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Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of Chlamydia porcine taqman-mgb probe

A technology of real-time fluorescence quantification and porcine chlamydia, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial measurement/inspection, etc., to achieve cost reduction, low cost, and simple instruments

Active Publication Date: 2015-09-23
重庆海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no real-time fluorescent quantitative PCR amplification technology using TaqMan-MGB probes to detect Chlamydia porcine ( C.cuis ) kit and detection method report

Method used

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  • Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of Chlamydia porcine taqman-mgb probe
  • Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of Chlamydia porcine taqman-mgb probe
  • Primers, kits and detection methods for real-time fluorescent quantitative PCR detection of Chlamydia porcine taqman-mgb probe

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, the design and screening of primer

[0034]Chlamydia porcine fluorescent quantitative PCR amplification primers and probes were designed based on the reference sequence of the MOMP gene of Chlamydia porcine published by GenBank, compared with MEGA5, the sequence was analyzed and primers and probes were designed in its conserved region. The probe design software Primer Express 3.0 was used to design 4 sets of fluorescent quantitative primers, which were synthesized by Yingjun (Shanghai) Co., Ltd. ABI 7500 FAST PCR instrument was used to monitor the amplification in the reaction process in real time, and the amplification of different primer sets The parameters such as the starting time of starting time, the time of entering the maximum amplification rate, the maximum amplification rate and the time required to reach the plateau stage were analyzed, and a set of fluorescent quantitative PCR amplification primers with the highest amplification rate and good sp...

Embodiment 2

[0040] Embodiment 2, the preparation of positive control substance

[0041] The nucleic acid of the porcine Chlamydia cell culture was extracted with a commercial kit, and the nucleic acid was identified by PCR and electrophoresis, and amplified with the PCR upstream primer SEQ ID NO.4 and the PCR downstream primer SEQ ID NO.5, and gel The recovery kit recovers the amplified bands. Carry out ligation reaction with pMD19-T vector at a ratio of 1:10, ligate overnight at 4°C, and transform DH5α bacteria. After resistance selection and positive PCR identification, re-sequence verification, and use a spectrophotometer to measure the OD value of its nucleic acid. Its 260 / 280 ratio is between 1.8 and 2.0. Thereby obtaining positive recombinant plasmid DNA.

Embodiment 3

[0042] Embodiment 3, the preparation of negative control substance

[0043] Genomic DNA was extracted from pig blood without Chlamydia porcine infection by using a commercial kit, and identified by PCR and electrophoresis.

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Abstract

The invention discloses a primer and kit for swine chlamydia (C.cuis) Taqman-MGB probe real-time fluorescence quantification PCR detection and a detection method. According to the detection method, two primers and one MGB probe are designed according to one area of MOMP in a target sequence, and swine chlamydia can be amplified specifically. The kit comprises an amplified reaction liquid tube, a positive control device, a negative control device and sterilization deionized water. By means of the primer and kit for swine chlamydia (C.cuis) Taqman-MGB probe real-time fluorescence quantification PCR detection and the detection method, a goal target sequence can be detected quickly, efficiently, specifically and sensitively through two amplified reactions and signal collection; moreover, operation is simple and convenient, expensive instruments and reagents are not needed, technical requirements for operators are not required, detection cost is low, and detection time is short.

Description

technical field [0001] The invention relates to the field of animal epidemic molecular biology detection methods and detection reagents, in particular to a primer, a kit and a detection method for real-time fluorescent quantitative PCR detection of a Chlamydia porcine Taqman-MGB probe. Background technique [0002] animal chlamydial disease ( Chlamydiosis ) is composed of various types of chlamydia ( Chlamydia ) is a very important class of natural foci infectious diseases that infect mammals, poultry and other animals. The disease is endemic and often causes great harm and economic losses. The latest taxonomic studies of Chlamydiaceae show that Chlamydiaceae ( Chlamydiaceae ) into the genus Chlamydia ( Chlamydia ) and Chlamydia spp. ( Chlamydophila) , including Chlamydia trachomatis ( Chlamydia trachomatis ), Chlamydia swine ( Chlamydia suis ), Chlamydia trachomatis ( Chlamydia muridarum ), Chlamydia abortus ( Chlamydophila abortus ), Chlamydia felis ( Ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
CPCC12Q1/04C12Q1/686C12Q2561/113C12Q2531/113C12Q2561/101
Inventor 李应国王昱聂福平杨俊肖进文王国民袁曾壮
Owner 重庆海关技术中心