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

A Salmonella detection method technology, applied in the field of safety inspection of poultry and its products, can solve the uninvented Salmonella typhimurium PCR detection method nucleic acid and primers, multiple PCR costs, high experimental technology, false positive PCR results and other problems, to achieve detection The effect of short time, simple result judgment and specific test result

Inactive Publication Date: 2012-08-01
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 specific PCR detection method for Salmonella typhimurium

[0015] Step 1, designing primer pairs for specific amplification of Salmonella typhimurium

[0016] Through bioinformatics analysis, a conserved and specific STM4493 gene was screened from the genome DNA sequence of Salmonella typhimurium, and it was used as a 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%-60%, and the product size range to 100-500bp, select the primer pair from the alternative primer pair, and the primer pair sequence is as follows (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, DNA template pr...

Embodiment 2

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

[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 C), Salmonella choleraesulus (S.Choleraesuls), Salmonella pullorum (S.Pullorosis), Salmonella duck (S.Anatis), Salmonella chicken (S.Gallinarum), Salmonella Dublin (S.Dublin), Salmonella enteritidis (S. Enteritidis), Salmonella Heidelberg (S. Heidelberg), Salmonella Senftenberg (S. Senftenberg) genomic DNA templates.

[0030] Step 2: Specificity evaluation test of PCR detection method for Salmonella typhimurium

[0031] According to the PCR reaction system in Step 3 of Example 1, take 1 μL of the DNA templates of each strain prepared in Step 1 as PCR reaction templates and add them to the PCR reaction system respectively, and use 1 μL of sterile double-distilled water as ...

Embodiment 3

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

[0040] Step 1, DNA template preparation

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

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

[0043] Take 2 μL of each gradient and add it into 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 3, result judgment

[0045] Take 5 μL of the PCR amplification product, perform electrophoresis analysis on 2% agarose gel, and observe under ultraviolet light irradiation. If there is a single amplification band at the ...

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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 a 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, which is the most common infection in infants and young children. The main clinical symptoms are fever, nausea, vomiting and diarrhea. The bacterium is a Gram-negative bacillus belonging to Salmonella group B, which does not form spores, has no capsule, but has flagella. The bacterium is widely distributed in nature, has strong resistance in the external environment, can reproduce rapidly at room temperature, is resistant to low temperature, dryness, and is not heat-resistant, and is sensitive to acid, ultraviolet rays and various chemical disinfectants. The bacterium can be divided into 608 phage typ...

Claims

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Application Information

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Patent Type & Authority Applications(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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