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Method for detecting egg drop syndrome viruses and kit for method

A technology for egg production decline and syndrome, applied in the field of molecular biology, can solve problems such as inaccurate quantification, false positives, cross-contamination, etc., and achieve high sensitivity, strong specificity, and accurate detection

Inactive Publication Date: 2014-04-23
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional PCR technology still has deficiencies in its application. One is that it cannot be accurately quantified, and the other is that it is easy to cross-contaminate and produce false positives.

Method used

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  • Method for detecting egg drop syndrome viruses and kit for method
  • Method for detecting egg drop syndrome viruses and kit for method
  • Method for detecting egg drop syndrome viruses and kit for method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The construction of embodiment 1 EDSV standard plasmid

[0050] 1 Experimental materials

[0051] 1.1 Strains, viruses and vectors

[0052] Egg drop syndrome virus (EDSV) NE4 strain, chicken infectious anemia virus (CIAV) CUX-1 strain, duck plague virus (DEV) CV strain, Marek's virus (MDV) 814 strain are preserved in our laboratory; DH5α Competent cells were prepared and preserved by our laboratory; pEASY-T1 cloning kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0053] 1.2 Main instruments and reagents

[0054] ABI7900HT fluorescent quantitative PCR instrument was purchased from Applied Biosystems (ABI); fluorescent quantitative PCR 2×Ex premix Taq kit and DNA fragment recovery kit were purchased from Dalian Bao Biological Engineering Co., Ltd. (TaKaRa); plasmid small extraction reagents The box was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0055] 2 Methods and results

[0056] 2.1 Primer and probe design

[0057] Acc...

Embodiment

[0065] Design and synthesis of primers and probes used in embodiment 2EDSV TaqMan fluorescent quantitative PCR detection method

[0066] According to the EDSV AV-127 strain gene (Accession No.Y09598.1) sequence registered in GenBank, the highly conserved hexon protein coding region gene was selected as the amplified region by sequence comparison, and the genetic markers for detecting EDSV were determined. Its nucleotide sequence is as shown in SEQ ID NO.1, further through sequence comparison design taqman probe and the matching primer design thereof, its nucleotide sequence is respectively as SEQ ID NO.4, SEQ ID NO.2, Shown in SEQ ID NO.3.

[0067] The primer sequence is:

[0068] Taq-EHFP: 5'-GACCGGTCACAAAAACTTCATCT-3',

[0069] Taq-EHRP: 5'-CACAATCCTGTTATTCACCCACATTT-3'.

[0070] The probe sequence is:

[0071] 5'FAM-CGCATCGTCCCGATCCAAACAGA-BHQ3'.

[0072] Although it is preferred that the primers and probes are completely complementary to the target sequence, those ski...

Embodiment 3

[0073] Example 3 Establishment of TaqMan Fluorescent Quantitative PCR Detection Method for Chicken Egg Drop Syndrome Virus

[0074] 1. The 20 μl reaction system of real-time fluorescent quantitative PCR is: Premix Ex Taq 2×buffer 12.5 μL, 10 μmol / L upstream primer Taq-EHFP and 10 μmol / L downstream primer Taq-EHRP 0.4 μL each, 10 μM probe 0.8 μL, ROX dye 0.4 μL, template (plasmid DNA) 2 μL, supplemented with ddH 2 0 to 20 μL.

[0075] The reaction program of real-time fluorescent quantitative PCR was: pre-denaturation at 95°C for 30s, 1 cycle; denaturation at 95°C for 5s, annealing at 60°C for 30s, 40 cycles.

[0076] 2. Preparation of system standard curve

[0077] The standard plasmid template prepared in Example 1 was subjected to fluorescent quantitative PCR according to the above conditions and system. By optimizing the conditions, accurately matching the template concentration, selecting the most suitable annealing temperature, and shortening the sample addition time, t...

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Abstract

The invention provides a method for detecting egg drop syndrome viruses and a kit for the method. By means of sequencing and comparing egg drop syndrome virus genes, a genetic marker for detecting the egg drop syndrome viruses, a real-time fluorescent quantitative PCR (polymerase chain reaction) primer and a probe are provided, and nucleotide sequences are respectively showed as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The invention further provides a real-time fluorescent quantitative PCR method for detecting the egg drop syndrome viruses and the detecting kit. The minimum detectable amount of the detection method reaches 10 copy / muL, the method has a good linear relation within a 1.0X102-1.0X108 copy / muL detection range, and R2 equals to 0.992. The method has the advantages of accurate detection, high sensitivity and specificity, simplicity, convenience and speediness, and has good specimen detection ability.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a target sequence for detecting chicken egg drop syndrome virus, fluorescent quantitative PCR primers and probes, and the invention also relates to using the target sequence to detect chicken egg drop syndrome virus methods and kits. Background technique [0002] Egg drop syndrome (EDS) is also known as egg drop syndrome-76 or egg drop syndrome-76 (Egg drop syndrome-76, EDS-76). Egg drop syndrome virus (EDSV) belongs to the Avian adenovirus Group III of the family Adenoviridae. The disease is characterized by recessive infection in chickens before sexual maturity, and clinical symptoms appear only after laying, which mainly causes clinical symptoms such as decreased egg production and incomplete egg shell formation, resulting in a huge loss of egg production. Chicken egg drop syndrome was initially limited to Europe, and then the disease occurred in Australia, Belgium, France, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12R1/93
Inventor 李刚马震原李文超
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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