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PCR-HRM primer for quickly detecting salmonella gallinarum and application thereof

A PCR-HRM and Salmonella technology is applied in the field of PCR-HRM primers for rapid detection of Salmonella typhimurium.

Inactive Publication Date: 2016-02-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods take a long time to detect, and need to go through procedures such as enzyme digestion and electrophoresis to determine the results, and the efficiency is too low in the purification of cultured Salmonella longus

Method used

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  • PCR-HRM primer for quickly detecting salmonella gallinarum and application thereof
  • PCR-HRM primer for quickly detecting salmonella gallinarum and application thereof
  • PCR-HRM primer for quickly detecting salmonella gallinarum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 is used to detect Salmonella gallinarum typhi HRM primers and reaction conditions

[0036] (1) Primers: According to the complete genome sequence of Salmonella published by NCBI and related information, it is found that Salmonella gallinarum typhi has a regular change at position 598 on the rfbS gene. G, some other serotypes and strains of other types do not contain this gene or have large changes, so specific amplification primers can be designed according to this site. Adopt Oligo7 primer design software, according to the Salmonella rfbS sequence issued by GeneBank (the GeneBank accession number of the rfbS sequence of Salmonella pullorum: LK931482; the GeneBank accession number of the rfbS sequence of Salmonella gallinarum typhi: AF442573; ) and the rfbS sequence obtained by sequencing the Salmonella typhi standard strain (the rfbS sequence is the same as that in GeneBank accession number: AF442573), and a primer pair was designed on both sides of the 598 s...

Embodiment 2

[0042] Embodiment 2 is used to detect the application method of the PCR-HRM primer of Salmonella gallinarum typhi

[0043] (1) Preparation and PCR-HRM analysis of Salmonella typhi standard substance

[0044] (1) Extraction of bacterial DNA:

[0045] Resuscitate the standard strain of Salmonella typhi (CICC21510), pick a single colony and shake the bacteria in 1mL LB medium for 12 hours, use the OmegaDNAkit kit to extract the bacterial DNA, follow the instructions to extract and purify the sample DNA, and store the extracted DNA at -20°C .

[0046] (2) Preparation of standard products:

[0047] In order to verify the feasibility of the primers and methods of the present invention, construct a positive sample at the same time, provide a positive template for subsequent clinical sample detection, use the extracted standard strain DNA as a template, use the upstream primer Sg-up described in Example 1, the downstream Primer Sg-low was used for ordinary PCR amplification.

[00...

Embodiment 3

[0066] Example 3 is used to detect the clinical application of Salmonella gallinarum typhi HRM primer

[0067] Liver samples were collected from clinically suspected Salmonella typhi-affected chickens, and a total of 15 samples were collected.

[0068] (1) Processing of clinical samples

[0069] Take 25g of the liver of the tested chicken, grind the sample into a sterile enrichment bag, add 225g of BPW (protein water) to pre-enrichment in a constant temperature shaker at 37°C at 100r / min for 6-8 hours, take 1mL of the pre-enrichment solution, Add 9 mL of selective enrichment solution TTB (sodium tetrathiosulfonate brilliant green), and incubate at a constant temperature of 37°C for 20 hours.

[0070] (2) Extraction of sample DNA

[0071] Take 1 mL of the selective pre-enrichment solution, and follow the instructions of the OmegaDNAkit kit to extract and purify the sample DNA, and store the extracted DNA at -20°C.

[0072] (3) PCR-HRM amplification

[0073] Using DNA as a t...

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Abstract

The invention discloses a PCR-HRM primer for quickly detecting salmonella gallinarum and application thereof. The PCR-HRM primer for quickly detecting salmonella gallinarum and the application are established for the first time. The PCR-HRM primer is convenient to operate, and during the detection, only saturated fluorescent dye needs to be added into a PCR; the clinical detection process is simplified, the detection time is greatly shortened; and a method has the advantages of being high in throughput and low in cost. True closed tube operation is achieved from the PCR process to HRM analysis, a PCR product does not need to be transferred to other devices in the whole process, cross contamination is avoided, and quantitative analysis can be finished. The PCR-HRM primer is high in specificity, salmonella gallinarum can be distinguished well, and the problem that a conventional detection method is low in salmonella gallinarum identification accuracy is solved. The PCR-HRM primer is high in sensitivity, and the lowest detection limit can be 42 copy numbers per microliter. The PCR-HRM primer has extensive application prospects in rapid diagnosis of salmonella gallinarum.

Description

technical field [0001] The invention belongs to the technical field of diagnosis and detection of pathogenic microorganisms, in particular to a PCR-HRM primer for rapid detection of Salmonella gallinarum typhi and its application. Background technique [0002] Salmonella (Salmonella) is one of the most common zoonotic pathogens. There are more than 2500 serotypes. It has a wide range of hosts and can widely survive in humans, warm-blooded and cold-blooded animals, as well as in food and the external environment. Many animals are pathogenic. In my country, 70% to 80% of bacterial food poisoning is caused by Salmonella, and 90% is animal food such as meat. Salmonella gallinarum (Salmonella gallinarum) is a serotype of Salmonella, which can cause poultry septicemia infectious disease, sick chickens can die quickly, mainly against poultry over 3 months old, mainly occurs in chickens, and can also infect turkeys and duck. Its symptoms are similar to those of pullorum. Therefore...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/42
CPCC12Q1/686C12Q2527/107C12Q2563/107
Inventor 廖明任行星张建民徐成刚詹泽强梁德媚
Owner SOUTH CHINA AGRI UNIV
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