Multiplex fluorescence quantitative PCR detection primer and probe combination and detection method for simultaneously detecting four duck-susceptibility viruses

A multiplex fluorescence quantitative and detection method technology, applied in the multiplex fluorescence quantitative PCR detection primer and probe combination and detection field, can solve the problems of high virus purification requirements, long time, lack of PCR sensitivity, etc., to achieve good repeatability, Detection of high sensitivity and specificity

Pending Publication Date: 2019-05-31
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These diseases often exist in the form of mixed infection clinically, and it is difficult to make a rapid differential diagnosis only by visual observation of symptoms, autopsy changes, and disease characteristics. imminent
[0003] Clinically, virus isolation and identification, serological detection, ELISA, immunoelectron microscopy, and conventional PCR have been applied to the detection of these viral infections, but traditional methods such as virus isolation and identification are time-consuming, and serological and ELISA detection is time-consuming and laborious. The limitations of factors such as the freshness of clinical disease materials, the degree of pollution, or the course of the disease, the lack of sensitivity of conventional PCR, and the high requirements for equipment and virus purification of immunoelectron microscopy have brought limitations to clinical diagnosis.
Clinically, these diseases are often a variety of mixed infections, and the differential diagnosis is even more difficult.

Method used

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  • Multiplex fluorescence quantitative PCR detection primer and probe combination and detection method for simultaneously detecting four duck-susceptibility viruses
  • Multiplex fluorescence quantitative PCR detection primer and probe combination and detection method for simultaneously detecting four duck-susceptibility viruses
  • Multiplex fluorescence quantitative PCR detection primer and probe combination and detection method for simultaneously detecting four duck-susceptibility viruses

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Experimental program
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Effect test

Embodiment 1

[0041] Primer design and screening

[0042] According to the conserved genes of each virus in GenBank, the fusion protein gene (F gene) of NDV, the matrix protein gene (M gene) of AIV, the nucleocapsid protein gene (VP1 gene) of DHAV, the envelope protein gene (E gene) of DTMUV ), using DNAMAN software for homology analysis, using Primer Premier5.0 software, designing specific probes in its conserved regions, and labeling FAM, JOE, ROX, Cy5 and other fluorescent emitting groups at the 5' end, and at the 3' BHQ1 or BHQ2 fluorescence quenching group is labeled at the end, and at the same time, according to the position of the probe, two or more primers are designed for each virus for screening, and primers with good specificity, no cross-reaction and good amplification efficiency are selected. The virus-specific primers and probes are listed in Table 1.

[0043] Table 1

[0044]

[0045]

Embodiment 2

[0047] Plasmid standard preparation

[0048] Step 1: Primer Synthesis

[0049] The 4 pairs of virus plasmid construction primers designed by the present invention are shown in Table 2, and were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0050] Step 2: Total viral RNA extraction

[0051] According to the instructions of the Viral RNA Extraction Kit kit, four kinds of viral RNA were extracted respectively, and the reverse transcription step was performed immediately.

[0052] Step 3: Reverse transcription PCR

[0053] Add 1 μl of NDV, AIV, DHAV, and DTMUV RNA to an RNase-free PCR tube, and use ThermoScientific RevertAidFirstStrand cDNASynthesisKit for RT-PCR reaction, including 4μl of 5xBuffer, 2μl of 10mM dNTP, 1μl of Random Primers, 1μl of 20U / μl Ribolock RNase, 200U / μl ReverAid M-MuLVRTase 1μl, DEPC water 10μl, total system 20μl, cDNA was obtained after the reaction.

[0054] Step Four: PCR Amplification

[0055] 50μl PCR reaction system: 5μl 10x taq buff...

Embodiment 3

[0061] Primer specificity verification

[0062] Use the standard plasmids constructed by the four viruses as templates to amplify the corresponding target fragments. Each reaction system is 20 μl, including 10.0 μl of 2x SYBR Green, 0.4 μl of upstream and downstream primers, 1 μl of a single virus plasmid template, and deionized water to make up 20μl; the reaction program is: 95°C for 3min, 95°C for 10s, 54°C for 30s, after 40 cycles, add a melting curve program: 95°C for 10s, 60°C for 30s, and 97°C for 1s. The specificity was 100%.

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Abstract

The invention discloses a multiplex fluorescence quantitative PCR detection primer and probe combination and detection method for simultaneously detecting four duck-susceptibility viruses. Sequences of primers include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11 respectively; sequences of probes include SEQ IDNO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12 respectively. The four duck-susceptibility viruses include the duck-derived avian influenza virus, the duck-derived newcastle disease virus, the duck hepatitis a virus and the duck tembusu virus. By means of the multiplex fluorescence quantitative PCR detection primer and probe combination and detection method, the four viruses can be detected simultaneously, pathogens can bequantitatively detected, the detection is quick and convenient, and the time and labor are saved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a combination of multiple fluorescent quantitative PCR detection primers and probes and a detection method for simultaneously detecting four duck susceptible viruses. Background technique [0002] my country is the largest producer and consumer of ducks in the world, and duck breeding accounts for more than 60% of the world's total. With the rapid development of the duck industry in recent years, the expansion of the breeding scale, the increase of mixed breeding models, the increased mobility of humans and livestock, and the decline in water quality caused by water pollution have created favorable conditions for the spread of the virus. Local epidemics The viral diseases in China have increased significantly, and new epidemics have occurred continuously, causing serious economic losses to the duck breeding industry. At present, duck-derived avian...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCY02A50/30
Inventor 宋素泉闫丽萍姚明
Owner NANJING AGRICULTURAL UNIVERSITY
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