Primer pair for detecting canine diarrhea viruses through one-step method, TaqMan probe, method and application

A diarrhea virus and primer pair technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, and resistance to vector-borne diseases, etc., can solve the problems of rapid identification and detection, cumbersome process, and long time-consuming, and achieve the best specificity and repeatability, improved sensitivity, and reduced cost

Active Publication Date: 2020-10-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of conventional etiological detection methods is not only time-consuming, but also cumbersome.

Method used

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  • Primer pair for detecting canine diarrhea viruses through one-step method, TaqMan probe, method and application
  • Primer pair for detecting canine diarrhea viruses through one-step method, TaqMan probe, method and application
  • Primer pair for detecting canine diarrhea viruses through one-step method, TaqMan probe, method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The first step is to analyze the whole genome sequences of CPV, CCoV, CAstV and CaKoV, and design primers and fluorescent probes. A set of primer pairs and probes are designed for each pathogen according to the conserved region without interference. The probes are respectively labeled with different fluorophores, where

[0040] The primer pair sequence and TaqMan probe sequence for detecting canine parvovirus (Canine Parvovirus, CPV) are:

[0041] The primer sequence of the upstream primer CPV-QF is SEQ ID NO: 1, which is 5'-TTCGGTAAACTTAACACCAAC-3',

[0042] The primer sequence of the downstream primer CPV-QR is SEQ ID NO: 2, which is 5'-CTGTATGTTAATATAGTCACCCA-3',

[0043] The sequence of the Taqman probe CPV-probe is SEQ ID NO: 3, which is 5'6-FAM-CTGCAATTTCTCTGAGCTTA-3'MGB;

[0044] The primer pair sequence and TaqMan probe sequence for detecting canine coronavirus (Canine Coronavirus, CCoV) are:

[0045] The primer sequence of the upstream primer CCV-QF is SEQ ID ...

Embodiment 2

[0072] The multiple real-time fluorescent quantitative PCR detection of embodiment 2CPV, CCoV, CAstV and CaKoV

[0073] Based on the primer pair and probe in Example 1, we performed the following verification operations.

[0074] Step 1. Collect the anal swab or feces of dogs with diarrhea, pretreat the disease materials, use the existing equipment in the laboratory to extract total RNA and DNA from diarrhea feces samples or intestinal tissues, reverse transcribe them into cDNA templates, and finally obtain The template is a mixture of DNA and RNA. Since CPV is a DNA virus and the other three are RNA viruses, the detection template needs to be a mixture of DNA and cDNA;

[0075] Step 2: Use the primer pair designed in the first step to amplify the target gene by ordinary PCR and connect to the pMD18-T vector to construct the target recombinant plasmid.

[0076] In step 3, the feasibility of the method is verified according to the qPCR method and the fluorescence acquisition c...

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Abstract

The invention discloses a primer pair for detecting canine diarrhea viruses through a one-step method, a TaqMan probe, a method and application. Exploratory research on multiplex fluorescent PCR is carried out in multiple aspects of the specificity of a primer probe, a key factor in a reaction system, quality control construction and the like; a detection fragment is amplified by nucleic acid or reverse transcription products of a sample to be detected, and connected to a pMD18-T vector; plasmids are successfully constructed and used as a template; a multiplex real-time fluorescent quantitative PCR method of canine parvovirus, canine coronavirus, canine astrovirus and canine kobuviruses is established; the patent allows that at most four viruses causing canine diarrhea can be simultaneously detected in one reaction tube; the detection sensitivity is up to 1*10<2> copies; the detection sensitivity and the specificity of four canine diarrhea viruses can be obviously improved; furthermore, the detection period is effectively shortened; and thus, the patent has important meanings.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer pair, a TaqMan probe, a method and application for one-step detection of canine diarrhea virus. Background technique [0002] Canine Parvovirus (CPV), Canine Coronavirus (CCoV), Canine Astrovirus (CAstV) and Canine Kobuviruses (CaKoV) have caused severe disease in pet dogs in my country and around the world. The morbidity and mortality of canine diarrhea remain high, and they are the main types of canine diarrhea virus diseases, which often cause serious economic and spiritual losses to the owners. In particular, CPV can cause 70% mortality in puppies and canine hemorrhagic enteritis; CCV mainly causes gastroenteritis; while the clinical manifestations and prevalence of CAstV are not yet clear, mainly associated with CPV infection; CaKoV is considered There is some association with canine viral diarrhea, which was first detected in dogs with acute gastroenteritis...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2561/101C12Q2545/114Y02A50/30
Inventor 粟硕王茹怡张文艳潘中洲李改茹
Owner NANJING AGRICULTURAL UNIVERSITY
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