Primer probe combination for detecting bovine respiratory viruses, kit and application

A technology of primer probe and respiratory tract, which is applied in the field of bovine respiratory virus detection, can solve the problems of time-consuming and labor-intensive PCR, low sensitivity and accuracy, mixed infection, etc., and achieve the goal of reducing nucleic acid pollution, high specificity and high throughput, and high detection sensitivity Effect

Pending Publication Date: 2021-11-23
湖南省动物疫病预防控制中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of clinical diagnosis, these four diseases have relatively similar clinical symptoms, and often have mixed infections, making their diagnosis and prevention difficult.
[0004] Conventional PCR is time-consuming, has low sensitivity and accuracy, and is prone to false positives

Method used

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  • Primer probe combination for detecting bovine respiratory viruses, kit and application
  • Primer probe combination for detecting bovine respiratory viruses, kit and application
  • Primer probe combination for detecting bovine respiratory viruses, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Screening and preparation of primers and probe sets

[0078] The performance of the primers determines the pros and cons of the detection effect of the kit. The disclosure screens multiple sets of primer-probe combinations for detection indicators BHV1 virus, BRSV virus, BPIV3 virus and BVDV virus, and finally screens a set of optimal primer-probe combinations. This example is intended to illustrate the screening process of primer-probe combinations.

[0079] Primer design: According to the gene sequences of BHV-1, BRSV, BPIV-3 and BVDV registered in GenBank, the gB gene of BHV-1, the F gene of BRSV, and the BPIV3

[0080] Table 2 is aimed at the primer probe group sequence of BHV-1, BRSV, BPIV-3 and BVDV virus

[0081]

[0082] The M gene of M gene and the 5'UTR sequence of BVDV were used as the target genes, and DNAMAN software was used to analyze the homology of the reference gene sequence. According to the analysis results, three pairs of primers wer...

Embodiment 2

[0091] Example 2 kits for multiple fluorescent PCR reaction-specific detection

[0092] (1) Establishment and optimization of multiplex fluorescent PCR reaction conditions

[0093] The best single-plex fluorescent PCR system obtained by screening is shown in Table 5. Use the best combination of primers and probes obtained in Example 1 to carry out fluorescent PCR reaction, select a 25 μL system to include: one-step reverse transcription 2×MIX: 12.5 μL, one-step reverse transcriptase: 1 μL, upstream and downstream primers (10 μmol / L) 0.8 μL each, probe: 0.5 μL each, standard positive plasmid DNA template 4.0 μL, add ddH 2 0 to 25 μL. On the basis of the single-plex fluorescent PCR reaction conditions in Example 1, the single-plex PCR amplification conditions were optimized to obtain the best single-plex fluorescent PCR reaction conditions, as shown in Table 5. After determining the optimal single-plex fluorescent PCR reaction conditions, establish and optimize the multiplex...

Embodiment 3

[0101] Example 3 Using the kit for detecting four kinds of bovine respiratory viruses to detect the sensitivity of multiple fluorescent PCR reactions

[0102] After measuring the concentration with a spectrophotometer to be 112.1ng / μL, the positive plasmid was carried out from 10 -1 to 10 -10 10-fold concentration gradient dilution, according to the optimized multiple PCR reaction system in the above-mentioned embodiment 2 (that is, the test kit for detecting four kinds of bovine respiratory viruses in the embodiment of the present disclosure) and reaction conditions, with the standard of different dilutions The positive plasmid was used as a template, and the reaction system was 25 μL, and the template was 3 μL for multiplex fluorescent PCR amplification. Amplification results such as Figure 4 , Figure 5 , Figure 6 and Figure 7 shown.

[0103] The results showed that the minimum detection amount of DNA of BHV-1, BRSV, BPIV-3 and BVDV was 3.363×10 -5 pg, 3.363×10 -...

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Abstract

The invention provides a primer probe combination for detecting bovine respiratory viruses, a kit and application. According to the primer probe combination for detecting the bovine respiratory viruses, the kit and the application provided by the invention, four main bovine respiratory viruses (BHV-1, BRSV, BPIV-3 and BVDV) can be rapidly, accurately and simultaneously identified on the basis of multiple fluorescent PCR; the detection sensitivity is high; the specificity is high; and the flux is high. The kit for detecting the bovine respiratory virus based on the multiple fluorescent PCR technology can be used for detecting pathogen nucleic acid molecules with high sensitivity and specificity. Compared with a common PCR detection method, the detection method of the kit is more sensitive and specific; and nucleic acid pollution in detection is reduced.

Description

technical field [0001] The disclosure relates to the technical field of bovine respiratory virus detection, in particular to a primer-probe combination, kit and application for detecting bovine respiratory virus. Background technique [0002] Bovine Respiratory Disease Complex (BRDC), also known as transport fever, has been frequently reported at home and abroad in recent years. It is widely distributed worldwide, mostly endemic or sporadic, with high morbidity and mortality. The economic loss caused by the cattle industry cannot be ignored. There are many kinds of pathogens that cause the disease, usually caused by single or mixed infection of one or several pathogens, such as bovine herpes virus, bovine respiratory syncytial virus, bovine parainfluenza virus or bovine viral diarrhea virus. [0003] Bovine herpes disease, bovine respiratory syncytial disease, bovine parainfluenza or bovine viral diarrhea is an acute, febrile, contact respiratory infectious disease that cau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107
Inventor 胡巧云王昌建何世成姜利霞唐小明林源彭志王卫国张坤谢怡灵
Owner 湖南省动物疫病预防控制中心
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