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Semi-nested reverse transcription-polymerase chain reaction (RT-PCR) typing primers and kit for identifying different serotypes of live vaccines for infectious bronchitis virus (IBV)

A technology of RT-PCR and bronchitis, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. It can solve the problems of long detection cycle, inability to obtain specific serum, and heavy workload. Achieve strong specificity and sensitivity, low cost, and overcome time-consuming effects

Inactive Publication Date: 2018-11-23
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic method for identifying strains of different serotypes is the serum neutralization test. Although this technique is a routine technique, it requires the use of positive sera specific for different serotypes. At present, it is not possible to obtain sera for the above four serotypes through commercialization. All specific sera of vaccine strains
Moreover, the detection cycle of this method often takes more than several weeks, which has the limitations of long detection cycle and heavy workload.

Method used

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  • Semi-nested reverse transcription-polymerase chain reaction (RT-PCR) typing primers and kit for identifying different serotypes of live vaccines for infectious bronchitis virus (IBV)
  • Semi-nested reverse transcription-polymerase chain reaction (RT-PCR) typing primers and kit for identifying different serotypes of live vaccines for infectious bronchitis virus (IBV)
  • Semi-nested reverse transcription-polymerase chain reaction (RT-PCR) typing primers and kit for identifying different serotypes of live vaccines for infectious bronchitis virus (IBV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Identification of M-type, 4 / 91-type, LDT3-A-type and QX-type infectious bronchitis virus vaccine strains Semi-nested RT-PCR method The first round of PCR primer (universal primer) design.

[0051] Refer to the M-type IBV vaccine H52 strain, H120 strain and Ma5 strain IBV S1 gene sequence registered in GenBank (GenBank accession numbers are: KF188436, AF352315, AY561713), 4 / 91 strain S1 gene sequence (GenBank accession number is KF377577), LDT3 - Design and screen specific general primers for the hypervariable region of the S1 gene sequence of the A strain (GenBank accession number is KR608272) and the QXL87 strain S1 gene sequence (GenBank accession number is MH743141), for M type, 4 / 91 type, LDT3-A Both the S1 gene sequences of type and QX vaccine strains could be effectively amplified. After repeated comparison and screening, 3 alternative upstream primers and 1 downstream primer were finally obtained (Table 2).

[0052] Table 2 Design of universal primers ...

Embodiment 2

[0064] Example 2 Identification of M-type, 4 / 91-type, LDT3-A-type and QX-type infectious bronchitis virus vaccine strains Semi-nested RT-PCR method Design of the second round of PCR primers (identification primers).

[0065] According to the hypervariable region sequences of vaccine strains of different serotypes amplified by the first round of PCR, after sequence comparison, the regions with significant differences among different serotypes but conserved within serotypes were screened to design M type and 4 / 91 type , LDT3-A and QX vaccine strain-specific identification primers were respectively used as upstream primers to pair with the general downstream primer IB-VacUni-R (SEQ ID NO.2) of the first round of PCR for semi-set PCR amplification. After repeated comparison and screening, the four serotype-specific primers finally obtained are shown in Table 3.

[0066] Table 2 Design of primers for the second round of semi-nested RT-PCR serotype-specific identification

[0067] ...

Embodiment 3

[0072] Example 3 Application of half-set RT-PCR of the present invention to detect and identify strains of commercialized vaccines

[0073] (1) Extraction of total RNA from the sample

[0074]Get chicken infectious bronchitis live vaccine (H52 strain), chicken infectious bronchitis live vaccine (H120 strain), chicken infectious bronchitis live vaccine (Ma5 strain), chicken infectious bronchitis live vaccine (4 / 91 strain) 1 bottle of chicken infectious bronchitis live vaccine (LDT3-A strain) and chicken Newcastle disease-infectious bronchitis dual live vaccine (LaSota strain + QXL87 strain) each, add 2 mL nuclease-free sterilization to each bottle of vaccine with a syringe Resuspend the lyophilized vaccine in ultrapure water, let it stand at room temperature for 5 minutes, take 100 μL of the suspension for each vaccine sample, and extract total RNA according to the instructions of the Trizol RNA extraction reagent.

[0075] (2) reverse transcription into cDNA

[0076] Add the...

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Abstract

The invention belongs to the technical field of biological detection, in particular relates to semi-nested reverse transcription-polymerase chain reaction (RT-PCR) typing primers and a kit for identifying different serotypes of live vaccines for infectious bronchitis virus (IBV). The typing detection primers provided by the invention comprise universal primers having sequences shown in SEQ ID 1-2,and serotype specific primers having sequences shown in SEQ ID 3-6. The invention also discloses the kit comprising the typing detection primers. The M-type, 4 / 91-type, LDT3-A-type and QX-type serotype live vaccines for the IBV are identified by utilizing the primers and the kit which are provided by the invention and by adopting a semi-nested RT-PCR method; compared with the conventional PCR methods, the semi-nested RT-PCR method has stronger specificity and sensitivity; the method also has the characteristics of being fast, high in efficiency and low in cost, can complete the sample detection within 5-6h, and overcomes the defect of longer time consumption of the traditional serum neutralization test method; furthermore, the method is suitable for the detection and analysis a great batch of samples.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to the RT-PCR typing detection of chicken infectious bronchitis virus live vaccine. 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 length of about 27.6 kb. The IBV genome encodes four key structural proteins: spike protein (S), membrane protein (M), nucleocapsid protein (N) and small membrane protein (E). [0003] The S protein is the main structural protein of IBV, located on the outermost surface of the virus, and is the main component of the outermost fiber of the virion. S protein has many important biological functions, which are related to virus adsorption to host cells and tissue tropism. After the S protein is translated, it is cleaved by the host cell protease to produce the N-terminal S1 protein ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11
CPCC12Q1/6848C12Q1/701C12Q2531/113
Inventor 张小荣吴艳涛曹永忠吴天琪廖凯苏晋胡亚歌
Owner YANGZHOU UNIV
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