PCR method for detecting duck viral enteritis and kit thereof

A duck viral enteritis and kit technology, applied in the field of molecular biology, can solve the problems of being unfavorable for rapid and accurate diagnosis, poor sensitivity, specificity, and time-consuming duck plague diagnosis, and achieve simple operation, strong specificity, Easy-to-use effects

Inactive Publication Date: 2012-03-07
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, it is difficult to determine the diagnosis by one method, and finally a comprehensive judgment must be made through the above-mentioned methods.
These methods take a long time to diagnose duck plague, have poor sensitivity and specificity, and are not conducive to rapid and accurate diagnosis of the disease.

Method used

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  • PCR method for detecting duck viral enteritis and kit thereof
  • PCR method for detecting duck viral enteritis and kit thereof
  • PCR method for detecting duck viral enteritis and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Duck plague chick embryonized attenuated vaccine strain was purchased from China Veterinary Drugs Supervision Institute, and the DNA extraction kit of Treasure Bioengineering (Dalian) Co., Ltd. was used to extract DNA;

[0046] A sequence of UL30 and UL31 in GenBank (accession number: EF417996) was selected as a template, and a pair of primers P1 and P2 were designed and synthesized with Primer 5.0 software. The theoretically amplified fragment was 446 bp. The positions and sequences of the primers were as follows:

[0047] P1 (37154-37173): 5'-CAAGGCTCTATTCGGTAATGAT-3'

[0048]P2 (37580-37599): 5'-GAAGGCGGGTATGTAATGTACA-3'

[0049] Synthesized by Dalian Bao Biological Engineering Co., Ltd.;

[0050] Press the reaction system of table 1;

[0051] Table 1 Composition of PCR reaction system

[0052]

[0053] According to the PCR reaction condition of table 2;

[0054] Table 2 PCR amplification program

[0055]

[0056]

[0057] At the same time, normal SPF c...

Embodiment 2

[0060] Carry out electrophoresis on 1.0% low-melting point agarose gel with the PCR product of duck plague chicken embryonized attenuated vaccine strain (referred to as duck embryo virus) in implementation 1, cut out the target band, and follow the instructions of the micro gel recovery kit Recover the target DNA; mix 4.5 μL of the purified target DNA, 0.5 μL of the T vector, and 5 μL of the connection solution, connect overnight at 16°C, transform DH5α competent cells, pick white colonies and inoculate 5mL of LB liquid medium, and shake in a water bath at 37°C Vibrated and cultured for 12 hours; the plasmid was extracted by alkaline lysis, and identified as a positive plasmid by Hind III / EcoR I double enzyme digestion, and sent to Shanghai Dingan Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in the sequence table SEQ ID NO.1. Sequencing results showed that the length of the PCR amplified fragment was 446bp, and the obtained sequence was compared with...

Embodiment 3

[0061] Example 3 Sensitivity Test

[0062] DNA was extracted from chicken embryo culture of duck plague chicken embryonized attenuated vaccine strain, and the nucleic acid mass concentration was determined to be 0.07 μg / μL on a nucleic acid detector, and the extracted DNA template was diluted 10 times with prepared TE (pH8.0), each Take 3 μL of template DNA for each dilution, detect according to Example 1, observe the positive band, calculate the sensitivity with the highest dilution of the template amount used for the positive expected band, and the minimum detection amount of the result is 2.1fg, see figure 2 .

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Abstract

The invention discloses a PCR method for detecting duck viral enteritis. The method comprises the following steps: gene fragments of duck viral enteritis UL30 and UL31 are selected; a pair of primers are determined through screening; PCR amplification is carried out by using virus DNA extracted from standard strain and various wild strains as a template to obtain about 446bp strips respectively; sequencing results are compared with those announced in UL30 and UL31 regions, homology reaches 100 percent, variation phenomenon is not found, and 6 duck susceptible viruses and 3 similar viruses do not have response. DNA extract of each tissue can be detected by the established PCR method, and DNA fragments with size about 446bp can be amplified with minimum detection capacity of 2.1fg (or copy). The method is applicable to clinical and port detection, has the advantages of high sensibility, quick specificity, convenient operation and easy standard; the assembled standard kit has the advantages of stable performance, convenient and quick operation and high amplification yield, is safer by using C-KCE strain as positive control, and establishes accurate determination standard.

Description

technical field [0001] The invention belongs to the field of molecular biology, specifically a PCR method and a kit for detecting duck viral enteritis by using molecular biology, in particular for the detection of chicken embryonized attenuated vaccine strains of duck plague. Background technique [0002] Duck viral enteritis (DVE), commonly known as "duck plague", "big head plague" or "rotten intestine plague". It is an acute, febrile and septic infectious disease of ducks, geese and other birds of Anseriformes. The epidemic is widespread and spreads rapidly, with high morbidity and mortality. [0003] In the past ten years, my country's duck industry has developed rapidly. According to the statistics of the United Nations Food and Agriculture Organization (FAO) in 2002, my country's duck population is about 661 million, accounting for 69.7% of the world's total. It can be seen that my country is the largest duck producer . Moreover, with an annual rate of 10% to 15%, new...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 潘风光刘静波周玉
Owner JILIN UNIV
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