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Group of real-time fluorescent QRT-PCR primers capable of detecting cattle, sheep akabane virus by one-step method, probe and usage method thereof

A technology of Akabane disease virus and probe, which is applied in the field of sheep Akabane disease virus and cattle detection, can solve the problems of insufficient sensitivity of cattle and sheep Akabane disease virus methods, achieve good promotion and application value, ensure safety, and solve sensitivity problems Effect

Active Publication Date: 2015-04-22
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the insufficient sensitivity of existing methods for detecting Akabane disease virus in cattle and sheep, and to provide a set of methods for detecting Akabane disease virus in cattle and sheep that are accurate, fast, specific, and more sensitive. Primers, probes and methods of use thereof

Method used

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  • Group of real-time fluorescent QRT-PCR primers capable of detecting cattle, sheep akabane virus by one-step method, probe and usage method thereof
  • Group of real-time fluorescent QRT-PCR primers capable of detecting cattle, sheep akabane virus by one-step method, probe and usage method thereof
  • Group of real-time fluorescent QRT-PCR primers capable of detecting cattle, sheep akabane virus by one-step method, probe and usage method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Design and synthesis of primers and probes

[0046] According to the conserved fragment sequence of Akabane disease S gene, a pair of specific primers Pf99 / AKAV (sequence shown in SEQ ID NO.1) and Pr184 / AKAV (sequence shown in SEQ ID NO.2) were designed using Primer Express 3.0 gene analysis software shown), and a probe Pb132 / AKAV (sequence shown in SEQ ID NO.3), the 5' of the probe is labeled with FAM, and the 3' is labeled with BHQ1.

[0047] SEQ ID NO.1:

[0048] 5'-GCAGCTCAACTTTACTGTTGCTAGA-3'

[0049] SEQ ID NO.2:

[0050] 5'-CACTTGGTTGTGGCGTCTTATGTA-3'

[0051] SEQ ID NO.3:

[0052] 5’- FAM- CCTCAACCAGAAGAAGGCCAAGATGG-BHQ1-3’

[0053] The synthesis of primers and probes was completed by Shanghai Yingjun Biotechnology Co., Ltd.

Embodiment 2

[0054] Example 2 Preparation of template

[0055] 1. Extraction of bovine and sheep Akabane disease virus RNA

[0056] Take 200 μL of inactivated cattle and sheep Akabane disease virus culture (the virus was donated by the MacArthur Institute of Agriculture in Australia), add it to a sterilized 1.5mL centrifuge tube, and follow the instructions of TIANGEN’s nucleic acid extraction kit to extract viral RNA , and the nucleic acid content was determined to be 158 ng / μL on a UV spectrophotometer. The obtained RNA can be used directly or frozen at -20°C for future use.

[0057] 2. Preparation of cDNA of cattle and sheep Akabane disease virus

[0058] According to the instructions of Reverse Transcriptase XL (AMV) reverse transcriptase from Treasure Bio (Dalian) Co., Ltd., the viral RNA was reverse-transcribed into cDNA and stored at -20°C.

[0059] 3. Preparation of plasmids containing bovine and sheep Akabane disease virus nucleic acids

[0060] (1) Design a pair of specific p...

Embodiment 3

[0068] Example 3 Establishment of one-step real-time fluorescent QRT-PCR reaction system

[0069] 1. Primer concentration optimization for real-time fluorescence QRT-PCR reaction

[0070] In order to establish the most suitable reaction system, the primer concentration was diluted as needed. The primer concentration is 100 μM, add 0.2 μL, 0.5 μL, 1 μL, 1.5 μL, and 2 μL primers respectively, perform real-time fluorescent QRT-PCR reaction, compare the CT value, and finally get the optimal primer volume of 0.2 μL 100 μM primers, the final concentration of primers is 400nM.

[0071] 2. Probe concentration optimization for real-time fluorescence QRT-PCR reaction

[0072] To establish the most suitable reaction system, the probe concentration was diluted as needed. The probe concentration was 100 μM, and 0.1 μL, 0.2 μL, 0.5 μL, 1 μL, and 1.5 μL probes were added respectively, and real-time fluorescent QRT-PCR reactions were performed, and the CT values ​​were compared to finally ...

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Abstract

The invention discloses a group of real-time fluorescent QRT-PCR primers capable of detecting cattle, sheep akabane virus by a one-step method, a probe and a usage method thereof. The nucleotide sequences of the described primers and the probe are respectively as shown in SEQIDNO.1-3. The method for detecting the cattle, sheep akabane virus is as follows: sample RNA is used as template, the above primers and probe are adopted to perform the one-step real-time fluorescent QRT-PCR amplification test of the sample RNA, after reaction, if a specific fluorescent amplification curve appears, the sample RNA is judged to contain the cattle, sheep akabane virus nucleotide, namely the sample is tested positive for the cattle, sheep akabane virus. A standard curved prepared according to analog standards can quantify the one-step real-time fluorescent QRT-PCR amplification test. The method for detecting the cattle, sheep akabane virus is safe, quick, high in specification and sensitivity, and provides technical support for detection of the cattle, sheep akabane disease in livestock husbandry and the inspection and quarantine work of the exit cattle and sheep.

Description

technical field [0001] The invention belongs to the technical field of virus molecular biology detection. More specifically, it relates to a set of one-step real-time fluorescent QRT-PCR primers, probes and usage methods for detecting bovine and sheep Akabane disease viruses. Background technique [0002] Akabane disease (AKAD), also known as Akabane spot disease, is a polymorphic viral infectious disease of cattle, sheep and goats caused by Akabane virus (AKAV). Symptoms of the disease mainly include miscarriage, premature birth, stillbirth, fetal malformation, mummification, etc., which can cause congenital arthrogryposis and hydrocephalus syndrome. The disease is an insect-borne infectious disease, and the vectors are blood-sucking mosquitoes and Culicoides. Akabane disease is a legally notifiable disease of the World Organization for Animal Health (OIE), and it is also one of the seven diseases that must be tested for cattle and sheep imported from abroad in my country...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 鱼海琼林志雄陈茹翟建新罗长保赵吟贾坤田纯见朱道中王宏
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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