Method for quickly detecting duck viral enteritis by real-time fluorescence quantitative PCR and kit thereof

A real-time fluorescence quantitative, duck virus enteritis technology, applied in the field of molecular biology, can solve problems such as environmental pollution, lack of management experience, improper epidemic prevention measures, etc., to eliminate false negative problems, improve compatibility, and stabilize biological characteristics Effect

Inactive Publication Date: 2011-05-04
吉林出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the introduction of breeding ducks or eggs through various channels, frequent exchanges in the domestic poultry product market, lack of management experience in intensive breeding, improper epidemic prevention measures, and serious environmental pollution, various diseases of ducks continue to occur and become more and more complicated. The more serious hazards are still viral infectious diseases, which account for 15% to 20% of duck deaths every year, seriously restricting the sustainable and stable development of my country's duck industry

Method used

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  • Method for quickly detecting duck viral enteritis by real-time fluorescence quantitative PCR and kit thereof
  • Method for quickly detecting duck viral enteritis by real-time fluorescence quantitative PCR and kit thereof
  • Method for quickly detecting duck viral enteritis by real-time fluorescence quantitative PCR and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Real-time fluorescent quantitative PCR rapid detection of duck viral enteritis positive quality control standard - preparation of recombinant plasmid pMD18-T-DEV76bp

[0054] Primers and Taqman probes were designed according to the conserved sequence DEVUL31 gene

[0055] Primer FP (37421-37440) 5'-ACGCTGTCCACGTCAGTTTG-3'

[0056] Primer RP (37475-37496) 5′-TGGCTCATGTTTGGCATTCTAC-3′

[0057] Probe probe(37448-37470) 5′-FCGGTCGCGCCTCACGACAAGTAP-3

[0058] The 5' end of the probe sequence is labeled with FAM (indicated by F), and the 3' end is labeled with TAQMAN (indicated by P);

[0059] Duck viral enteritis chick embryonized attenuated vaccine strain was purchased from China Veterinary Drug Control Institute, and genomic DNA was extracted with the DNA extraction kit of Treasure Bioengineering (Dalian) Co., Ltd.;

[0060] Press the table:

[0061]

[0062] reaction system;

[0063] The reaction conditions are: 94°C for 2min; 94°C for 30s, 58°C for 30s,...

Embodiment 2

[0071] Example 2 Using the recombinant plasmid pMD18-T-DEV76bp as a template, routine PCR verification primer specificity test

[0072] Use the recombinant plasmid pMD18-T-DEV76bp as a template, and use the following primers:

[0073] FP (37421-37440) 5′-ACGCTGTCCACGTCAGTTTG-3′

[0074] RP (37475-37496) 5′-TGGCTCATGTTTGGCATTCTAC-3′

[0075] According to the reaction system:

[0076] Routine PCR verification primer specificity test target gene amplification system

[0077]

[0078] PCR reaction program according to the table

[0079]

[0080] Perform conventional PCR amplification; get a band that matches the expected size (76bp), the band is specific, and the primers do not have dimers, such as image 3 ;

[0081] Perform electrophoresis on the PCR amplified product on 1% low-melting point agarose gel, and excise the target band; recover the target DNA according to the instructions of the Micro Gel Recovery Kit; purify the target DNA 4.5 μL, T vector 0.5 Mix μL and...

Embodiment 3

[0083] Example 3 Preparation of quantitative positive quality control standard pMD18-T-DEV76bp

[0084] The Escherichia coli competent cell DH5α containing the recombinant plasmid pMD18-T-DEV76bp frozen in Example 2 was transferred to the + 60μg / ml LB liquid medium, 250r / min shaking enrichment culture overnight, transfer the cultured overnight bacterial solution to LB liquid medium, 250r / min shaking enrichment culture for 2-3 hours, and then use TaKaRa company plasmid extraction The kit extracts the plasmid; take 10 μL of the plasmid sample, dilute it with 1990 μL of double-distilled water, take 70 μL and transfer it to a 1.0 cm quartz cuvette, and measure A260 on a UV spectrophotometer; the amount of DNA in the sample can be calculated according to the absorbance value of A260 Content, that is, 1 OD value is equivalent to 50ug / ml double-stranded DNA; then,

[0085] According to the formula: Calculate the plasmid concentration,

[0086] According to the formula: Calculat...

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Abstract

The invention provides a method for quickly detecting duck viral enteritis by real-time fluorescence quantitative PCR. The method selects a gene fragment of duck viral enteritis UL31 to design a primer and a probe, and order-checking results on an amplified fragment proves that homologies of the amplified fragment and the gene fragment are both 100 percent without a cross reaction, thereby eliminating problems of virus variation and false negative caused by region factors, and improving detectable positive rate and compatibility of the method. The method constructs a quantitative positive quality control standard (recombinant plasmid pMD18-T-DEV76bp), establishes a standard curve of fluorescence quantitative detection, and can minimally detect 2.1*10 degrees copy / [mu]liter. The assembled standard kit reacts under a standard reaction condition, and determines according to accurate quality control standard and determination standard. The method has the advantages of strong specificity, high sensibility, safe quantitative positive quality control stand, simple operation and low cost, is applicable to large-scale quarantine inspection, port quarantine inspection, epidemiological investigation and laboratory quick diagnose of field duck viral enteritis.

Description

technical field [0001] The invention belongs to the category of molecular biology, specifically a method and a kit for rapidly detecting duck viral enteritis by using fluorescent PCR technology. Background technique [0002] 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%, newly-built large-scale farms are gradually increasing, and the annual output value of duck products is close to 30 billion yuan, and they are exported to the European Union, Southeast Asia, Japan, South Korea and other places. With the further development of the duck industry, the space for duck products will continue to expand, so the duck industry has huge potential. However, due to t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 孟日增石建平肖成蕊刘晶赵庆松刘和平
Owner 吉林出入境检验检疫局检验检疫技术中心
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