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Reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains

A bronchitis and infectious technology, which is applied in the field of RT-PCR method for identifying epidemic strains of chicken infectious bronchitis virus and vaccine strains, can solve the problems of inability to distinguish vaccine viruses and wild virus strains, time-consuming and labor-intensive, etc., and achieves good results. Specificity, low cost, and good sensitivity

Inactive Publication Date: 2012-08-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isolation and identification of viruses requires clinical samples to be processed and then inoculated with a suitable carrier (usually SPF chicken embryos) for preliminary identification by observing characteristic chicken embryo lesions (dwarf embryos). Perform serotyping, therefore time consuming
Conventional RT-PCR methods have obvious limitations due to the widespread use of M41-like live vaccines, which cannot distinguish vaccine viruses from field strains

Method used

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  • Reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains
  • Reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains
  • Reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 is designed to be used for identifying the primer combination of chicken infectious bronchitis virus epidemic strain and vaccine strain

[0033] According to the highly conserved segment of the genome of chicken infectious bronchitis virus registered on GenBank and the highly different regions of genotypes in the genome of chicken infectious bronchitis virus vaccine strains and epidemic strains (GenBank accession numbers are FJ888351, AY851295, EU817497) Design the following 4 primer sequences:

[0034] Upstream primer P1: 5'-GACCACAGTCACGCACAA-3';

[0035] Downstream primer P2: 5'-AATCTCCTCGGGTTCATC-3';

[0036] Downstream primer P3: 5'-AACTACACTCAACAATGGA-3'; and

[0037] Downstream primer P4: 5'-CCTGATTCTTCTTGATAC-3'.

Embodiment 2

[0038] Embodiment 2 identifies the RT-PCR method of chicken infectious bronchitis virus epidemic strain and vaccine strain

[0039] 1 Processing of clinical samples

[0040] 1.1 Experimental reagents and main instruments

[0041] Main reagents: sterile saline.

[0042] Main instruments: 4°C desktop centrifuge; vortex shaker; grinder.

[0043] 1.2 Experimental steps

[0044] (1) Tissue sample processing: take 1 mg of organ tissue sample, add 1 ml of sterilized saline, grind and suspend with a grinder, centrifuge the tissue suspension at 3000 rpm for 30 min, and take the supernatant for detection.

[0045] (2) Treatment of cotton swab samples: add 1 ml of sterilized physiological saline to the vortex shaker to suspend the tracheal or cloacal swab samples, then centrifuge as above to obtain the supernatant for detection.

[0046] 2 Extraction of total RNA from samples

[0047] 2.1 Experimental reagents and main instruments

[0048] Main reagents: Trizol reagent; DEPC treate...

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Abstract

The invention provides a reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains by multi-primer combination. The method comprises the following steps of sample total ribose nucleic acid (RNA) extraction, sample complementary deoxyribose nucleic acid (cDNA) obtained through reverse transcription, target segment amplification through multi-primers and gel electrophoresis and result judgment. The method is applicable to the fast verification detection of ordinary vaccine strains and main epidemic wild strains of avian IBV, and has the characteristics of high specificity, high sensitivity, high efficiency and low cost, the verification of IBV and other viruses is realized, and meanwhile, the attribution of strain genotype is verified. The establishment of the method has very important significance on both molecular variation mechanisms of the avian IBV and molecular epidemiology analysis.

Description

technical field [0001] The invention relates to RT-PCR detection technology, in particular to the RT-PCR method for identifying the epidemic strain and vaccine strain of chicken infectious bronchitis virus. Background technique [0002] Chicken infectious bronchitis (Avian Infectious Bronchitis, IB) is an acute, highly contagious upper respiratory tract disease of chickens, the pathogen is Infectious Bronchitis Virus (Infectious Bronchitis Virus, IBV). The main difficulty in the prevention and control of IBV vaccines is that there are many serotypes of the virus and the lack of effective cross-protection among different serotypes. Therefore, it is very important to effectively distinguish IBV vaccine strains from circulating wild strains and to clarify their serotypes in the production of the disease. [0003] At present, the methods for pathogenic detection of infectious bronchitis virus mainly include virus isolation and identification and RT-PCR method. Isolation and id...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张国中唐娜龚一秦秀慧
Owner CHINA AGRI UNIV
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