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Gene chip, kit and method for detecting three viruses for immunosuppression disease of chickens

A gene chip and immunosuppression technology, which is applied in the field of gene chips for detecting three kinds of immunosuppressive disease viruses in chickens, can solve the problems that high specificity and large batches of samples cannot be detected.

Active Publication Date: 2015-04-01
北京翎羽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing ALV, CIAV and REV detection methods also have various defects, and cannot meet high specificity and large-scale sample detection, while gene chip technology has unique advantages in terms of high throughput and accuracy

Method used

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  • Gene chip, kit and method for detecting three viruses for immunosuppression disease of chickens
  • Gene chip, kit and method for detecting three viruses for immunosuppression disease of chickens
  • Gene chip, kit and method for detecting three viruses for immunosuppression disease of chickens

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Embodiment 1. The design of probe and primer and the preparation of gene chip

[0101] Step 1. Design of Specific Probes

[0102] 459 complete cDNA sequences of the reference strains of CAV, REV, and ALV A, C, D, and J subgroups were collected from Genbank. 459 sequences were built according to species, and 13 reference strain sequences were selected for each subgroup after comparison, among which the GenBank number of the CAV reference strain sequence was M55918; the REV reference strain sequence GenBank number was DQ387450, NC006934; The reference strain sequence GenBank number of ALV A subgroup is HM452339, M19113, M14901, HM452340, AB617819; the reference strain sequence GenBank number of ALV C subgroup is V01197; the reference strain sequence GenBank number of ALV D subgroup is D10652; The reference strain sequence GenBank numbers of ALV J subgroup are AY027920 and GU982307.

[0103] These sequences were compared with the Clustal W program of DNAStar software to ...

Embodiment 2

[0116] Embodiment 2. Preparation of the DNA template of the sample to be tested

[0117] The CAV tissue virus (that is, the plasmid carrying CAV cDNA) preserved in our laboratory was extracted with the DNA extraction kit from QIAGEN, and the nucleic acid concentration was finally detected, and stored at -20°C for future use.

[0118] The corresponding target sequences of REV and ALV (subgroups A, C, D, and J) have been synthesized by Shanghai Sangon Biotechnology Co., Ltd. and connected to the PCU57 vector plasmid. Take the bacterial liquid culture containing the synthetic plasmid, and use Tiangen Company’s plasmid small The test kit extracts the plasmid from the bacterial culture, and finally detects the nucleic acid concentration, and stores it at -20°C for later use.

Embodiment 3

[0119] Example 3. Optimizing the Multiplex PCR Amplification System

[0120] The purpose of multiplex PCR is to amplify the target sequence with as few primers as possible, so as to reduce the experimental time and steps, and improve the detection efficiency; the two multiplex PCR systems are: system 1, which can simultaneously amplify ALV A and C in the same system The target sequences of the four subgroup strains of , D, and J; System 2: The target sequences of two strains of CAV and REV can be amplified simultaneously in the same system.

[0121] In this study, the gene sequence of a reference strain was selected as a representative sequence in ALV, CAV, and REV, and the concentration was adjusted to 1ng / μL, and then mixed in equal volumes, and the above primers were used for PCR amplification respectively. The initial concentration of primers Both are 10 μmol / L. Through repeated experiments and optimization, the finally determined reaction system and reaction conditions ar...

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Abstract

The invention discloses a gene chip, a kit and a method for detecting three viruses for immunosuppression disease of chickens, and relates to the field of animal medical diagnosis. The gene chip is characterized in that specific probe sequences of four subgroups A, C, D and J of an ALV (Avian leucosis virus) as well as a CAV (Chicken Anemia Virus), and an REV (Reticuloendotheliosis virus) are arranged on a substrate of the chip. The invention further provides the kit for detecting the four subgroups A, C, D and J of the ALV, the CAV and the REV. The chip, kit and detecting method, provided by the invention can be used for detecting various pathogens quickly and accurately in a high-flux manner, so that infectious diseases can be diagnosed correctly within a relatively short period to be prevented from spreading.

Description

technical field [0001] The invention relates to veterinary medical diagnosis, in particular to a gene chip, a kit and a method for detecting three types of immunosuppressive disease viruses in chickens Background technique [0002] In recent years, my country's poultry industry has developed very rapidly and has become one of the pillar industries of my country's animal husbandry. However, with the rapid development of the poultry industry, various internal and external factors are seriously threatening the healthy development of the poultry industry, among which disease has become the number one enemy threatening the poultry industry. Although the prevention and control of major poultry infectious diseases has achieved certain results through the use of vaccination and drug control, various diseases will continue to occur in vaccinated chicken flocks in the production practice of chicken farms in various places. One of the causes of neglect is poultry immunosuppressive dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C12R1/93
CPCC12Q1/6837C12Q1/70C12Q2565/501
Inventor 杨兵苏霞陈小玲徐福州周宏专王金洛朱瑞豪杨丽聪
Owner 北京翎羽生物科技有限公司
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