Fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis

Abstract

The invention discloses a fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis. The method specifically comprises the following steps of(1) taking the PirAVp gene of VpAHPND as a detection target gene and designing a specific primer and a TaqMan-MGB probe; (2) extracting the DNA of the VpAHPND, constructing a recombinant plasmid, preparing a standard substance, and storing at -20 DEG C for use; (3) performing a quantitative PCR, and establishing a standard curve and a standard equation between the logarithm of the initial templatecopy number and the threshold cycle number; (4) detecting the to-be-detected sample with the primer and the probe in the step (1),and based on the measured threshold cycle number, according to the standard curve, calculating the copy number of the VpAHPND in the to-be-detected sample. The Fluorescent quantitative PCR method for detecting the VpAHPND for non-diagnostic purposes established by theinvention has the advantages of high sensitivity, strong specificity, good reproducibility, rapid quantification and the like, and can be used for clinically detecting the VpAHPND of shrimp samples and water samples.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a fluorescent quantitative PCR method for detecting acute hepatopancreatic necrosis vibrio parahaemolyticus. Background technique [0002] Since 2009, Acute hepatopancreas necrosis disease (AHPND) has been widespread in shrimp farming worldwide, mainly harming Litopenaeus vannamei, Penaeus monodon and Fenneropenaeus chinensis), resulting in a sharp drop in the production of prawns, which has brought huge economic losses to the shrimp farming industry. The prawn farming industry in my country, which is dominated by Litopenaeus vannamei farming, has also suffered heavy losses. The cause of the disease has no obvious symptoms and no obvious precursors before death. Most of the prawns die at the bottom of the pond and are not easy to be found. It is commonly known as "stealing death disease". Since the disease mainly occurs 7-30 days after the shrimps are stocked, so Als...

AI Technical Summary

Problems solved by technology

[0004] However, in actual detection work, one-step PCR is not sensitive enough, and it is difficult to detect latently infected samples; nested PCR is highly sensitive, but requires two rounds of PCR and electrophoresis, which is cumbersome to operate and takes a long time to detect; LAMP is sensitive and fast, but easy False positives occur; TaqMan probe fluorescence quantitative PCR is not sensitive due to the length of the probe is too long

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