Ternary PCR (polymerase chain reaction) kit for canine distemper virus, enteritis parvovirus and Aleutian disease virus

A technology of enteritis parvovirus and canine distemper virus, which is applied in the detection field of mink virulent virus, can solve the problems of time-consuming and laborious operation, low sensitivity, and insufficient specificity, and achieve high safety, high specificity and sensitivity in use , The effect of no biological safety hazard ingredients

Inactive Publication Date: 2011-09-14
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The conventional diagnostic methods for these three diseases are virus isolation and identification, hemagglutination test, agar diffusion test, convective immunoelectrophore...

Method used

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  • Ternary PCR (polymerase chain reaction) kit for canine distemper virus, enteritis parvovirus and Aleutian disease virus
  • Ternary PCR (polymerase chain reaction) kit for canine distemper virus, enteritis parvovirus and Aleutian disease virus
  • Ternary PCR (polymerase chain reaction) kit for canine distemper virus, enteritis parvovirus and Aleutian disease virus

Examples

Experimental program
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Effect test

Embodiment 1

[0047] 1 Materials and methods

[0048] 1.1 Virus strains CDV3, MEVB and ADV-G were all provided by the Institute of Special Products of the Chinese Academy of Agricultural Sciences; M-MLV reverse transcriptase, rTaq DNA polymerase, dNTPS, DNA Marker DL2000, pMD18-T vector, etc. were all purchased from Bao Bioengineering (Dalian) Co., Ltd., gel recovery kits were purchased from Shanghai Huashun Biotechnology Co., Ltd., and viral RNA extraction reagents were purchased from Invitrogen.

[0049] 1.2 Construction of positive control recombinant plasmid

[0050] According to the CDV3 virus strain N gene design detection CDV virus-specific primers CDV-P1 5'-GATAAAGCATGTCATTATAGTCCTAA-3'; CDV-P2 5'-CTTGAGCTTTCGACCTTC-3', the length of the amplified fragment is 335bp. Vero cells were used to proliferate CDV3 seed virus, and after culturing for 3 days, the total RNA was extracted with reference to the virus RNA extraction reagent (Invitrogen Company), and the CDV 335 fragment was ampl...

Embodiment 2

[0069] Detection of Virus Samples Using a Triple-PCR Method for Canine Distemper Virus, Enteritis Parvovirus and Mink Aleutian Virus

[0070] 1 Materials and methods

[0071] 1.1 Virus strains CDV3, MEVB and ADV-G were all provided by the Institute of Special Products of the Chinese Academy of Agricultural Sciences; M-MLV reverse transcriptase, rTaq DNA polymerase, dNTPS, DNA Marker DL2000, etc. were purchased from Bao Biological Engineering (Dalian) Co., Ltd. The company purchased viral RNA extraction reagents from Invitrogen.

[0072] 1.2 Preparation of viral nucleic acid template (RNA and DNA extraction)

[0073] Refer to the viral RNA extraction reagent (Invitrogen Company) to extract the total RNA of the virus strain CDV3, and use M-MLV reverse transcriptase to perform reverse transcription, and extract its cDNA to be used as a template for canine distemper virus PCR. The DNA of MEVB and ADV-G strains was directly lysed by boiling water, that is, it was placed in a wate...

Embodiment 3

[0080] Detection of Canine Distemper Virus in Clinical Samples Using a Triple-PCR Method for Canine Distemper Virus, Enteritis Parvovirus and Mink Aleutian Virus

[0081] 1 Materials and methods

[0082] 1.1 Virus strain CDV3 was provided by the Institute of Special Products of the Chinese Academy of Agricultural Sciences; M-MLV reverse transcriptase, rTaq DNA polymerase, dNTPS, DNA Marker DL2000, etc. were purchased from Bao Biological Engineering (Dalian) Co., Ltd., and viral RNA extraction reagents were purchased from Invitrogen .

[0083] 1.2 Preparation of viral nucleic acid template (RNA extraction)

[0084] Refer to the viral RNA extraction reagent (Invitrogen Company) to extract the total RNA of virus strain CDV3 and canine distemper clinical samples, and use M-MLV reverse transcriptase to perform reverse transcription, and extract its cDNA to be used as canine distemper virus PCR template.

[0085] 1.3 Triple PCR reaction

[0086] The nucleic acid extracted from th...

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Abstract

The invention discloses a ternary PCR (polymerase chain reaction) kit, which can simultaneously detect mink canine distemper virus, enteritis parvovirus and Aleutian disease virus. The ternary PCR kit comprises DNA (deoxyribonucleic acid) polymerase, dNTPs (deoxynucleotide triphosphates), primers, PCR buffer solution and double-distilled water; and the ternary PCR kit is characterized in that theprimers comprise three pairs of the primers, wherein the sequences of the first pair of the primers are 5'-GATAAAGCATGTCATTATAGTCCTAA-3' and 5'-CTTGAGCTTTCGACCTTC-3', the sequences of the second pairof the primers are 5'-AACTGGGCGGAGCCTAAA-3' and 5'-CAGAGCGAAGATAAGCAG-3', and the sequences of the third pair of the primers are 5'-GAAACCACGGTGGAGACA-3' and 5'-AAGAGTTGACCTGGAGGG-3'. By adopting themultiple PCR kit, whether the infection or the multi-infection of the mink canine distemper virus, the enteritis parvovirus and the Aleutian disease virus exists or not can be simultaneously detectedin a same reaction system, thereby having higher specificity and sensitivity, saving time and reagent and providing an effective tool for fast and early diagnosis of a mink group.

Description

technical field [0001] The invention discloses a triple PCR kit capable of simultaneously detecting mink distemper virus, enteritis parvovirus and Aleutian virus, belonging to the detection field of mink venomous virus. Background technique [0002] Canine distemper (Canine distemper, CD) is a highly contagious and fatal infectious disease of canines, mustelidae and some raccoons caused by avian canine distemper virus (CDV) of the family Paramyxoviridae Morbillivirus (Document 1). The characteristic is that the virion has an envelope with a diameter of about 150 nm, including a linear, negative-sense single-stranded RNA genome with a size of about 15.0 kb, and the virus has only one serotype. The disease is highly contagious, and the mortality rate can be as high as 80%. In the early stage of the disease, it is a biphasic fever type, with inflammation of the mucous membranes of the eyes, nose, and digestive tract, followed by bronchitis, catarrhal pneumonia, and gastroenter...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 易立程世鹏王建科杨莘许红丽
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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