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A kind of multiple PCR detection kit for diagnosing fadv/mdv/alv/rev4

A virus and upstream technology, applied in the field of molecular biology diagnosis of animal diseases in veterinary medicine, can solve problems that have not been reported

Active Publication Date: 2019-10-01
XIANYANG VOCATIONAL TECHN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] But so far, the technology that can directly diagnose the infection of group I avian adenovirus, Marek's disease virus, chicken lymphocytic leukemia virus and avian reticuloendotheliosis virus through a single PCR test has not been reported.

Method used

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  • A kind of multiple PCR detection kit for diagnosing fadv/mdv/alv/rev4
  • A kind of multiple PCR detection kit for diagnosing fadv/mdv/alv/rev4
  • A kind of multiple PCR detection kit for diagnosing fadv/mdv/alv/rev4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] (1) Design and preparation of primers

[0054] Referring to the group I avian adenovirus, Marek's disease virus, chicken lymphocytic leukemia virus and avian reticuloendotheliosis virus genome sequences published on GenBank, combined with the related virus gene sequences determined by our research group for several years, DNAStar biological The software comprehensively analyzed and compared, and designed 8 primers for the above 4 virus genes. The sequences of the primers are as follows:

[0055] FADV-F upstream primer: 5'-ATGACTGCGCTTACTCCCGA-3';

[0056] FADV-R downstream primer: 5'-ACCCAGATAGTTGGTACC-3';

[0057] MDV-F upstream primer: 5'-CAGCTGCGTATTTTCCCCG-3';

[0058] MDV-R downstream primer: 5'-AATTACTTGGTCTTTAACC-3';

[0059] ALV-F upstream primer: 5'-TGTAGTGTTATGCAATACTC-3';

[0060] ALV-R downstream primer: 5'-GGGTATGTTGGCTCCCTGCA-3';

[0061] REV-F upstream primer: 5'-AATGTGGGAGGGAGCTCCGG-3';

[0062] REV-R downstream primer: 5'-ACATTTTCCCTCATTGGA-3'.

[...

Embodiment 2

[0072] (1) Design and preparation of primers

[0073] With embodiment 1.

[0074] (2) Treatment of tissue disease samples and extraction of DNA solution

[0075] 1) Take 3-5 g of tissue disease materials from suspected cases, such as enlarged liver and spleen, cut them into paste, add 5 times aseptic PBS, grind them thoroughly with a tissue grinder, collect the suspension, 4 ° C, 12 000 r / min Centrifuge for 10 minutes, absorb the supernatant, and set aside.

[0076] 2) Take 100 μL of the supernatant of suspected case tissue disease material, put it into a 1.5 mL sterile EP tube, add 800 μL of lysate, mix it upside down, let it stand for lysis for 10 minutes, add 600 μL of ice-cold absolute ethanol, mix it upside down, and let it stand Precipitate for 10 min, centrifuge at 4°C and 12,000 r / min for 10 min, discard the supernatant, wash once with 75% ethanol, discard the ethanol, invert the EP tube to dry naturally in the air, and use 40 μL of 8 mmol·L -1 Dissolve the NaOH sol...

Embodiment 3

[0083] (1) Design and preparation of primers

[0084] With embodiment 1.

[0085] (2) Treatment of serum samples and extraction of DNA solution

[0086] 1) Take wing vein blood from chickens of suspected cases, coagulate naturally, separate serum after precipitation of serum, and set aside.

[0087] 2) Take 100 μL of the isolated serum to be tested and put it into a 1.5 mL sterile EP tube, add 800 μL of lysate, mix it upside down, let it stand for lysis for 10 minutes, add 600 μL of ice-cold absolute ethanol, mix it upside down, let it settle for 10 minutes, and keep it at 4°C , Centrifuge at 12000r / min for 10min, discard the supernatant, wash once with 75% ethanol, discard the ethanol, invert the EP tube to dry naturally in the air, and use 40μL of 8mmol·L -1 The NaOH solution was dissolved to obtain the DNA solution of the isolated serum to be tested, which was set aside.

[0088] 3) Simultaneously process the negative control sample solution: draw 100 μL negative control...

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Abstract

The invention belongs to a quadruple PCR detection kit for diagnosis FAdV / MDV / ALV / REV. The kit is mainly used for identifying quadruple polymerase chain reaction of group I fowl adenoviruses, Marek's disease viruses, viruses of lymphoid leucosis of chicken and reticuloendotheliosis viruses, and comprises lysate, amplified reaction mixed liquor, a negative contrast and a positive contrast. Four pairs of primers are designed for genomes of the group I fowl adenoviruses, the Marek's disease viruses, the viruses of lymphoid leucosis of chicken and the reticuloendotheliosis viruses, a quadruple polymerase chain reaction diagnosis method is established, and the kit for identifying quadruple polymerase chain reaction diagnosis is assembled. The kit is rapid and sensitive in detection and good in specificity, and can detect four types of viral diseases which cause hepatosplenomegaly of poultry at a time; results are visible and accurate; the time and the labor are saved; diagnosis costs are greatly reduced; and the quadruple PCR detection kit is suitable for general survey diagnosis of large-range neoplastic diseases of the poultry.

Description

technical field [0001] The invention belongs to the technical field of molecular biology diagnosis of animal epidemic diseases in veterinary medicine, and mainly relates to a multiplex PCR detection kit for diagnosing FAdV / MDV / ALV / REV4, which is mainly used for identifying group I adenovirus, Marek's disease virus, chicken Lymphoblastic leukemia virus and avian reticuloendotheliosis virus 4-plex polymerase chain reaction. Background technique [0002] Since the second half of 2015, a new disease has appeared in chicken farms in some provinces of my country, such as Henan, Anhui, Liaoning, Hebei, Beijing, Shandong, Shanxi, Jiangsu, and Guangdong. The disease is characterized by pericardial effusion and liver disease. It is characterized by swelling and is called "pericardial effusion-hepatitis syndrome". At present, it is believed that its pathogen is group I fowl adenovirus (Fowl adenovirus, FAdV). FAdV particles have a diameter of 70-90nm, no capsule, and an icosahedral sym...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686
Inventor 朱小甫吴旭锦
Owner XIANYANG VOCATIONAL TECHN COLLEGE
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