Triple real-time fluorescent PCR detection primers, probes, detection kits and detection methods for four bacteria

Abstract

The invention discloses primers, probes, a test kit and a test method for triple real-time fluorescence PCR detection of Salmonella choleraesuis, Salmonella paratyphi, Salmonella typhi, and Salmonella gallinarum. According to the present invention, a triple real-time PCR method is used, wherein a consensus sequence of Salmonella choleraesuis and Salmonella paratyphi is used for the detection of Salmonella choleraesuis and Salmonella paratyphi, a specific sequence of Salmonella typhi is used for the detection of Salmonella typhi, and a specific sequence of Salmonella gallinarum is used for the detection of Salmonella gallinarum. Whether a sample is contaminated by Salmonella choleraesuis, Salmonella paratyphi, Salmonella typhi, and Salmonella gallinarum is determined via a real-time fluorescence PCR amplification, and then Salmonella choleraesuis and Salmonella paratyphi are distinguished via a single fluorescent PCR amplification; the detection is rapid, the process of preparing sample and issuing test results can be finished in 31hours, the results are reliable, and sensitivity and specificity are high.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to triple real-time fluorescent PCR detection primers, probes and detection methods for Salmonella choleraesuis, Salmonella paratyphi, Salmonella typhi and Salmonella gallinarum. Kits and assay methods. Background technique [0002] Salmonella (Salmonella) belongs to the Enterobacteriaceae family and is an important intestinal pathogenic bacterium for humans. It can also reproduce or cause disease in the intestinal tract of animals. At present, there are more than 2,500 serotypes of Salmonella known, and more than 200 serotypes have been found in my country. There are two main categories of illnesses caused by Salmonella. One is typhoid and paratyphoid, which are caused by typhoid or paratyphoid Salmonella, and occasionally typhoid-like infections are caused by other Salmonella, such as Salmonella choleraesuis and Salmonella enteritidis. Salmonella gallinarum (Salmonella g...

AI Technical Summary

Problems solved by technology

With the development of molecular biology techniques, there have been reports targeting invA, 16srRNA, SpvC, invB, fimA, agfA, SEFl4, sefA, sdf, ssaQ, fimY, etc. as target genes, using common PCR or real-time fluorescent PCR to detect Salmonella. However, the PCR method using the above sequence as the target fragment can only detect a part of the Sa serotype or its virulence gene, and cannot simultaneously obtain the serotype and toxin secretion type of the detected Salmonella.

At present, there is also the commercialized Bax system produced by DuPont. This system uses fluorescent PCR technology to detect Salmonella at a subordinate level. It has high specificity, but it cannot type Salmonella.

Camila et al. designed primers for ompC, SdfI, ViaB, and Spy as the target genes, and established a multiplex PCR detection method for Salmonella (Salmonellaspp), SE (Enteritidis), typhoid Sa (Typhi), ST (Typhimurium), which was applied to Salmonella in chicken For detection, KIM et al. also designed multiple pairs of primers based on the specific sequences of Salmonella typhimurium and Salmonella typhimurium, and established a method for multiplex PCR detection of Salmonella typhimurium and Salmonella typhimurium, and all obtained good results. However, the above methods need to be determined by gel electrophoresis. As a result, the operation is more cumbersome

EdelO'Regan et al. used flic, sefA, sdf, and acek genes to design primer probes, and used multiple fluorescent PCR methods to detect Enteritidis, Gallinarum, Typhimurium, Kentucky, and Salmonella Dublin, but sefA was the target gene and could not accurately distinguish Enteritidis, Gallinarum, and Dublin, flic For the target fragment, it is impossible to distinguish between Typhimurium and Kentucky. David Fde et al. used ordinary PCR and optical PCR to detect Salmonella choleraesuis and Salmonella paratyphi C, but this study did not systematically analyze the amplification efficiency of the fluorescent PCR amplification system. Evaluation

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