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Liquid-phase chip detection kit for simultaneously detecting BVDV, IBRV and BPIV, and special primer and coupling probe thereof

A kit and probe technology, applied in the field of liquid phase chip detection technology and its kit, can solve the problem of leukopenia, etc., and achieve the effect of simple operation, high sensitivity and high throughput

Active Publication Date: 2019-12-24
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no direct relationship between cytopathic and strain virulence, but almost all virulent BVDV strains are non-cytopathic, which can cause a large decrease in white blood cells in peripheral blood, which is the main cause of acute BVDV infection

Method used

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  • Liquid-phase chip detection kit for simultaneously detecting BVDV, IBRV and BPIV, and special primer and coupling probe thereof
  • Liquid-phase chip detection kit for simultaneously detecting BVDV, IBRV and BPIV, and special primer and coupling probe thereof
  • Liquid-phase chip detection kit for simultaneously detecting BVDV, IBRV and BPIV, and special primer and coupling probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Designing the labeled primers and coupling probes for detecting BVDV / IBRV / BPIV viruses with liquid-phase chip technology

[0053] Selection of BVDV detection sequence: Select conservative 5'UTR gene sequences for BVDV type I and type II viruses (SED ID NO: 10 and 11 in the sequence table, respectively), which can simultaneously amplify BVDV type I and type II For viruses, according to the design principles of liquid-phase labeled primers and coupling probes, the coupling probes and labeled primers are designed to obtain multiple sets of primers. Based on the experimental results, the final optimal primer sequence information is as follows:

[0054] BVDV-F (upstream primer): 5'-AGCAACAGTGGTGAGTTCGTTGGA-3' (SED ID NO: 1 in the sequence listing).

[0055] BVDV-R (downstream primer): 5'-biotin-GCCCTCGTCCACGTGGYATC TCG-3' (SED IDNO: 2 in the sequence listing).

[0056] BVDV-P (coupled probe): 5'-amino (amino)-liner C12 (12 carbons)-AGTACAGGGTAGTCGTCARTGGTTCG-3' (SED ID NO...

Embodiment 2

[0065] Example 2. Using the coupling probe of the present invention to couple the luminex magnetic ball and verify the coupling efficiency

[0066] 1. Coupling of coupling probe and luminex magnetic ball

[0067] Select 44 / 54 / 14 luminex magnetic spheres and the corresponding BVDV / IBRV / BPIV coupling probes for coupling. The coupling process is as follows:

[0068] (1) Dilute the coupled probe, and dilute the synthesized coupled probe with 5'-labeled amino group to 100μmoL;

[0069] (2) Return the luminex magnetic ball to room temperature, and mix upside down, suck 100μL (1.25×10 6 ) Into a 1.5mL sterile enzyme-free centrifuge tube, centrifuge at 12000rpm for 2min, and aspirate the supernatant;

[0070] (3) Add 50μL of 0.1M MES pH4.5 solution stored at 4℃ and mix by pipetting;

[0071] (4) Add 5 μL of 0.1 mM coupled probe and pipette to mix;

[0072] (5) Prepare the EDC solution now, take about 10 mg of EDC dry powder, and dissolve it in a 1.5 mL sterile enzyme-free centrifuge tube with 1 m...

Embodiment 3

[0085] Example 3. Qualitative detection of bovine viral diarrhea virus (BVDV), bovine infectious rhinotracheitis virus (IBRV) and bovine parainfluenza virus type 3 (BPIV-3) using the method of the present invention

[0086] 1. Extract the genomic nucleic acid of the sample to be tested

[0087] Extract genomic RNA / DNA from the inactivated bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and bovine parainfluenza virus cell cultures (positive control) and the sample to be tested. The specific method includes the following steps (Axyprep TM Body Fluid Viral DNA / RNA Miniprep Kit, AXYGEN company):

[0088] (1) Preparation of AXYGEN kit: According to the kit instructions, prepare isopropanol containing 1% glacial acetic acid in advance and add the specified concentration of absolute ethanol to the reagents Buffer W1A and Buffer W2.

[0089] (2) Add 200 μL of the sample to be tested into a 1.5 mL centrifuge tube, and add 200 μL of Buffer V-L, vortex and mix well, and then...

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Abstract

The invention provides a liquid-phase chip detection kit for simultaneously detecting bovine viral diarrhea-mucosal disease virus, infectious bovine rhinotracheitis virus and bovine parainfluenza virus as well as a special primer and a coupling probe thereof. The kit disclosed by the invention is simple and convenient to detect BVDV, IBRV and BPIV, strong in specificity, high in sensitivity and good in repeatability, and can be applied to the fields of virus detection, vaccine production, calf product quality inspection and the like.

Description

Technical field [0001] The invention belongs to the detection of veterinary animal pathogens in the field of biological detection technology, and in particular relates to a specific detection of bovine viral diarrhea virus (BVDV), bovine infectious rhinotracheitis virus (IBRV) and bovine parainfluenza virus type 3 ( BPIV) liquid-phase chip detection technology and its kits. Background technique [0002] Liquid chip detection technology [0003] Liquid-phase chip detection technology is a high-throughput detection technology. It was originally developed by Luminex in the United States. This technology combines flow cytometry, fluorescent coded microspheres, lasers, digital signal processing, and traditional biochemical technologies. A multi-functional analysis platform that can realize simultaneous qualitative and quantitative analysis of multiple indicators. This technology has the characteristics of high throughput, high sensitivity, good repeatability and sample saving. Since ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6837C12N15/11
CPCC12Q1/701C12Q1/6837C12Q2531/113C12Q2563/143C12Q2563/149Y02A50/30
Inventor 王艳杰陈君彦刘建奇王秀明魏学峰刘国英范秀丽张贵刚
Owner JINYUBAOLING BIO PHARMA CO LTD
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