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Avian paramyxovirus PCR universal detection primer, diagnostic reagent and kit

A general-purpose detection and diagnostic reagent technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as undetectable serotypes, and achieve easy observation, simple operation, and good versatility sexual effect

Pending Publication Date: 2022-01-21
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most assays target only one serotype of avian paramyxovirus and may fail to detect emerging serotypes (eg, APMV-14 to -22)

Method used

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  • Avian paramyxovirus PCR universal detection primer, diagnostic reagent and kit
  • Avian paramyxovirus PCR universal detection primer, diagnostic reagent and kit
  • Avian paramyxovirus PCR universal detection primer, diagnostic reagent and kit

Examples

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Effect test

Embodiment 1

[0021] This example aims to describe the design and synthesis of general detection primers for avian paramyxovirus PCR.

[0022] In this embodiment, with reference to the nucleotide sequences of the L genes of each serotype of avian paramyxovirus in Genbank, a pair of PCR primers were designed according to their shared conserved sequences. The expected amplified fragment size was 349bp, and the designed primer sequences were as follows:

[0023] Upstream primer: 5'-TTTATAACAACTGACCTGCAAAAGTA-3'; as shown in SEQ ID No.1;

[0024] Downstream primer: 5'-CTTGATTGTCACCTTGTACCATGCAG-3'; as shown in SEQ ID No.2.

[0025] The above primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0027] This example aims to provide a process for using the primers described in Example 1 to realize the two-step RT-PCR detection of avian paramyxovirus.

[0028] 1. Determination of two-step RT-PCR detection of avian paramyxovirus

[0029] (1) Virus proliferation

[0030] The avian paramyxovirus type 1 strain was inoculated into 9-11-day-old SPF chicken embryos through the allantoic cavity of chicken embryos, incubated in a 37°C incubator, and aseptically collected chicken embryos that died at any time and survived after 72 hours. Allantoic fluid was stored in a -80°C refrigerator.

[0031] (2) Extraction of viral RNA

[0032] Take 200 μL of viral allantoic fluid, add 600 μL Trizol lysate, and shake slowly for 5-10 minutes; add 120 μL of chloroform, shake vigorously for 15 seconds, let stand for 15 minutes, and centrifuge at 12000 g / min at 4°C for 15 minutes; transfer the supernatant to the tube, add After mixing 300 μL of isopropanol upside down, place it at room temper...

Embodiment 3

[0040] This Example 3 aims to verify the effect of using the primers described in Example 1 for RT-PCR detection of avian paramyxovirus through experiments.

[0041] 1. Specificity experiment of detection method

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Abstract

The invention discloses an avian paramyxovirus PCR universal detection primer, a diagnostic reagent and a kit. The sequences of the primer are shown as SEQ ID NO. 1 and SEQ ID NO. 2. All serotype avian paramyxovirus infection of poultry and wild birds can be detected, a strip generated by electrophoresis is single and bright, and a result is easy to observe.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a PCR universal detection primer for avian paramyxovirus, a diagnostic reagent and a kit. Background technique [0002] Avian paramyxoviruses (APMV), also known as avian mumps viruses (Avian avulaviruses, AAvV), belong to the Avulavirinae subfamily of the Paramyxoviridae family. Avian paramyxovirus can infect a variety of birds worldwide, with a wide host spectrum and various clinical symptoms. So far, 22 serotypes (APMV-1 to -22) of avian paramyxovirus have been identified. Among them, the infection of APMV-1, -2, -3, -6 and -7 can lead to poultry disease. APMV-3 and -4 mainly infect wild birds, the main host of APMV-6 is wild duck, and APMV-7 is mainly isolated from pigeons. APMV-1 is Newcastle disease virus (NDV). Newcastle disease is a highly contagious infectious disease with high mortality that occurs in poultry and wild birds. It is mainly manifested as dys...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2521/107C12Q2565/125
Inventor 刘新鑫尹仁福王梦君闫巍文成珊育柴浩然高超
Owner JILIN UNIV
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