and economical benefits.

Active Publication Date: 2017-08-04
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In actual operation, people find that the traditional method has many problems such as time-consuming and labor-intensive

Method used

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  • Specific primer and multiplex-PCR detection kit for detecting spoilage pathogenic bacteria
  • Specific primer and multiplex-PCR detection kit for detecting spoilage pathogenic bacteria
  • Specific primer and multiplex-PCR detection kit for detecting spoilage pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The multiplex PCR detection kit for detecting staphylococcus saprophyticus and macrococcus caseolyticum in low-temperature meat products of the present invention comprises:

[0039] 1.2×PCR TaqMix 5 tubes (250μl / tube);

[0040] 2. Primer pair 1: Upstream primer Mid-F 1 branch (50 μl / branch); downstream primer Mid-R 1 branch (50 μl / branch)

[0041] 3. Primer pair 2: 1 downstream primer Duf-F (50 μl / recipe); 1 downstream primer Duf-R (50 μl / recipe)

[0042] 4. One positive control DNA of Staphylococcus saprophyticus (250 μl / bottle); one positive control DNA of E. caseinosa (250 μl / bottle)

[0043] 5. Negative control DNA 1 branch (250μl / cart);

[0044] 6. One bottle of sterilized deionized water (1000μl / cartridge).

[0045] The above-mentioned kit can detect 50 samples and store them at -20°C with a shelf life of 1 year.

[0046] Among them, 2×PCR TaqMix is ​​a commercial reagent;

[0047] Primer pair 1: Staphylococcus saprophyticus

[0048] Upstream primer Mid-F: 5'-...

Embodiment 2

[0058] The method for using the multiplex PCR kit for detecting Staphylococcus saprophyticus and Eclococcus caseolyticum in low-temperature meat products of the present invention is as follows:

[0059] 1. Extract total DNA from pure bacterial cultures, low-temperature meat products (such as sausages, bacon, salted duck, etc.), spoilage food and other disease materials. DNA extraction can use commercial kits.

[0060] 2. The 2×PCR TaqMix in the kit, primer pair 1 (upstream primer Mid-F, downstream primer Mid-R), primer pair 2 (downstream primer Duf-F, downstream primer Duf-R), sterile deionized Water and the extracted DNA template were prepared into a PCR reaction solution, and the total system was 50 μl. At the same time, use the positive control DNA in the kit as a template to amplify the positive control; use the negative control DNA in the kit as a template to amplify the negative control. The specific preparation method of the PCR reaction solution is shown in Table 1: ...

Embodiment 3

[0073] Specific detection of the kit of the present invention

[0074] Extract 10 strains of Staphylococcus squirrel, 5 strains of Staphylococcus aureus, 5 strains of Enterococcus faecalis, 5 strains of Enterococcus faecium, 10 strains of Escherichia coli, 10 strains of Salmonella, 10 strains of Pasteurella, and 5 strains of duck with bacterial DNA extraction kit The genomic DNA of Riemerella pneumoniae was used as a template to perform PCR detection with the kit of the present invention. The results showed that Staphylococcus squirrel, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Salmonella, Pasteurella, and Riemerella anatipestifer were all negative, indicating that the specificity of the kit was good.

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Abstract

The invention discloses a multiplex-PCR detection kit for detecting main spoilage pathogenic bacteria (staphylococcus saprophyticus and macrococcus caseolyticus) in low-temperature meat products. The invention provides a primer group for detecting staphylococcus saprophyticus and macrococcus caseolyticus, and the primer group comprises a primer pair 1 and a primer pair 2. The invention designs the two pairs of primers for specific amplification aiming at the specific segments of staphylococcus saprophyticus and macrococcus caseolyticus, the amplification segments are respectively staphylococcus saprophyticus-201bp and macrococcus caseolyticus-392bp, and a sample genome can be correspondingly judged through multiplex PCR and electrophoresis detection. Through the two pairs of specific primers and one-step multiplex PCR, the staphylococcus saprophyticus and macrococcus caseolyticus in the low-temperature meat product can be identified, and the multiplex-PCR detection kit has the characteristics of being rapid, low-cost, simple to operate, and easy to judge in results.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a specific primer for detecting putrefactive pathogenic bacteria and a multiplex PCR detection kit. Background technique [0002] Low-temperature meat products refer to a type of meat products that are processed and cooked by steaming, boiling, smoking, roasting, etc. The processing technology is carried out under normal pressure, and the temperature is often lower than 100°C, and the central temperature of the meat products is only 72-85°C, so the low-temperature Meat products have moderate protein denaturation during processing, and have the characteristics of tight meat structure, high elasticity, good chewing feeling, tenderness and juiciness. At the same time, they overcome the poor taste, poor flavor and loss of nutrients in high-temperature meat products. The disadvantage is that the original nutrition and flavor of meat products are kept to the maximum exte...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04C12N15/11C12R1/44C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 郑福英陈启伟宫晓炜刘永生
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI