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Kit for RT-PCR typing detection of avian infectious bronchitis virus

A technology of RT-PCR and bronchitis, applied in the field of chicken infectious bronchitis virus RT-PCR typing detection kit, can solve the problems of prevention and control difficulties, and achieve high sensitivity, strong specificity, convenient and quick use Effect

Active Publication Date: 2016-04-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The emergence of IBV mutant strains makes the existing vaccines unable to protect chickens well against wild virus attacks. IB outbreaks in chicken farms occur from time to time, which brings great difficulties to the prevention and control of IBV

Method used

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  • Kit for RT-PCR typing detection of avian infectious bronchitis virus
  • Kit for RT-PCR typing detection of avian infectious bronchitis virus
  • Kit for RT-PCR typing detection of avian infectious bronchitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] [Example 1] RNA extraction

[0055] 1 Detection sample processing

[0056] 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 for 3 times, then centrifuged at 12,000g for 10min, and 200μl of the supernatant was taken into a new sterilized centrifuge tube.

[0057] Processing of swab samples: Add 1ml of sterilized PBS to a fresh pharyngeal or cloacal swab, shake and stir well, then discard the swab (collect the liquid in the swab as much as possible). Centrifuge the extract at 12,000 g for 10 min, and take 200 μl of the supernatant into a new sterilized centrifuge tube.

[0058] Allantoic fluid treatment: collect virus-infected chick embryo allantoic fluid and centrifuge at 12,000 g for 10 min...

Embodiment 2

[0061] [Example 2] Reverse transcription reaction (RT reaction)

[0062] Take 1 μg of the above total RNA and add 1 μl Randomprimer, then add DEPC water to 12 μl. After ice bathing at 65°C for 5 minutes, add 4 μl of 5×buffer, 2 μl of 10mMdNTP, 1 μl of RNase inhibitor, and 1 μl of M-MuLV to the system, mix well, then carry out RT reaction at 25°C for 5 minutes, 42°C for 1h, and 70°C for 5 minutes to obtain reverse transcription product. The concentration of reverse transcription products was also measured by UV spectrophotometer. Store at -20°C for later use.

Embodiment 3

[0063] [Example 3] PCR reaction

[0064] 1. Primer Design

[0065] The S1 gene sequences of 400 representative IBV strains recorded in GeneBank were selected for comparative analysis. According to the results of comparison analysis, its conserved sequence was selected, and a pair of IBV general primers (general-F and general-R) and 4 / 91 type strain-specific identification primers (4 / 91-F and 24 / 91-R); in order to further identify respiratory and renal IBV, the full sequences of 30 classic IBV strains recorded on GENBANK were selected (including H120, M41, H52 and other respiratory strains, as well as BJ, SC021202, GX-YL5 and other nephrotype strains) were compared, and a phylogenetic tree was drawn based on the S1 gene sequence. Typed according to the distant relationship of the phylogenetic tree, selected the conserved sequence of the nsp12 gene of the nephrotype strain and designed a pair of identification primers (kidney-F and nephrotype-R) of the nephrotype strain. The ...

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Abstract

The invention relates to the technical field of biological detection, and aims at providing a kit for RT-PCR typing detection of an avian infectious bronchitis virus. The kit comprises the following three pairs of primers including the universal primers designed according to an S1 gene segment of the infectious bronchitis virus, the 4 / 91 type virulent strain specific primers and the kidney type virulent strain specific primers designed according to an nsp12 gene segment, and the sequences of the three pairs of the primers are shown in SEQ ID NO:6-11. According to the kit, the purpose of performing typing detection on the IBV can be achieved only through one time of an RT-PCR reaction, and identification detection on the IBV virulent strains of the kidney type, the 4 / 91 type and the respiration type through the one-time RT-PCR method is achieved for the first time in the industry; the advantages of being convenient and rapid to use, high in sensibility and specificity and the like are achieved, and a novel effective detection means is supplied to clinical rapid identification diagnosis of the IBV, epidemiological investigation and IBV prevention and control.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a chicken infectious bronchitis virus RT-PCR typing detection kit. Background technique [0002] Chicken infectious bronchitis (Infectiousbronchitis, IB) is an acute, highly contagious infectious disease of chickens caused by infectious bronchitis virus (InfectiousbronchitisVirus, IBV). Characterized by dyspnea, nephritis and decreased egg quality, the disease is widespread all over the world and is one of the most serious respiratory infectious diseases in the poultry industry. [0003] IBV belongs to the Nidovirilaes family Coronaviridae subgenus Coronavirus gamma coronavirus subgenus, is the representative virus of this subgroup, and is also the earliest isolated coronavirus. Chicken infectious bronchitis virus is widely prevalent in the world, and different viruses have different affinity for tissues, mainly including respiratory type, renal type, intestinal typ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2565/125
Inventor 廖敏雷静周继勇方祥年
Owner ZHEJIANG UNIV
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