Fluorogenic quantitative PCR detection reagent and preparation method and application thereof

A fluorescent quantitative and detection reagent technology, applied in the field of animal disease quarantine and detection, can solve problems such as economic losses in the poultry industry

Active Publication Date: 2016-08-31
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since 2015, poultry with serotype 4 (FAdV-4) in subgroup C species of I subgroup characterized by pericardial effusion, inclusion body hepatitis, nephriti

Method used

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  • Fluorogenic quantitative PCR detection reagent and preparation method and application thereof
  • Fluorogenic quantitative PCR detection reagent and preparation method and application thereof
  • Fluorogenic quantitative PCR detection reagent and preparation method and application thereof

Examples

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preparation example Construction

[0041] Such as figure 1 As shown, the preparation method of the fluorescence quantitative PCR detection reagent of the embodiment of the present invention comprises the following steps:

[0042] S101: Screen out the FAdV-4 hexon protein gene which is highly conserved and has FAdV-4 type-specific gene sequence, design a pair of specific primer pairs and a TaqMan MGB probe for detecting the FAdV-4 hexon protein gene, Named respectively as FAdV-4-F(P1), FAdV-4-R(P2) and FAdV-4-MGB-FAM-probe (ProbeP3);

[0043] S102: Preparation of template DNA: with the inactivated FAdV-4 chicken embryo chorioallantoic membrane virus as a positive control, and the normal SPF chicken embryo allantoic membrane as a negative control, the swabs, trachea, heart, Liver, kidney, spleen, and pericardial effusion samples to extract total DNA;

[0044] S103: extracting total RNA by Trizol method;

[0045] S104: Reverse transcription, each tube of reverse transcription reaction system contains the follow...

Embodiment 1

[0050] Embodiment 1, for the establishment of primers and probes and detection methods for the detection of the 4th serotype virus (FAdV-4) TaqMan MGB fluorescence quantitative PCR (FQ-PCR) of avian adenovirus 1 subgroup C

[0051] 1.1 Materials

[0052] 1.1.1 Strains

[0053] The inactivated avian adenovirus subgroup I subgroup C serotype 4 virus (FAdV-4) was isolated and identified by Henan Animal Disease Prevention and Control Center; the other 11 serotype viruses (FAdV-4) 1-3, FAdV-5-12) were purchased from the China Veterinary Drug Control Institute; other control viruses, bacteria or positive recombinant plasmids were provided by the Animal Disease Prevention and Control Center of Henan Province.

[0054] 1.1.2 Instruments and reagents

[0055] Fluorescence PCR instrument, product of American ABI Company, model ABI VⅱA 7; PCR amplification instrument, product of German Biometra Company; gel imaging analysis system, product of American Alpha Innotech Company; constant t...

Embodiment 2

[0093] Example 2, application of TaqMan MGB fluorescence quantitative PCR (FQ-PCR) detection technology for avian adenovirus I subgroup C serotype 4 virus (FAdV-4).

[0094] According to the avian adenovirus I subgroup C kind 4th serotype virus (FAdV-4) FQ-PCR detection method established in Example 1 of the present invention and the PCR method established in this study, prepare detection reagents, with inactivated FAdV-4 Chicken embryo chorioallantoic membrane virus was used as a positive control, and normal SPF chicken embryo allantoic membrane was used as a negative control. 47 samples of clinically suspected FAdV-4 infection (sample number: 1-47) were tested by application. The detection results of the method were compared and analyzed, and compared with the sequencing results. The results showed that 17 samples were positive for FAdV-4 gene by using the FAdV-4 FQ-PCR method provided by the present invention, and the numbers were 8-10, 16, 18, 20, 32-38, 41, 42, 45, 47 res...

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Abstract

The invention discloses a fluorogenic quantitative PCR detection reagent for FAdV-4TaqMan MGB and a preparation method and application of the fluorogenic quantitative PCR detection reagent. The fluorogenic quantitative PCR detection reagent comprises a pair of specific primers and a fluorescence probe. The nucleotide sequences of the specific primers are SEQ ID NO.1-2, and the nucleotide sequence of the fluorescence probe is SEQ ID NO.3. The aviadenovirus I subgroup C-race forth serotype virus (FAdV-4)TaqMan MGB fluorogenic quantitative PCR detection primers and probe and an FQ-PCR detection method established based on the primers and probe have the advantages of being fast, specific, sensitive, high in flux and the like, detection can be completed within 1 h to 2 h, and the requirement for large-batch and fast detection of aviadenovirus I subgroup C-race forth serotype viruses can be met. An effective tool is provided for fast detection, infection source surveying, transmission environment and pathogeny tracing of aviadenovirus, and great significance is achieved for prevention and control of the viruses.

Description

technical field [0001] The invention belongs to the technical field of animal epidemic quarantine detection, and in particular relates to a TaqMan MGB fluorescent quantitative PCR detection reagent for avian adenovirus serotype 4 (FAdV-4) and a preparation method and application thereof. Background technique [0002] Avian adenoviruses are divided into subgroup I, subgroup II, and subgroup III adenoviruses. Subgroup Ⅰ (Fowl adenovirus group Ⅰ, FAdVI) is a virus isolated from the respiratory tract of chickens, turkeys, geese and quails. The viruses of this subgroup are divided into 5 species, A, B, C, D, and E, with a total of 12 serotypes , There can be no cross-protection between viruses of each serotype. In the past many years, FAdVI mostly replicated in the body without becoming ill. After the animal was infected with the virus, it was a recessive infection, and it co-acted on poultry with other pathogens. However, since 2015, poultry with serotype 4 (FAdV-4) in subgrou...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 闫若潜吴志明班付国赵雪丽王华俊王东方马震原谢彩华曹伟伟王淑娟刘梅芬朱前磊仲伟平张和平安利民陈少渠周婷婷杜少甫
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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