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Probe primer group for rapid differential diagnosis of APMV (avian paramyxovirus)-1 and APMV-4, kit and detection method

A technology of APMV-1-F and APMV-1-R, which is applied to the determination/inspection of microorganisms, DNA/RNA fragments, microorganisms, etc., can solve the problems of long detection cycle, time-consuming and laborious, and unfavorable timely diagnosis and control of epidemic diseases. Achieve high repeatability, wide dynamic range, and high specificity

Pending Publication Date: 2018-09-14
POULTRY INST SHANDONG ACADEMY OF AGRI SCI SHANDONG SPECIFIC PATHOGEN FREEN CHICKS RES CENT
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Problems solved by technology

Although the traditional pathogen isolation method is effective, the disadvantage is that the detection cycle is too long, at least 3 days, which is time-consuming and laborious, which is not conducive to the timely diagnosis and control of epidemic diseases

Method used

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  • Probe primer group for rapid differential diagnosis of APMV (avian paramyxovirus)-1 and APMV-4, kit and detection method
  • Probe primer group for rapid differential diagnosis of APMV (avian paramyxovirus)-1 and APMV-4, kit and detection method
  • Probe primer group for rapid differential diagnosis of APMV (avian paramyxovirus)-1 and APMV-4, kit and detection method

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Embodiment 1

[0043] 1 method

[0044] 1.1 Design and synthesis of probes and primers

[0045] The popular strain sequences of APMV-1 serotype and APMV-4 serotype were downloaded from NCBI, and compared with BioEdit software, the conserved region sequences of the strain sequences were obtained. Primer ExPress 3.0 software was used to design real-time fluorescent quantitative PCR primers and TaqMan probes. The probes were labeled with FAM fluorescein at the 5' end and MGB fluorescein at the 3' end, as shown in Table 1.

[0046] Table 1 Amplification primers and probes

[0047]

[0048] 1.2 RNA extraction quality inspection and reverse transcription

[0049]Add 1mL Trizol to 0.2mL viral allantoic fluid and mix well, then add 200μL chloroform, mix well, and centrifuge at 12000rpm for 5-7min. Take the supernatant to a 1.5mL Eppendorf tube, add 600μL chloroform, mix well, and centrifuge at 12000 rpm for 5min. Take the supernatant to a 1.5mL Eppendorf tube, add 500uL isopropanol, mix well...

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Abstract

The invention relates to a probe primer group for rapid differential diagnosis of APMV (avian paramyxovirus)-1 and APMV-4, a kit, and a detection method and application of the kit, and belongs to thebiotechnology field. The probe primer group is shown as SEQ ID NO: 1-6 in the table. The probe primer group has the advantages of high sensitivity, high specificity, high repeatability, wide dynamic range and the like, thereby becoming useful and strong technology in the fields such as basic research and applied research. By the fluorescence probe quantitative PCR (polymerase chain reaction) amplification technology, rapid differential diagnosis of different APMV serotypes is realized; detection results can be judged rapidly and visually, and the time of from sample detection to report resultgeneration is only 2-3 hours; detection sensitivity and specificity are high, and the virus content of infected tissues can be quantified initially. Rapid differential diagnosis of the APMV-1 and theAPMV-4 can be realized, and the probe primer group is quite suitable for detection in clinical and primary laboratories.

Description

technical field [0001] The invention relates to a probe primer set, a kit, a detection method and an application for rapid differential diagnosis of APMV-1 and APMV-4, and belongs to the field of biotechnology. Background technique [0002] Avian Paramyxovirus (APMV) belongs to the family Paramyxoviridae, subfamily Paramyxovirinae, and the genus Avulavirus. There are nine serotypes of APMV, namely APMV-1 to APMV-9. Among them, APMV-1 is mainly Newcastle disease virus, commonly known as fowl plague virus, which is the most comprehensive and in-depth research on a type of avian paramyxovirus. The genome of avian paramyxovirus is single-stranded negative-strand RNA, and the genome encodes six main structural proteins, including nucleoprotein (NP), phosphoprotein (P), membrane protein (M), fusion glycoprotein (F), blood Lectin neuraminidase protein (HN) and large protein (L). At present, there are many studies on APMV-1, including biological characteristics, pathogenicity, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2521/107C12Q2563/107
Inventor 袁小远张玉霞王友令孟凯
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI SHANDONG SPECIFIC PATHOGEN FREEN CHICKS RES CENT
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