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Infectious bronchitis virus QX type strain identifying and detecting kit

A technology for bronchitis and chicken infectivity, applied in the field of chicken infectious bronchitis virus QX strain identification and detection kits, can solve the problems that QX strain infection cannot provide protection, economic loss of chicken industry, etc., and achieve rapid detection method Efficient effect

Active Publication Date: 2018-12-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional vaccines do not protect against infection with the QX strain
Therefore, the prevalence of QX strains has caused great economic losses to the chicken industry.

Method used

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  • Infectious bronchitis virus QX type strain identifying and detecting kit
  • Infectious bronchitis virus QX type strain identifying and detecting kit
  • Infectious bronchitis virus QX type strain identifying and detecting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] [Example 1] RNA extraction

[0056] 1 Detection sample processing

[0057] Tissue sample processing: Take the tissues that are easy to separate IBV, such as the trachea and kidney of dead chickens, cut them into pieces with scissors, add sterile PBS at a volume ratio of 1:3, and grind them thoroughly. The homogenized tissue suspension was repeatedly frozen and thawed at -20°C (or lower)-room temperature three times, then centrifuged at 12,000 rpm for 10 minutes, and 200 μl of the supernatant was taken into a new sterilized centrifuge tube.

[0058] Processing of swab samples: add 1ml of sterilized PBS to a fresh throat or cloacal swab, shake and stir well, then discard the swab (collect the liquid in the swab as much as possible). The extract was centrifuged at 12,000 rpm for 10 min, and 200 μl of the supernatant was taken into a new sterilized centrifuge tube.

[0059] Allantoic fluid treatment: collect the allantoic fluid from virus-infected chicken embryos and cent...

Embodiment 2

[0062] [Example 2] Reverse transcription reaction (RT reaction)

[0063] (4) The total RNA extracted in the above step is used as a template to perform a reverse transcription reaction, and the system of the reverse transcription reaction is:

[0064] 11 μl total RNA template, 4 μl 5× reaction buffer, 2 μl 10 mM dNTP Mix, 1 μl 0.2 μg / μl random primer, 1 μl 20 U / μl RNase inhibitor, 1 μl 200 U / μl reverse transcriptase, total 20 μl. After mixing, react at a constant temperature of 42°C for 1 hour, and then stop the reaction at a constant temperature of 70°C for 10 minutes to obtain cDNA. Store at -20°C

Embodiment 3

[0065] [Example 3] PCR reaction

[0066] 1. Primer Design

[0067] A total of 212 S1 gene sequences of IBV QX strains and some classic QX strains popular in my country in recent years were collected from GeneBank. At the same time, a total of 49 S1 gene sequences of non-QX strains of different genotypes were selected from GeneBank. Use DNAMAN software to carry out sequence comparison of these 261 S1 genes, and use the primer design software Premier Primer 5 to design a pair of primers (QX-F, QX-R) according to the conserved region of the QX type strain S1 gene, and this pair of primers is in Non-QX-type strains are not conserved. General primers (Tong-F, Tong-R, Table 1) refer to Ling Jifei's master's thesis. The primers were synthesized by Hangzhou Qingke Zixi Biotechnology Co., Ltd. and purified by PAGE. The primer information is shown in Table 1.

[0068] Table 1 Kit typing primer information

[0069]

[0070] 2. Preparation of positive standard

[0071] The IBV-H...

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Abstract

The invention relates to the technical field of bio-detection and aims to provide an infectious bronchitis virus QX type strain identifying and detecting kit. The kit comprises two pairs of primers which are separately universal primers for amplifying the infectious bronchitis virus and primers for specifically amplifying the QX gene type strain, wherein the sequences of the primers for specifically amplifying the QX gene type strain are as shown in SEQ ID NO: 5 and SEQ ID NO: 6. An effective RT-PCR detection method is established by means of the specific primers of QX genotype and a purpose of parting detection of the QX genotype can be achieved by the primary RT-PCR reaction is achieved. According to the kit, the two pairs of primers are added into a same PCR system in a same PCR reaction condition, so that IBV in a clinical sample or a chick embryo allantoic fluid can be detected effectively, and IBV of the QX genotype can be identified specifically. The detection method is quick, efficient and accurate, and has very good meaning in guiding selection of vaccines and quick control of IB.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a kit for identifying and detecting chicken infectious bronchitis virus QX strain. Background technique [0002] Chicken infectious bronchitis (Infectious Bronchitis, IB) is an acute, highly contagious infectious disease caused by chicken infectious bronchitis virus (Infectious Bronchitis virus, IBV). The disease can affect the respiratory system, genitourinary system and digestive system of chickens. It is one of the important infectious diseases that seriously endanger the poultry industry in the world. [0003] Avian infectious bronchitis virus belongs to the genus Coronaviridae of the family Coronaviridae of the order Nidoviridae. Its genome is a non-segmented positive-strand RNA virus with a total length of about 27.6 kb. The viral genome sequence from 5' end to 3' end is 5'-1a-1b-S1-S2-3a-3b-3c-M-5a-5b-N-poly(A)-3'. IBV contains four main structural protein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2565/125C12Q2521/107
Inventor 周继勇陶春豪廖敏颜焰曹尚尚
Owner ZHEJIANG UNIV
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