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Fluorescent quantization PCR (Polymerase Chain Reaction) method for detecting dog-cat leptospira as well as primer and detection kit thereof

A leptospira, fluorescence quantitative technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low antibody titer, difficult to detect, difficult to cultivate, etc., to achieve good repeatability Effect

Inactive Publication Date: 2011-01-05
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the bacteria is not easy to cultivate. Although the blood of early infected animals contains pathogens, the antibody titer is very low, and the traditional serological detection is not easy to detect.

Method used

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  • Fluorescent quantization PCR (Polymerase Chain Reaction) method for detecting dog-cat leptospira as well as primer and detection kit thereof
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  • Fluorescent quantization PCR (Polymerase Chain Reaction) method for detecting dog-cat leptospira as well as primer and detection kit thereof

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Embodiment 1

[0029] The present invention uses the reference standard strain jaundice hemorrhagic group Lai type Lai strain (56601) in my country to extract the above-mentioned DNA by using the phenol-chloroform method. Determine the concentration and purity of DNA.

[0030] According to the gene nucleotide sequence of 16s rRNA in Genbank, specific primers were designed and synthesized by Shanghai Yingjun Biotechnology Co., Ltd. The primers for 16s rRNA are: P16F-CAA TACTCA GCG GCG AAC; P16R-TTT TTG AGA TTA GCT CCC C, the size is 616bp. Accquire Taq HF high-fidelity enzyme (purchased from Invitrogen) was used to amplify the 16s rRNA gene, with a total reaction volume of 50 μL, including: 20 pM P16F and P16R primers, 100 ng DNA template, 25 μL 2× high-fidelity enzyme Buffer and 1 μL high-fidelity enzyme. The parameters of the PCR reaction were: heat melting at 94°C for 30s, then annealing at 54°C for 30s, extension at 72°C for 60s, a total of 30 cycles of the above steps, and extension at 7...

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Abstract

The invention provides a fluorescent quantization PCR (Polymerase Chain Reaction) method for detecting dog-cat leptospira, as well as a primer and a detection kit thereof. A 16s rRNA gene sequence is used as a design primer, including an upstream primer of 5-ACGAAAGCGTGGGTAGTG-3' and a downstream primer of 5'-GCGGTCTACTTAATCCGT-3'. The invention establishes an SYBR Green I fluorescent quantization PCR method for identifying the dog-cat leptospira based on the 16s rRNA gene. Under the optimal reaction conditions, the reaction efficiency is 0.98, the acquired related coefficient R value of a standard curve reaches 1.000, the linear range of the standard curve reaches 8 orders of magnitude from 1 to 1*107 copies, and at least about 1 target copy, i.e. 1.2 fg DNA can be detected. The SYBR Green I fluorescent quantization method established by the invention has the advantages of strong specificity, high sensitivity and wide quantization scope.

Description

Technical field: [0001] The invention relates to a fluorescence quantitative PCR method for rapidly detecting Leptospira in dogs and cats, and also relates to primers and a detection kit used in the method. It belongs to the technical field of biopharmaceuticals. Background technique: [0002] Leptospira (referred to as "Leptospira"), belonging to the genus Leptospira (Leptospira) of the family Leptospira in the order Spirochetes, is divided into pathogenic L. interrogans and saprophytic hyperbolic Leptospira (L. biflexa). There are many natural hosts of Leptospira, among which the most harmful to humans as the source of infection are mainly rodents, frogs and domestic animals (pigs, cattle and dogs). As pet dogs are currently the closest companion animals to humans, it is particularly important to carry out the detection and prevention of leptospirosis in companion animals. After leptospirosis infects humans or pets, it can cross-infect almost all warm-blooded animals th...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/64C12N15/11
CPCY02A50/30
Inventor 丁铲陈鸿军于圣青宋翠萍仇旭升胡青海韩先干
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI