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Nucleic acid, kit and detection method for detecting pigeon salmonella paratyphi

A detection kit and salmonella detection technology, applied in the biological field, can solve the problems of low accuracy of results, cumbersome operation steps, and undiscovered problems, and achieve the effects of accurate results, high sensitivity, and simple operation

Active Publication Date: 2018-11-06
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In my country, some scholars have successively used Salmonella typhimurium fimA, fimY, fljB and other genes as the target genes to carry out specific PCR detection of Salmonella typhimurium, but the conventional PCR method for bacterial isolation and identification is cumbersome and the accuracy of the results is low.
Find through literature retrieval to prior art, have not yet found and pigeon Salmonella paratyphi PCR detection method of the present invention, the nucleic acid sequence from pigeon origin relevant report

Method used

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  • Nucleic acid, kit and detection method for detecting pigeon salmonella paratyphi
  • Nucleic acid, kit and detection method for detecting pigeon salmonella paratyphi
  • Nucleic acid, kit and detection method for detecting pigeon salmonella paratyphi

Examples

Experimental program
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Effect test

Embodiment 1

[0033] The establishment of embodiment 1 pigeon paratyphi Salmonella specific PCR detection method

[0034] Step 1, designing primer pairs for specific amplification of Salmonella paratyphi

[0035]Through bioinformatics analysis, the conserved and specific FliC gene was screened from the genomic DNA sequence of Salmonella typhimurium, and it was used as the target gene for detection of Salmonella paratyphi. The gene DNA sequence was input into the software DNASTAR to design the amplification primer pair, and set GC% ranges from 40% to 60%, and the product size ranges from 400 to 700bp. From the candidate primer pairs, through a large number of experiments and adaptive adjustments to the primers, the final verification is selected as a specific primer pair, upstream of Salmonella paratyphi The sequence of the primer Fli1 and the downstream primer Fli2 of Salmonella paratyphi are shown below, and the primers can be synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The ...

Embodiment 2

[0050] Example 2 Pigeon Paratyphi Salmonella PCR detection method specificity evaluation experiment

[0051] Step 1, DNA template preparation

[0052] The strains closely related to Salmonella paratyphi were selected as reference strains, and the reference strains were Salmonella typhimurium (S.Typhimurium), Salmonella gallinarum typhi (S.Gallinarum), Salmonella pullorum (S.Gallinarum), Salmonella choleraesuis (S. Choleraesuls), Salmonella enteritidis (S.Enteritidis), Escherichia coli (E.Coli), these strains can be purchased from China Veterinary Microbiology Culture Collection Center (CVCC) or China Medical Bacteria Collection Center (CMCC). Genomic DNA templates of Salmonella typhimurium, Salmonella gallinarum typhi, Salmonella pullorum, Salmonella choleraesuis, Salmonella enteritidis, and Escherichia coli were respectively extracted according to step 2 of Example 1.

[0053] Step 2, specificity evaluation test of PCR detection method for Salmonella paratyphi in pigeons

...

Embodiment 3

[0061] Example 3 Pigeon paratyphi Salmonella PCR detection method sensitivity evaluation experiment

[0062] Step 1, DNA template preparation

[0063] According to step 2 of Example 1, the DNA genome template of Salmonella paratyphi was extracted respectively. After detection by OD260 / 280, the concentration of Salmonella paratyphi DNA solution was 39.5 ng / μL, and 10-fold gradient dilution was performed with sterile water, and a total of 7 dilutions were made. gradient.

[0064] Step 2: Sensitivity evaluation test of PCR detection method for Salmonella paratyphi in pigeons

[0065] Take 1.5 μL of each gradient and add to the PCR reaction system, according to the third method of step 1 of Example 1, but only add the sequences of the upper and lower primers of Salmonella paratyphi pigeonii, such as the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, to the DNA template Perform PCR amplification detection.

[0066] Step 3, result judgment

[0067] Take 8 μL of the PCR amplific...

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Abstract

The invention provides a nucleic acid, a kit and a detection method for detecting pigeon salmonella paratyphi. The invention provides a primer pair for detecting pigeon salmonella paratyphi and an internal reference primer pair for detecting salmonella. The size of the amplified fragments is 613 bp for pigeon salmonella paratyphi and 463 bp for salmonella. A rapid and accurate PCR detection methodfor detecting pigeon salmonella paratyphi is established in the invention, and the method can accurately detect clinical suspicious strain samples, distinguish other common pathogenic salmonella serotypes, and the amplified results of non-salmonella pathogenic bacteria are negative. The detection method provided by the invention has the advantages of high specificity, high sensitivity, short detection time, simple operation, easy judgment and low requirements on equipment, and only needs half a day to complete the detection and plays a very important role in the diagnosis and control of epidemic situation, thereby minimizing economic losses and harm to public health.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid for detecting Salmonella paratyphi in pigeons, a kit containing the nucleic acid and a detection method. Background technique [0002] Salmonella is an important food-borne pathogen and an important pathogen that threatens public health and safety. There are more than 2,600 serotypes. This bacterium can cause various infectious diseases of poultry and mammals, and can also cause human Disease is an important zoonotic pathogen. Among them, Salmonella typhimurium (Salmonella typhimurium) belongs to Salmonella group B. Salmonella typhimurium (Copenhagen variant) is the pathogenic bacterium of pigeon paratyphoid fever, which is highly pathogenic to young pigeons. However, there are few domestic researches on the diagnosis of salmonella from pigeons, and the detection of this bacterium mainly relies on traditional culture. method (see national standard GB / T478...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12N15/11C12R1/42
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143
Inventor 张莉陈小玲章振华先宏杨兵陈葵许健樊艺丹闫伟卢秋翰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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