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TaqMan fluorogenic quantitative PCR kit and detection method for porcine deltacoronavirus

A fluorescence quantitative and coronavirus technology, applied in the field of molecular biology detection of viruses, can solve the problems that the specificity and sensitivity of detection cannot meet the requirements of porcine delta coronavirus diagnosis and the low positive detection rate of viruses, and achieve good results. Specificity, good inter-assay and intra-assay reproducibility, and high sensitivity results

Inactive Publication Date: 2018-01-09
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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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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  • TaqMan fluorogenic quantitative PCR kit and detection method for porcine deltacoronavirus

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Embodiment

[0024] Embodiment: A TaqMan fluorescent quantitative PCR kit and detection method for porcine delta coronavirus.

[0025] 1. Primer and probe design

[0026] According to the whole genome sequence of porcine delta coronavirus (PDCoV) published by GenBank, the conserved N gene sequence was found by sequence comparison with DNA Star software, and two pairs of primers and two probes were designed using the probe design software Primer Express5.0 , see Table 1 for specific information. Primers and probes were synthesized by Beijing Huada Gene Services Co., Ltd.

[0027] Table 1 PDCoV primers and probes

[0028]

[0029] 2. Extraction of PDCoV RNA

[0030] PDCoV RNA was extracted according to the RNeasy Mini kit (50) QIAGen operating instructions. Specific operation: Take 350 μL of the virus treatment solution and add it to a 1.5 mL centrifuge tube, add an equal volume of RLT and 3.5 μL of β-mercaptoethanol, let it stand on ice for 5-10 minutes, then quickly add 700 μL of abs...

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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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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCY02A50/30
Inventor 刘新生张永光王永录方玉珍周鹏肖帅
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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