Reagent kit for detecting peste des petits ruminant vaccine poison by pyrophosphoric acid sequencing technology

A technology for pyrosequencing and Peste des petits ruminants, which is applied in the field of pyrosequencing, and achieves the effects of short time consumption, convenient construction of standardized operation procedures, and small sample volume.

Inactive Publication Date: 2015-04-08
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been many reports on the diagnosis of PPRV and the exclusion of rinderpest virus (RPV) interference by PCR, RT-PCR, ELISA and other methods, but there are few reports on the method of distinguishing and detecting PPRV vaccine strains from virulent strains

Method used

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  • Reagent kit for detecting peste des petits ruminant vaccine poison by pyrophosphoric acid sequencing technology
  • Reagent kit for detecting peste des petits ruminant vaccine poison by pyrophosphoric acid sequencing technology
  • Reagent kit for detecting peste des petits ruminant vaccine poison by pyrophosphoric acid sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design of primers for detection of Peste des petits ruminants vaccine virus

[0037] By comparing and analyzing the PPRV N gene sequences published in the NCBI Gene Bank (GenBank) of the National Center for Bioinformatics in the United States, the specific sequence of the N gene of the Ⅱ series vaccine strain (Nigeria75 / 1 strain) is determined to be 1~ 250bp gene, according to the specific sequence, use Assay Design SW software to design specific 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 based on the analysis results - PCR and sequencing work.

[0038] According to the specific region sequence of Peste des petits ruminants vaccine virus N gene designed by the present invention, specific primers for the virus of Peste des petits ruminants vaccine can amplify a specific single band for the virus RNA of Peste des petits ruminants vaccine, and the l...

Embodiment 2

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

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

[0045] Using inactivated PPRV Nigeria75 / 1 vaccine 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 supernatant of the inactivated vaccine virus culture solution and add it to a sterilized 1.5ml Eppendorf tube, then add 1.0mL Trizol, 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, centri...

Embodiment 3

[0063] Example 3 Using Pyrosequencing Technology to Detect Sheep Blood Samples

[0064] Take 2 blood samples of sheep immunized with the Peste des petits ruminants vaccine, 2 blood samples of diseased sheep infected with Peste des petits ruminants virus and 2 blood samples of healthy sheep, and use the method in Example 2 to detect the samples.

[0065] 1. Extraction of RNA from sheep blood samples

[0066] The QIAGEN QLAamp Viral RNA Mini Kit (K2904) was used to extract genomic RNA from goat blood samples. The kit operation steps are as follows:

[0067] (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).

[0068] (2) Add 140 μL goat blood supernatant to the centrifuge tube containing buffer AVL-carrier RNA. Vortex thoroughly for 15 seconds to ensure lysis efficiency.

[0069] (3) Stand at room temperat...

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Abstract

The invention provides a reagent kit for detecting peste des petits ruminant vaccine poison by a pyrophosphoric acid sequencing technology. The reagent kit comprises a peste des petits ruminant specific primer and a pyrophosphoric acid sequencing primer, and the nucleotide sequences of the peste des petits ruminant specific primer and the pyrophosphoric acid sequencing primer are respectively shown as SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. The RNA of a to-be-tested sample is extracted, and RT-PCR amplification is carried out by the specific primer. If the length of a primer-amplified fragment is 180bp, pyrophosphoric acid sequencing reaction is carried out. If a sequencing fragment is identical to an N gene target fragment (SEQ ID NO.1), peste des petits ruminant vaccine poison is determined. If at least one basic group of the sequencing fragment is different from the target fragment, the sample is judged as a negative one. The reagent kit can be used for the rapid detection of the peste des petits ruminant vaccine poison and has the characteristics of high throughout and low cost. A product can be directly used for sequencing, secondary treating is not required, the operation is very simple, the quantity of required sample is small, and the application prospects are great.

Description

technical field [0001] The invention relates to pyrosequencing technology, in particular to a kit for detecting the virus of Peste des petits ruminants vaccine by using the 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 been spreading to all parts of the world, posing a greater threat to countries without the disease, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/70C12Q2565/301
Inventor 王彩霞林祥梅吴绍强张永宁冯春燕
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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