Animal Torque Teno virus rapid detection kit and detection method using loop-mediated isothermal amplification technology

A loop-mediated isothermal, detection kit technology, applied in microorganism-based methods, biochemical equipment and methods, microorganism determination/inspection, etc., can solve the problem of temperature amplification gene technology detection animal TorqueTenovirus kit and detection method, There are no problems such as animal TorqueTenovirus LAMP diagnosis method, which achieves the effect of simple identification, high sensitivity and high sensitivity

Active Publication Date: 2013-09-11
重庆海关技术中心 +1
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

There have been many reports on the detection of pathogens at home and abroad, using the LAMP method to detect various animal diseases and microorganisms, but in the veterinary clinical diagnosis method, there is no LAMP diagnosis method for animal Torque Teno virus.
However, there is no report on the kit and detection method for the detection of Torque Teno virus in animals by loop-mediated isothermal amplification gene technology

Method used

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  • Animal Torque Teno virus rapid detection kit and detection method using loop-mediated isothermal amplification technology
  • Animal Torque Teno virus rapid detection kit and detection method using loop-mediated isothermal amplification technology
  • Animal Torque Teno virus rapid detection kit and detection method using loop-mediated isothermal amplification technology

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Experimental program
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Effect test

Embodiment 2

[0058] Embodiment 2, design and screening of primers of the present invention

[0059] According to the genome full-length sequence of known TTV1 (AY823990.1) and TTV2 (AY823991.1) nucleotide sequence, utilize ClustW software to carry out multiple alignment, design specific primers with LAMP primer design software Primer Explorer V4.0 software, They are marked as: SEQ ID NO.1...SEQ ID NO8. All primers were synthesized by Bao Biological Engineering (Dalian) Co., Ltd. The TTV virus DNA was extracted with a commercially available virus nucleic acid extraction kit, amplified, and primers with non-specific amplification were excluded. Get the primers as follows:

[0060]

Embodiment 3

[0061] Embodiment 3, the preparation of positive control substance

[0062] The viral DNA of positive porcine TTV1 and TTV2 was extracted with a commercially available nucleic acid extraction kit, and then amplified by PCR. The amplified lengths were 449 bp for TTV1 and 468 bp for TTV2, respectively. The amplified target band was recovered, ligated with the pMD19-T vector, ligated overnight at 4°C, transformed into JM109 competent cells, and the recombinant plasmid was extracted from those positive for resistance screening, shaking bacteria, and PCR. Sequence verification, obtain the clone containing the target fragment, mix the positive plasmids of the two serotypes in equal proportions, aliquot each 50 μL, and control the concentration at 80-100 ng / μL.

Embodiment 4

[0063] Embodiment 4, the preparation of negative control substance

[0064] Commercially available commercial nucleic acid extraction kits were used to extract the DNA of the negative sera not containing porcine TTV1 and TTV2, respectively. Dilute with sterilized deionized water, control the concentration at 80-100ng / μL, and pack into 50μL each.

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Abstract

The invention relates to the field of animal disease molecular biology detection methods and detection agents, in particular to an animal Torque Teno virus rapid detection primer, a kit and a detection method using a loop-mediated isothermal amplification (LAMP) technology. Multiple comparisons are carried out by using ClustalW according to two different serotype gene sequences of an animal Torque Teno virus, and conserved zones of the sequences are analyzed, an inner primer and an outer primer are respectively designed by adopting LAMP primer design software, and detection and identification of TTV1 and TTV2 serotypes are realized rapidly specifically. The animal Torque Teno virus detection method disclosed by the invention has the advantages of rapidness, high sensitivity, strong specificity, convenience for operation, low requirement of equipment, time and labor saving, easiness in observing results, no complex post-treatment, and wide application range.

Description

technical field [0001] The invention relates to an animal TTV kit, which belongs to the field of animal epidemic molecular biology detection methods and detection reagents, in particular to a loop-mediated isothermal amplification technique (LAMP) detection kit for animal TTV virus. The present invention also applies the kit to a biological detection method for isothermal amplification detection of animal TTV virus. Background technique [0002] TTV (Torque Teno virus) virus is a non-enveloped single-stranded circular spherical DNA virus, now classified as Circoviridae (Circoviridae) Anellovirus (Anellovirus), including two different serotypes, namely TTV1 and TTV2. The virus was first isolated from the serum of a patient with non-A-non-G hepatitis after blood transfusion by Japanese scholar Nishizawa et al. in 1997, and was named after the patient's abbreviation (TT). Because of the coincidence of TTV and transfusion transmitted virus (transfusion transmitted virus, TTV),...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 聂福平聂奎李应国王国民黄鹤杨俊肖进文李贤良吴晓薇王昱向海洋
Owner 重庆海关技术中心
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