PCR (polymerase chain reaction) detection method for specificity of salmonella typhimurium

A Salmonella and detection method technology, applied in the field of safety inspection of poultry and its products, can solve the problems such as nucleic acid and primers, multiple PCR cost, high experimental technology, false positive PCR results, etc. that have not been invented for Salmonella typhimurium PCR detection method, and achieve detection The effect of short time, simple result judgment, and specific test results

Inactive Publication Date: 2014-07-02
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some scholars have used Salmonella typhimurium fimA, fimY, fliC, fljB and other genes as the target genes to carry out specific PCR detection on Salmonella typhimurium, but due to the low specificity of the target site, the PCR results produce false positives, which makes it difficult to judge
In addition, some scholars designed 4 pairs of primers based on the 16S DNA, spvC, invB, and fimA genes of Salmonella typhimurium to detect Salmonella typhimurium by multiplex PCR. high
[0003] Find through literature retrieval to prior art, as yet invention and the Salmonella typhimurium PCR detection method of the present invention, nucleic acid and the relevant report of primer

Method used

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  • PCR (polymerase chain reaction) detection method for specificity of salmonella typhimurium
  • PCR (polymerase chain reaction) detection method for specificity of salmonella typhimurium
  • PCR (polymerase chain reaction) detection method for specificity of salmonella typhimurium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Establishment of a specific PCR detection method for Salmonella typhimurium

[0015] Step 1: Design a primer pair that specifically amplifies Salmonella typhimurium

[0016] Through bioinformatics analysis, the conserved and specific STM4493 gene was screened from the genomic DNA sequence of Salmonella typhimurium, and it was used as the target gene for detecting Salmonella typhimurium. The gene sequence is shown in SEQ ID NO: 1;

[0017] Input the DNA sequence of the STM4493 gene into the software Primer Premier 5.0 to design the amplification primer pair, set the GC% range to 40% to 60%, and the product size range to 100 to 500bp, and select the primer pair from the alternative primer pairs. The primer pair sequence is as follows (The primers were synthesized by Shanghai Jikang Biotechnology Co., Ltd.)

[0018] Upstream primer: 5-'AAATGTGCTTGAGGCGTTAG-3' (SEQ ID NO: 2);

[0019] Downstream primer: 5-' CGTGCGGCGATGTTAGTT-3' (SEQ ID NO: 3)

[0020] Step 2, ...

Embodiment 2

[0027] Example 2 Specificity evaluation experiment of Salmonella typhimurium PCR detection method

[0028] Step 1, DNA template preparation

[0029] Salmonella typhimurium (S.Typhimurium), Salmonella paratyphi A (S.Paratyphi A), Salmonella paratyphi B (S.Paratyphi B), Salmonella paratyphi C (S.Paratyphi) were extracted according to step 2 of Example 1. C), Salmonella Gallinarum (S.Choleraesuls), Salmonella Pullorosis (S.Pullorosis), Salmonella Duck (S.Anatis), Salmonella Gallinarum (S.Gallinarum), Salmonella Dublin (S.Dublin), Salmonella Enteritidis (S. Enteritidis), S. Heidelberg (S. Heidelberg), S. Senftenberg (S. Senftenberg) genomic DNA template.

[0030] Step 2, specificity evaluation test of Salmonella typhimurium PCR detection method

[0031] According to the PCR reaction system in step 3 of Example 1, take 1 μL of the DNA template of each strain prepared in step 1 as the PCR reaction template and add it to the PCR reaction system respectively, and use 1 μL of sterile...

Embodiment 3

[0039] Example 3 Sensitivity evaluation experiment of Salmonella typhimurium PCR detection method

[0040] Step 1, DNA template preparation

[0041] According to step 2 of Example 1, the DNA genome templates of Salmonella typhimurium were extracted respectively, and the concentration of Salmonella typhimurium DNA solution was 24.85 μg / ml by OD260 / 280 detection, and 10-fold gradient dilution was made with sterile water, and a total of 6 gradients were diluted.

[0042] Step 2: Sensitivity evaluation test of Salmonella typhimurium PCR detection method

[0043] Take 2 μL of each gradient and add it to the PCR reaction system, and perform PCR amplification detection on the DNA template according to the method in step 3 of Example 1.

[0044] Step three, result judgment

[0045] Take 5 μL of PCR amplification product, conduct electrophoresis analysis on 2% agarose gel, and observe under ultraviolet light irradiation. If the electrophoresis result shows a single amplification ban...

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Abstract

The invention discloses a PCR (polymerase chain reaction) detection method for specificity of salmonella typhimurium. The PCR detection method includes steps: designing amplifier set sequences SEQ ID NO:2 and SEQ ID NO:3 according to a conservative and specific STM (scanning tunneling microscope) 4493 gene sequence SEQ ID:1 in a salmonella typhimurium complete genome sequence; extracting a DNA (deoxyribose nucleic acid) sample, and realizing amplification by a PCR method; and detecting an amplified product by means of gel electrophoresis and judging whether the sample contains the salmonella typhimurium or not. Particularly, if a single amplification band appears at 456bp in an electrophoresis result, the sample contains the salmonella typhimurium; and if the corresponding amplification band does not appear, the sample does not contain the salmonella typhimurium. When applied to detect the salmonella typhimurium, the PCR detection method has the advantages of short detection time, low cost, capability of detecting specificity of the detection result and simplicity in judging the result.

Description

technical field [0001] The invention relates to a PCR detection method in the field of safety inspection of poultry and its products and a nucleic acid and primer pair therein, in particular to a specific PCR detection method for Salmonella typhimurium. Background technique [0002] Salmonella Typhimurium (Salmonella Typhimurium) is one of the common serotypes of Salmonella pathogenic. It is the most common infection in infants and young children. The clinical symptoms are mainly fever, nausea, vomiting and diarrhea. The bacterium is a gram-negative bacillus belonging to Salmonella group B, which does not form spores and has no capsule, but has flagella. The bacteria are widely distributed in nature, have strong resistance in the external environment, can multiply rapidly at room temperature, are resistant to low temperature, dryness, and heat resistance, and are sensitive to acids, ultraviolet rays and various chemical disinfectants. The bacteria can be divided into 608 ph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
Inventor 程安春黄静玮汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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