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Triple fluorescent quantitative PCR kit for simultaneously detecting bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus and application method of triple fluorescent quantitative PCR kit

A technology for bovine viral diarrhea and bovine rotavirus, applied in bovine diarrhea pathogen detection kits, bovine coronavirus and bovine viral diarrhea virus kits, and simultaneous detection of bovine rotavirus, can solve the problem that cannot meet the needs of multiple viruses Requirements for differential diagnosis of sexually transmitted pathogens, complex detection technology, unsuitable batch detection, etc., to achieve stable detection efficiency, high amplification efficiency, and save detection time

Inactive Publication Date: 2021-05-07
北京三元集团畜牧兽医总站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the detection techniques of nucleic acid hybridization and polyacrylamide gel electrophoresis are complex and costly, and are not suitable for clinical detection
RT-PCR, nano-PCR, fluorescent quantitative PCR and other detection technologies have good specificity and high detection sensitivity, and can be used for detection of tissue, blood, stool and other samples; however, since RT-PCR and nano-PCR are not suitable for batch detection, in production The application is still limited; at the same time, the RT-PCR method needs electrophoresis detection after detection, and the detection sensitivity is low. It is gradually replaced by real-time fluorescent quantitative PCR in rapid detection.
[0011] Fluorescent quantitative PCR method is a fast, accurate and high-throughput virus detection technology, which has been widely used in the rapid diagnosis of pathogens such as hepatitis C and AIDS in recent years; however, the existing methods for bovine rotavirus, coronavirus, or bovine viral diarrhea The virus detection methods are single-plex fluorescent quantitative PCR detection. In actual production applications, single-plex fluorescent quantitative PCR detection technology cannot meet the clinical needs of rapid, accurate, and high-throughput differential diagnosis of various viral pathogens that cause calf diarrhea.

Method used

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  • Triple fluorescent quantitative PCR kit for simultaneously detecting bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus and application method of triple fluorescent quantitative PCR kit
  • Triple fluorescent quantitative PCR kit for simultaneously detecting bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus and application method of triple fluorescent quantitative PCR kit
  • Triple fluorescent quantitative PCR kit for simultaneously detecting bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus and application method of triple fluorescent quantitative PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus triple real-time fluorescent quantitative PCR kit composition and reaction conditions

[0055] A triple fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus, including primers and probes, reverse transcriptase, standard positive RNA template, TaqDNA polymerase, and fluorescent quantitative PCR reaction solution;

[0056] (1) The content of each component of the fluorescent quantitative PCR kit - the reaction system is shown in Table 1.

[0057] Table 1

[0058] μL / reaction N responses Volume (μL) PCR reaction solution 12.5 10 125 reverse transcriptase 1 10 10 DNA polymerase (5u / μL) 1.5 10 15 BRV-1 Primers and Probes 3 (including upstream and downstream primers and probes) 10 30 BVDV-2 Primers and Probes 3 (including upstream and downs...

Embodiment 2

[0075] Example 2 Fluorescence Quantitative Kit Repeatability and Stability Detection

[0076]This embodiment is to detect the stability and repeatability of the triple fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus in embodiment 1. Using the RNA standard with known template concentration as a template, set up two concentration gradients PI and P2, and set up a negative control at the same time, and conduct intra-assay and inter-assay repeatability experiments. The method is: select three batches of primers and probes in June, September, and November 2019, named 2019-I, 2019-II, and 2019-Ⅲ, and each batch of reagents detects 2 different concentrations of templates 3 times, Seven replicates were set for the same reagent and the same template concentration in the same experiment, and the obtained CT values ​​were analyzed to obtain the intra-assay and inter-assay errors. In principle, only when ...

Embodiment 3

[0088] Example 3 Application of triple real-time fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus in the detection of calf diarrhea samples

[0089] In this embodiment, the triple fluorescent quantitative PCR kit for simultaneous detection of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus in Example 1 is used for the detection of pathogens in cattle farm calf diarrhea samples. The specific testing process and results are as follows:

[0090] (1) Extract and purify sample RNA. RNA was extracted from 100 microliters of diarrhea samples by the magnetic bead method and dissolved in 50 microliters of nuclease-free water.

[0091] (2) Prepare the reaction system according to Table 9.

[0092] Table 9 Fluorescent quantitative PCR reaction system for detection of clinical diarrhea samples

[0093]

[0094] (3) The reaction conditions of the fluorescent quantitative PCR kit ar...

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Abstract

The invention provides a triple fluorescent quantitative PCR kit for simultaneously detecting a bovine rotavirus, a bovine coronavirus and a bovine viral diarrhea virus and an application, and belongs to the technical field of virus detection. The triple fluorescent quantitative PCR kit comprises three groups of specific primers and three corresponding probes aiming at the bovine rotavirus, the bovine coronavirus and the bovine viral diarrhea virus. The primer and the probe for detecting the three viruses have the advantages of strong specificity and high reaction efficiency. The triple real-time fluorescent quantitative PCR kit has the characteristics of high amplification efficiency, strong specificity, stable detection effects and good repeatability, and is suitable for dairy cow diarrhea pathogen detection, regular monitoring, dairy cow diarrhea epidemiological investigation and the like. The kit disclosed by the invention can be used for simultaneously detecting three dairy cow diarrhea pathogens in a real-time fluorescent quantitative PCR reaction system, so that the detection time is saved, the detection cost is reduced, a plurality of samples can be simultaneously detected, and the detection efficiency is high.

Description

technical field [0001] The invention relates to a bovine diarrhea pathogen detection kit, in particular to a kit for simultaneously detecting bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus, and belongs to the technical field of virus detection. Background technique [0002] Bovine rotavirus, coronavirus and bovine viral diarrhea virus are important pathogens causing bovine viral diarrhea. With the large-scale and intensive development of my country's dairy farming industry, bovine diarrhea, especially neonatal calf diarrhea, has become one of the important diseases that plague the dairy farming industry, seriously endangering the health of the cow herd and hindering the development of the dairy farming industry in China. [0003] Bovine rotavirus (Bovine Rotavirus, BRV) disease is an acute gastrointestinal infectious disease caused by rotavirus in a variety of young animals and infants, characterized by mental fatigue, anorexia, vomiting, diarrhea, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2563/107C12Q2521/107C12Q2545/113Y02A50/30
Inventor 陈华林张丹刘邓
Owner 北京三元集团畜牧兽医总站
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