TaqMan fluorogenic quantitative PCR kit and detection method for porcine deltacoronavirus

Abstract

The invention discloses a TaqMan fluorogenic quantitative PCR kit and a detection method for porcine deltacoronavirus. The kit comprises specific primers and a probe, wherein primer sequences are as follow: the upstream primer is 5'-ACGTCGTAAGACCCAGCATC-3' and the downstream primer is 5'-CCCACCTGAAAGTTGCTCTC-3', and a probe sequence is 5'-FAM-GTATGGCTGATCCTCGCATCATGGC-BHQ1-3'. A gene part fragment648bp of the porcine deltacoronavirus is expanded, a lowest detection limit of the TaqMan real-time fluorogenic quantitative PCR disclosed by the invention is 26.6copies/mu L, and the detection results of the TaqMan real-time fluorogenic quantitative PCR to swine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine kobuvirus, porcine reproductive and respiratory syndromevirus as well as foot and mouth disease virus are all feminine. The TaqMan probe real-time fluorogenic quantitative PCR method for detecting the porcine deltacoronavirus (PDCoV) disclosed by the invention has the advantages of strong specificity, high sensitivity and good inter-batch and intra-batch repeatability. When the TaqMan probe real-time fluorogenic quantitative PCR method disclosed by theinvention are used for detecting 194 clinical pig manure samples, a result shows that a PDCoV positive rate is 22.1% and is about one time higher than a 11.9% positive rate of detecting the clinicalpig manure samples by general RT-PCR.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection of viruses, and in particular relates to a TaqMan fluorescent quantitative PCR kit and detection method for porcine delta coronavirus. Background technique [0002] Porcine delta coronavirus (PDCoV), also known as porcine D-coronavirus, mainly infects the entire small intestine of pigs, causing enteritis accompanied by diarrhea and vomiting, and the morbidity and mortality of suckling pigs is as high as 50%-100%. At present, the detection methods for porcine delta coronavirus at home and abroad are mainly ELISA method, RT-PCR and fluorescent quantitative PCR detection method. The clinical incidence is relatively similar, which increases the difficulty of detection and identification of PDCoV. Among the many PDCoV detection methods, RT-PCR method has become one of the main methods for detecting PDCoV research due to its high sensitivity and specificity, but the positive detecti...

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Problems solved by technology

[0003] The purpose of the present invention is to provide a kind of porcine delta coronavirus TaqMan fluorescent quantitative PCR kit and detection method, aiming to solve the problem of low virus positive detection rate, detection specificity and sensitivity during existing porcine delta coronavirus RT-PCR detection Sex has not yet met the question of porcine deltacoronavirus clinical sample diagnosis

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