Nano multiplex PCR method for distinguishing four kinds of serotype avian adenovirus I group

A poultry adenovirus and serotyping technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of tediousness, need for special instruments, poor sensitivity, etc., and achieve high sensitivity, good stability, and specificity strong effect

Active Publication Date: 2018-03-27
QINGDAO AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the defects in existing avian adenovirus group I typing methods, which are cumbersome, require special instruments, and have poor sensitivity, and seek to provide a fast, simple and accurate method for distinguishing four serotypes of avian adenoviruses. Group I nano-multiplex PCR method

Method used

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  • Nano multiplex PCR method for distinguishing four kinds of serotype avian adenovirus I group
  • Nano multiplex PCR method for distinguishing four kinds of serotype avian adenovirus I group
  • Nano multiplex PCR method for distinguishing four kinds of serotype avian adenovirus I group

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Synthesis of nanometer multiplex PCR primers:

[0030] According to the 4,8a, 8b and 11 type avian adenovirus group I typing detection primer sequences designed in Table 1, synthesize various type-specific primers; Type 4 avian adenovirus group I primer (F: 5'-CAARTTCAGRCAGACGGT-3' R:5'-AAGAGGCCCGGGCAATGC-3'); Type 8a adenovirus I group primer (F:5'-CAARTTCAGRCAGACGGT-3'R:5'-AATGTTTGACGAGCTGATGGG-3'); Type 8b avian adenovirus group I primer (F :5'-CAARTTCAGRCAGACGGT-3'R:5'-ATGCTGCAGCTGTTGCCGTAG-3'); 11 type avian adenovirus I group primers (F:5'-CAARTTCAGRCAGACGGT-3'R:5'-ACTGCCGTCGTCTCGTCTAAG-3');

[0031] 2. Extraction of genes:

[0032] Take 4, 8a, 8b, and 11 types of avian adenovirus group I standard strains (purchased from the China Veterinary Drug Administration) cell grinding solution 400μL in a 1.5mLEP tube, add 400μL chloroform, 600μL lysate, mix well and precipitate at 4°C for 10min Afterwards, centrifuge at 12000r / min for 10min, take 600μL of supernatant,...

Embodiment 2

[0042] Embodiment 2, nanometer multiplex PCR specificity test:

[0043] The cDNAs of infectious bronchitis virus (IBV), egg drop syndrome virus (EDSV), H9 subtype avian influenza virus (AIV-H9) and Newcastle disease (NDV) were detected by using the nanometer multiplex PCR method established above. Validate the specificity of the method.

[0044] Such as image 3 Shown, M: 2000maker, 1: four kinds of serotypes avian adenovirus group I positive sample mixture, 2: IBV, 3: EDSV, 4: AIV-H9, 5: NDV.

[0045] No bands were amplified in the test results, and only the specific bands of the four serotypes of avian adenovirus-I group were amplified, indicating that the primers had good specificity.

Embodiment 3

[0046] Embodiment 3, sensitivity test:

[0047] The positive plasmid established by the hexon whole gene of the standard strain of avian adenovirus group I of type 4, 8a, 8b and 11 was used as a template, and the 10-fold serial dilution was carried out to determine the minimum detection concentration of nanometer multiplex PCR and ordinary multiplex PCR.

[0048] Such as Figure 4 As shown, multiplex PCR concentration gradient: M: 2000maker, 1: original concentration plasmid, 2: 10×10 1 1-fold diluted plasmid, 3:10×10 2 1-fold diluted plasmid, 4:10×10 3 Double diluted plasmid, 5:10×10 4 1-fold diluted plasmid, 6:10×10 5 1-fold diluted plasmid, 7:10×10 6 1-fold diluted plasmid, 8:10×10 7 Dilute the plasmid one-fold. The minimum concentration of ordinary multiplex PCR detection is: FAdV-4 is 5.14×10 7 Copy·μL -1 , FAdV-8a is 2.87×10 7 Copy·μL -1 , FAdV-8b is 1.71×10 6 Copy·μL -1 , FAdV-11 is 4.35×10 7 Copy·μL -1 .

[0049] Such as Figure 5 As shown, nanometer m...

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Abstract

The invention relates to a nano multiplex PCR method for distinguishing four kinds of serotype avian adenovirus I group. Various types of specific primers are designed and synthesized according to hexon gene sequences of 4, 8a, 8b and 11 type avian adenovirus I group disclosed by GenBank; after gene extraction, nano multiplex PCR amplification is performed, with the PCR reaction conditions of pre-degeneration for 5min at 95 DEG C and 30s at 94 DEG C, annealing for 1min at 53 DEG C and 1min49s at 72 DEG C, and 30 circulation; extension for 10min at 72 DEG C; and after nano multiplex PCR amplification reaction is ended, a PCR product is taken, and the size of an amplification fragment is observed by taking DL 2000 as a Maker and adopting 1% agarose gel electrophoresis, so as to determine theserotype of virus. According to the method disclosed by the invention, the detection time can be obviously shortened; by adding nano particles, under the condition of detecting four kinds of serotypeviruses, the sensitivity is at least increased by 10 times in comparison with common multiplex PCR; and the method disclosed by the invention is strong in specificity, high in sensitivity and good instability, and can quickly, sensitively and accurately classify serotype avian adenovirus I group which is popular mainly in China.

Description

Technical field: [0001] The invention relates to a nanometer multiplex PCR method, in particular to a nanometer multiplex PCR method for distinguishing four serotypes of poultry adenovirus group I, and belongs to the technical field of animal virus detection. Background technique: [0002] Group I of avian adenovirus (Fowl adenovirus, FAdV) mainly includes 12 serotypes. Changjing Li et al. (2016) sequenced and analyzed the Hexon gene of isolated strains of Group I of fowl adenovirus in my country from 2006 to 2016, indicating that The main prevalent serotypes are 4, 8a, 8b and 11. The high infection rate and high mortality of avian adenovirus group I to broiler chickens have caused serious economic losses to the chicken industry. These four serotypes of avian adenoviruses all caused severe hepatic inflammation in broiler chickens, and the clinical symptoms caused by them were very similar, which brought difficulties to the clinical detection of different serotypes of avian ad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2537/143C12Q2563/149
Inventor 王建琳江之瑶尹燕博王守春
Owner QINGDAO AGRI UNIV
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