Kit for detecting peste des petits ruminants virus by utilizing pyrophosphoric-acid sequencing technology

A technology of pyrosequencing and Peste des petits ruminants, which is applied in the field of pyrosequencing, can solve the problems of no treatment methods, etc., and achieve the effect of simple operation, small sample volume, and convenient construction of standardized operation procedures

Inactive Publication Date: 2014-03-26
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no effective treatment for this disease, and it can only be prevented effectively. Therefore, diagnosis and monitoring technology has become an important means of controlling the disease.

Method used

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  • Kit for detecting peste des petits ruminants virus by utilizing pyrophosphoric-acid sequencing technology
  • Kit for detecting peste des petits ruminants virus by utilizing pyrophosphoric-acid sequencing technology
  • Kit for detecting peste des petits ruminants virus by utilizing pyrophosphoric-acid sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 detects the design of the primers of Peste des petits ruminants virus

[0037] By comparing and analyzing the PPRV N gene sequences published in various countries and regions in the NCBI Gene Bank (GenBank) of the National Center for Bioinformatics in the United States, the highly conserved sequence of the PPRV N gene was determined, and the specific sequence was designed using Assay Design SW software Universal RT-PCR primers and pyrosequencing primers, select the primer set with a higher score for Blast analysis, and select a set of optimal primers for subsequent RT-PCR and sequencing work based on the analysis results.

[0038] The general primer of Peste des petits ruminants virus designed according to the specific region sequence of Peste des pets ruminants virus N gene can amplify a specific single band for the RNA of Peste des pets ruminants virus, and the length of the RT-PCR amplified fragment is 78bp. Then, pyrosequencing primers were designed acc...

Embodiment 2

[0043] Example 2 Detection of Peste des petits ruminants virus by pyrosequencing technology

[0044] 1. Extraction of RNA from Peste des petits ruminants vaccine

[0045] Using inactivated PPRV Nigeria75 / 1 virus (purchased from China Veterinary Drug Administration) as raw material, RNA was extracted by TRIZOL method. The specific steps are:

[0046] (1) Take 200uL of the inactivated virus culture supernatant and add it to a sterilized 1.5ml Eppendorf tube, then add Trizol 1.0mL, mix repeatedly, and place on ice for 5min.

[0047] (2) Add 200uL chloroform, cover the cap carefully, shake the Eppendorf tube vigorously for 15s, and let it stand at room temperature for 5min. 4°C, 12000r / min, centrifuge for 15min, the solution is divided into 3 layers, the upper aqueous phase contains RNA.

[0048] (3) Transfer the water phase to a new Eppendorf tube, add 500uL isopropanol, mix well, and place at -20°C for 15min. 4°C, 12000r / min, centrifuge for 10min, after centrifugation, gel-l...

Embodiment 3

[0063] Example 3 Detection of Sheep Blood Samples Using Pyrosequencing Technology

[0064] 1. Extraction of RNA from sheep blood samples

[0065] The QIAGEN QLAamp Viral RNA Mini Kit (K2904) was used to extract sheep blood genomic RNA. The kit operation steps are as follows:

[0066] (1) Add carrier RNA to buffer AVL according to the instructions, take 560 μL buffer AVL (containing carrier RNA) into a 1.5ml centrifuge tube (the amount can be adjusted proportionally according to the actual sample volume).

[0067] (2) Add 140 μL goat blood supernatant to the centrifuge tube containing buffer AVL-carrier RNA. Thoroughly mediate and mix for 15 seconds to ensure lysis efficiency.

[0068] (3) Stand at room temperature for 10 minutes to inactivate potential contamination and RNase.

[0069] (4) Centrifuge briefly, and throw the droplets on the lid and side walls to the bottom of the tube.

[0070] (5) Continue to add 560 μL of absolute ethanol (96% to 100%) into the tube, and f...

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Abstract

The invention provides a kit for detecting peste des petits ruminants virus by a pyrophosphoric-acid sequencing technology. The kit comprises a general primer for detecting peste des petits ruminants and a pyrophosphoric-acid sequencing primer, and the nucleotide sequences are respectively shown in SEQ ID NO.2-4. The kit adopts the following steps of by extracting the RNA of a sample to be detected, carrying out RT-PCR amplification by the general primer; if the length of the amplified fragment of the primer is 78bp, carrying out pyrophosphoric-acid sequencing reaction; if the sequencing fragment is completely the same as N gene target fragments (SEQ ID NO.1), determining to be the peste des petits ruminants virus; if one or more than one basic groups of the sequencing fragment are different from those of the target fragment, determining that the sample is negative. The kit provided by the invention can be used for rapidly detecting the peste des petits ruminants virus, has the characteristics of high flux and low cost, and has the advantages that the products can be directly used for sequencing, secondary treatment is not needed, the operation is extremely convenient, and the needed sample amount is less, so that the application prospect is good.

Description

technical field [0001] The invention relates to pyrosequencing technology, in particular to a kit for detecting Peste des Petits Ruminants virus by using pyrosequencing technology. Background technique [0002] Peste des petits ruminants (PPR) is a highly contagious animal disease caused by Peste des petits ruminants (PPRV) of the genus Morbillivirus in the Paramyxoviridae family. The disease mainly infects domestic and wild small ruminants, and goats are especially susceptible. Its pathogenicity and molecular characteristics are very similar to rinderpest (RP), and it is defined as a category A animal disease by the World Organization for Animal Health (OIE). The foreign trade economy of countries affected by the disease, especially in Africa, the Middle East, the Near East and South Asia, has brought huge losses. Since the outbreak of the disease, it has continuously spread to all parts of the world, and the threat to countries without the disease, especially to countries...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/70C12Q1/6869C12Q2565/301
Inventor 王彩霞吴绍强林祥梅吕继洲袁向芬邓俊花
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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