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Primer group for identifying peste des petits ruminant virus wild strain and vaccine strain and application thereof

A technology of Peste des petits ruminants and vaccine strains, applied in the field of biotechnology detection, can solve the problems of difficult differential diagnosis and inability to distinguish vaccine-immunized sheep, etc., and achieve the effect of high practical value and high sensitivity

Inactive Publication Date: 2013-06-05
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The attenuated vaccine can produce complete protection after immunization of sheep. However, since there is only one serotype of PPR virus, it is impossible to distinguish the sheep infected with PPR virus from the vaccinated sheep serologically, which brings great difficulties to the differential diagnosis.

Method used

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  • Primer group for identifying peste des petits ruminant virus wild strain and vaccine strain and application thereof
  • Primer group for identifying peste des petits ruminant virus wild strain and vaccine strain and application thereof
  • Primer group for identifying peste des petits ruminant virus wild strain and vaccine strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Preparation of a differential diagnosis kit for Peste des petits ruminants virus vaccine strain and wild strain

[0050] 1. Synthesis of primers

[0051] Artificially synthesize the following 2 pairs of primers:

[0052] H1a: 5'-CCTGGATAGAGAACGCTTGGTC-3'

[0053] H2b: 5'-CCGAGAGTCAATGATTGCAGCT-3'

[0054] H3c: 5'-CGACGAAGGACCTGAGGTACATA-3'

[0055] H5e: 5'-TAGGGGATGGCTCGGAGGC-3'

[0056] 2. Preparation of positive control

[0057] The RNA of Peste des petits ruminants virus Nigeria 75 / 1 vaccine strain was extracted, PCR was amplified with PPRH3cT7 and PPRH5e primers, and the PCR product was purified and identified by sequencing. Take 200ng of the PCR product for in vitro transcription according to the instruction of Riboprobe In vitro Transcription Systems. The product was digested with DNase I, precipitated with 3M NaAc ethanol, dissolved in an appropriate amount of DEPC-treated water, and then it was the artificial RNA of the vaccine strain. The conce...

Embodiment 2

[0063] Example 2. Application of the differential diagnosis kit for Peste des petits ruminants virus vaccine strains and wild strains

[0064] Take 1 sick sheep diagnosed with Peste des petits ruminants virus infection, 1 sheep immunized with Peste des petits ruminants virus vaccine and 1 healthy sheep, take oral and nasal swabs respectively, and use the kit prepared in Example 1 to test the samples.

[0065] 1. Sample processing

[0066] Put the collected oral and nasal swabs into a 2ml screw centrifuge tube, add 1ml PBS, roll, centrifuge at 3000rpm for 5min, take 200ul supernatant and extract nucleic acid with a commercial RNA extraction kit (QIAamp Viral RNA Kit, Qiagen) , the resulting nucleic acid solution can be directly used as a template.

[0067] The mouth and nose swabs of sick sheep were used for the above treatment, and 5 μL of each nucleic acid template was taken into three 0.2 mL centrifuge tubes as group I reaction tubes. The snout and nose swabs of immunized ...

Embodiment 3

[0074] Example 3, Sensitivity Detection of Peste des Petits Ruminants Virus Vaccine Strains and Wild Strains Differential Diagnosis Kit

[0075] 1. Preparation of positive standard

[0076] With the step 2 of embodiment 1, the preparation final concentration is 10 6 The vaccine strain artificial RNA of copy / μL is used as the positive standard of the vaccine strain, and the final concentration of preparation is 106 Copy / μL wild strain artificial RNA was used as the wild strain positive standard.

[0077] 2. Preparation of serial dilutions of positive standard

[0078] The wild strain positive standard and the vaccine strain positive standard were serially diluted with DEPC-treated water, and the prepared concentrations were 2×10 5 copies / μL, 2×10 4 copies / μL, 2×10 3 copies / μL, 2×10 2 copies / μL, 2×10 1 copies / μL and 2×10 0 Serial dilutions in copies / μL.

[0079] Detection group I: In six 0.2mL centrifuge tubes, add 2.5μL each to a concentration of 2×10 5 copies / μL of th...

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Abstract

The invention discloses a diagnostic kit for identifying a peste des petits ruminant virus wild strain and a vaccine strain and an application thereof. The diagnostic kit provided by the invention comprises two pairs of primers, i.e. a primer pair which is combined with an H gene in a peste des petits ruminant virus Nigeria 75 / 1 vaccine strain genome (GenBank Accession Number X74443) and a primer pair which is combined with the H gene in a peste des petits ruminant virus Tibet 07 wild strain genome (GenBank Accession Number JF939201). The diagnostic kit for identifying the peste des petits ruminant virus wild strain and the vaccine strain has high detection sensitivity, can detect 200 replicated target ribonucleic acids (RNA) to the minimum extent, has good specificity, is simple and convenient to operate, can effectively identify peste des petits ruminant virus vaccine immune sheep and wild virus infected sheep, and is in particular suitable for monitoring and detecting peste des petits ruminants.

Description

technical field [0001] The invention belongs to the technical field of biotechnology detection, and in particular relates to a primer set for distinguishing wild strains and vaccine strains of Peste des petits ruminants virus and its application. Background technique [0002] Peste des petits ruminants is a severe and contagious disease infecting goats, sheep and wild small ruminants caused by peste des petits ruminants virus (PPRV) of the Paramyxoviridae family Morbillivirus genus. Peste des petits ruminants is mainly distributed in Central and North Africa, the Middle East, West Asia and South Asia. Peste des petits ruminants (PPR) virus was introduced into the Ngari region of Tibet in 2007, causing serious losses to the local sheep industry. Although the disease has been brought under control, Peste des petits ruminants is still prevalent in India, Bhutan, Nepal and other neighboring countries, coupled with the frequent cross-border migration and activities of wild anima...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 包静月王志亮刘春菊王清华
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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