Method for detecting canine distemper virus by using one-step RT-PCR method

A technology of RT-PCR and canine distemper virus, which is applied in the field of detecting canine distemper virus by one-step RT-PCR method, can solve the problems of long time-consuming, poor sensitivity and specificity, and low accuracy of CDV detection, and achieves cost-effectiveness. Low, high sensitivity, easy to operate effect

Inactive Publication Date: 2011-02-16
ZHENGZHOU HOUYI PHARMA
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  • Summary
  • Abstract
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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for detecting canine distemper virus using a one-step RT

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  • Method for detecting canine distemper virus by using one-step RT-PCR method

Examples

Experimental program
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Embodiment 1

[0036] Example 1 Extraction of liquid sample total RNA

[0037] 1) Pretreatment of liquid samples (nasal fluid, ocular conjunctival secretion and saliva)

[0038] Use cotton swabs to collect nasal fluid, ocular conjunctival secretions, and saliva samples from infected animals, put the collected cotton swabs upside down in a centrifuge tube, add sample diluent (0.01mol / LPBS (pH7.2-7.4)) and let it stand at 4°C. Place for 2 hours, centrifuge at 2000-3000r / min for 10 minutes, take 200μl of the supernatant, put it in a 1.5ml Eppendorf tube, clearly mark it, and put it at -20℃ for testing;

[0039] 2) Extraction of total RNA from the sample

[0040] Take 200 μl of the sample to be tested in a 1.5ml Eppendorf tube, add 800 μl Trizol, mix well, let stand for 5 minutes; add 200 μl analytical pure chloroform for 30 seconds, centrifuge at 12000 r / min for 10 minutes at 4°C; take 400-600 μl of the aqueous phase, add etc. The volume of isopropanol was gently inverted and mixed,...

Embodiment 2

[0042] Example 2 Detection of CDV in liquid samples by one-step RT-PCR

[0043] 1. Design the amplification primers for one-step RT-PCR

[0044] Design a pair of specific primers for the N gene of CDV in GeneBank, the sequence is as follows:

[0045]Upstream primer CDV 1: 5'-GGTCGGAGAATTTAGAATGAAC-3';

[0046] Downstream primer CDV 2: 5'-CCAAGAGCCGGATACATAG-3';

[0047] Primers were synthesized by Shanghai Handsome Biotechnology Co., Ltd.

[0048] 2. Establishment of RT-PCR reaction system

[0049] 1) Add 20μl DEPC-H to the total RNA pellet 2 O to dissolve it, take 10μl RNA solution, add 5.0μl of 2.5mmol / LdNTPs, 5.0μl of 10×Taq Buffer, MgCL 2 10μl, 20pmol / μl primer CDV 1 2.0μl, 20pmol / μl primer CDV 2 1.0μl, 5U / μl Ex-Taq 0.5μl, 200U / μl M-MuLV reverse transcriptase 1.0μl, 40U / μl RNasin 1.0 μl, DEPC-H 2 O 14.5 μl, the population of the reaction was 50 μl.

[0050] At the same time, the total RNA of the positive control and the total RNA of the negative control were u...

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Abstract

The invention discloses a method for detecting canine distemper virus by using a one-step reverse transcription-polymerase chain reaction (RT-PCR) method. The method comprises the following steps of: preprocessing a sample for the RT-PCR detection; extracting total ribonucleic acid (RNA); obtaining a specific amplification product by one-step RT-PCR reaction; and performing 1 percent agarose gel electrophoresis identification on the amplification product. The method of the invention has the characteristics of high flexibility, high specificity, simple and convenient operation procedure, low contamination rate and reliable detection result; the acquired sequence information is directly used for analyzing genetic derivation relation of an epidemic strain; and the method is suitable for etiological diagnosis and epidemiological research of canine distemper.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a method for detecting canine distemper virus by using a one-step RT-PCR method. Background technique [0002] Canine distemper is a highly contagious, acute infectious disease of dogs caused by canine distemper virus. CDV belongs to the genus Morbillivirus (MV) of Paramyxoviridae in classification, and has close antigenic relationship and common characteristics with measles virus and rinderpest virus, and is exactly the same as its ultrastructure and structure, and is similar to Newcastle disease and other viruses. Paramyxoviruses are very similar. Canine distemper virus is a highly contagious pathogen that spreads easily among susceptible dogs and mink. The virus exists in various organs and tissues such as liver, spleen, lung, kidney, brain and lymph nodes, and the virus is excreted through tears, nasal fluid, saliva, urine and exhaled air. Canine distemper can oc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/70
Inventor 吴红云李建正范金红
Owner ZHENGZHOU HOUYI PHARMA
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