Egg drop important virus loop-mediated multiplex PCR (polymerase chain reaction) quick detection primers

A technology for dropping egg production and detecting primers, applied in microorganisms, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of slow recovery of egg laying and failure of laying birds, and avoid mutual interference between primers. The effect of saving testing costs and ensuring testing efficiency

Inactive Publication Date: 2016-06-15
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

After 4 weeks, laying birds slowly resume eg

Method used

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  • Egg drop important virus loop-mediated multiplex PCR (polymerase chain reaction) quick detection primers
  • Egg drop important virus loop-mediated multiplex PCR (polymerase chain reaction) quick detection primers

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Embodiment 1

[0025] The preparation of embodiment 1 capture probe

[0026] In the present invention, the specific capture probes used to detect 3 kinds of viruses that cause poultry egg production decline are deduced and obtained by the following method:

[0027] 1.1 According to the 100kd gene of Egg Drop Syndrome Virus (EDSV), the capsid protein gene of Avian Tembusu Virus (ATV) and the hemagglutinin protein gene of H9 Subtype Avian Influenza Virus (H9AIV) published in GenBank Conserved region sequence Conserved region and partial sequence of enhanced green fluorescent protein (EGFP), 3 pairs of primers were designed for the preparation of capture probes (see Table 1 for primer information).

[0028]

[0029] 1.2. Use the green fluorescent protein particle (pIRES2-EGFP, Takara Biotechnology Co., Ltd., Dalian) as a template, and use the above three pairs of primers to amplify. The PCR reaction system is 50 μL: 5 μL of 10xFastPfuBuffer, 1 μL of FastPfuDNApolymerase (2.5u / μL), 1 μL of d...

Embodiment 2

[0030] Example 2: Establishment of loop-mediated multiplex PCR detection method

[0031] 2.1 According to the nucleic acid consensus sequences of the three virus-specific capture probes, design a pair of reverse amplification primers. The upstream primer is SeqIDNo.4 (rEGFPF): 5'-CTCGGCGCGGGTCTTGTAGTT-3', and the downstream primer is SeqIDNo.5 (rEGFPR ): 5'-CGAGGACGGCAGCGTGCAGCT-3'. Using these primers to amplify the three circularized probes, amplified fragments of different sizes can be obtained, the lengths of which are egg drop syndrome virus 436bp, avian Tembusu virus 480bp and H9 subtype avian influenza virus 681bp.

[0032] 2.2 Collect duck livers or oviducts that show decreased egg production. After homogenization according to conventional methods, dilute 1:3 with double-antibody-containing sterile PBS buffer (pH7.2), divide into 2 parts, and freeze-thaw repeatedly 3 times. One aliquot was centrifuged at 8000r·min-1 for 10min, and the supernatant was extracted with ...

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Abstract

The invention provides egg drop important virus loop-mediated multiplex PCR (polymerase chain reaction) quick detection primers. The method comprises the following steps: preparing a specific trapping probe for nucleic acids of three avian egg drop important viruses (egg drop syndrome virus, avian Tembusu virus and avian influenza virus H9 subtype), hybridizing with the corresponding viral nucleic acid, cyclizing under the action of ligase, carrying out PCR (polymerase chain reaction) amplification detection by using a pair of probe-specific universal detection primers, and determining the infection pathogen according to the size of the obtained amplification product. The primers can be used for detecting a single virus, and can also be used for simultaneous differential diagnosis of three different viruses. Thus, the primers have the characteristics of high efficiency, high specificity, high sensitivity and low cost, are suitable for pathogen detection and analysis on abundant clinical samples in case of egg drop of the laying poultry, and provide an important technical means for early-stage quick differential diagnosis and molecular epidemiological analysis for determining egg-drop-causing pathogens.

Description

technical field [0001] The invention relates to primers for rapid detection of multiplex PCR mediated by an important viral loop for egg production decline, and belongs to the field of preventive veterinary medicine. Background technique [0002] In recent years, the decline in egg production caused by infectious diseases during the breeding of breeders and laying poultry in my country has become more and more serious. The main manifestations are the decline in egg production and egg quality. The emergence of soft-shell eggs, sand-shell eggs, thin-shell eggs, deformed eggs and small eggs seriously affects the production performance of breeding (egg) poultry and causes huge economic losses. Existing studies have shown that many pathogens can lead to a decline in egg production in breeding (egg) poultry, such as avian Tembusu virus (ATV), egg drop syndrome virus (EDSV), H9 subtype avian influenza virus (H9AIV), poultry Paramyxovirus type 1 (APMV-1) and Salmonella and other bac...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6862C12Q1/701C12Q2600/124C12Q2600/158C12Q2600/16C12Q2521/501C12Q2537/143
Inventor 傅光华傅秋玲黄瑜程龙飞施少华陈红梅万春和陈翠腾林建生
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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