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Universal kit for detecting different genotypes of infectious bronchitis viruses

A bronchitis and infectious technology, applied in the field of molecular biology, can solve the problems of technical difficulties in detecting IBV and the generation of mutant strains, and achieve the effects of high specificity, convenient operation and good specificity

Active Publication Date: 2017-02-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are certain differences in the sequence homology among many serotypes of IBV, and new mutant strains are constantly produced, which brings technical difficulties to the detection of IBV

Method used

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  • Universal kit for detecting different genotypes of infectious bronchitis viruses
  • Universal kit for detecting different genotypes of infectious bronchitis viruses
  • Universal kit for detecting different genotypes of infectious bronchitis viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 is used to detect the design of the universal primer of different genotype infectious bronchitis virus

[0034] Refer to the IBV-YN (GenBank: F893452) genome sequence in the NCBI database and screen the primers. Among the many candidate primers, after repeated screening and comparison tests to exclude possible non-specific matches between the primers and other species sequences, after repeated screening and verification, 4 sets of candidate primer pairs were finally obtained, and the four sets of Primer pairs were tested and the optimal primer pair was selected. Primer 1, 2, 3, and 4 primer pairs in Table 1 are all primers for the same target gene, and their positions are adjacent. The electrophoresis results of each primer amplification were as follows: figure 1 shown. Choose the primer pair 1 with the brightest amplification target band and no non-specific bands as the best primers. The upstream primer is between 26235nt-26254nt of GenBank accession nu...

Embodiment 2

[0038] Example 2 The exploration of the optimal annealing temperature of the RT-PCR detection method for detecting infectious bronchitis virus

[0039] 1. Pretreatment of test samples

[0040] (1) Tissue sample processing: take 100 mg of organ tissue samples, add 0.5 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 and analysis.

[0041] (2) Treatment of cloacal or oropharyngeal swab samples: add 0.5 ml sterile saline to the swab sample and oscillate to suspend with a vortex shaker, centrifuge the sample suspension at 3000 rpm for 30 min, and take the supernatant for detection.

[0042] 2. Extraction of total RNA from samples

[0043] Extraction was performed according to the instructions of Trizol RNA Extraction Kit (Invitrogen).

[0044] 3. Reverse transcription into cDNA

[0045] Add the following components into a 0.2ml centrifuge tube: 4 μl of RNA solution, 1 μl of...

Embodiment 3

[0053] Embodiment 3 Detects the RT-PCR detection method of infectious bronchitis virus to explore the optimal number of cycles

[0054] For the pretreatment of the test sample, the extraction of the total RNA of the sample, and the reverse transcription into cDNA, refer to the corresponding method in Example 2. Using the conditions determined in Examples 1 and 2, 6 different cycle numbers (28, 29, 30, 31, 32 and 33) were designed to perform RT-PCR on IBV.

[0055] After mixing gently, carry out the following reactions with different cycle numbers: 94°C pre-denaturation for 5 minutes, 94°C for 45s, 57°C for 45s, 72°C for 50s, and different cycle numbers (28, 29, 30, 31, 32 and 33), at the end of the cycle, extend at 72°C for 10 min. Electrophoresis results such as image 3 As shown, the target band amplified with 30 cycles is similar in brightness to more cycles, and there are no non-specific bands, which can save time and improve detection efficiency, so it is selected as th...

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Abstract

The invention provides a universal kit for detecting different genotypes of infectious bronchitis viruses, and belongs to the technical field of RT-PCR detection. The kit contains a pair of specific primers, and the nucleotide sequences of the specific primers are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively. The invention further provides a method for detecting the different genotypes of infectious bronchitis viruses. The kit and the detection method have the advantages of high specificity, high sensitivity, high efficiency, good universality and low cost and can be used for conducting rapid differential diagnosis on clinical disease materials in 6.5 h, and a technical means is provided for early rapid diagnosis on the IBVs and development of molecular epidemiological investigation to better guide prevention and control over the disease in poultry raising production.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a general kit for detecting different genotypes of infectious bronchitis virus and its application. Background technique [0002] Infectious bronchitis virus (IBV) belongs to the family Coronaviridae and belongs to the genus Coronavirus. It is an enveloped single-stranded positive-sense RNA virus with a genome of about 27.6kb. The IBV genome encodes four essential structural proteins: Spike (S), Membrane (M), Nucleocapsid (N) and Small Membrane (E). N protein is a phosphorylated core polypeptide located in the membrane, with a molecular weight of about 46ku and a phosphorylated molecular weight of about 51ku. The gene sequence of N protein is relatively conservative, and its main function is to combine with viral RNA to form nucleocapsid, which can specifically bind to guide RNA, and can induce cell-mediated immune response and cross-reactive ELISA antibody. [0003] There are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 张国中赵静冯金玲徐美玉任颖超
Owner CHINA AGRI UNIV
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