Animal chlamydia TaqMan-MGB probe multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers, kit and method

A technology for real-time fluorescence quantification and animal chlamydia, which is applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems of detection kits and detection methods for three types of chlamydia, and achieve identification Simple, highly specific effect

Active Publication Date: 2015-05-27
重庆海关技术中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no reports on the kits and detection methods for the detection of three types of Chlamydia using the TaqMan-MGB probe fluorescence quantitative PCR amplification technology

Method used

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  • Animal chlamydia TaqMan-MGB probe multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers, kit and method
  • Animal chlamydia TaqMan-MGB probe multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers, kit and method
  • Animal chlamydia TaqMan-MGB probe multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers, kit and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, design and screening of primers

[0047] Primers and probes for fluorescent quantitative PCR amplification of three kinds of Chlamydia (including Chlamydia abortus, Chlamydia abortus, Chlamydia psittaci) were designed based on the reference sequences of MOMP genes of three Chlamydia published by GenBank, compared with MEGA5, and analyzed sequence and design primers and probes in its conserved regions. The probe design software Primer Express 3.0 was used to design 12 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. The starting time of the amplification, the time to enter the maximum amplification rate, the maximum amplification rate and the time required to reach the plateau were analyzed, and three groups of fluorescent quantitative PCR amplification with the highest amplification rate and good specifici...

Embodiment 2

[0059] Embodiment 2, the preparation of positive control substance

[0060] The kit was used to extract the nucleic acid of the cultured cells of Chlamydia abortus, the nucleic acid was identified by PCR and electrophoresis, and PCR upstream primer SEQ ID NO.10 and PCR downstream primer SEQ ID NO.11 were used to amplify according to the conventional PCR amplification method, The amplified bands were recovered using a gel extraction kit. According to the ratio of 1:10 and the pMD19T vector, the ligation reaction was carried out, ligated overnight at 4°C, and transformed into DH5α bacteria. After resistance selection and PCR identification were positive, it was sequenced and verified, and the OD value of its nucleic acid was measured by a spectrophotometer to make it 260 The ratio of / 280 is between 1.8 and 2.0. Obtain positive recombinant plasmid DNA of Chlamydia abortus. Domestic animal Chlamydia and Chlamydia psittaci positive recombinant plasmid DNAs were prepared accordin...

Embodiment 3

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

[0062] The DNA of the epithelial tissue without the above three kinds of Chlamydia was extracted with a kit, and identified by PCR and electrophoresis.

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Abstract

The invention discloses animal chlamydia TaqMan-MGB probe multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers, kit and method. The multiplex TaqMan-MGB probe real-time fluorescent PCR method can perform quick detection on Ch. Abortus, Ch.pecorum and Ch.psittaci. On the basis of the conserved region of the three chlamydia target sequences MOMP, the detection method designs three pairs of primers and three TaqMan-MHB probes. The method can quickly, efficiently, specifically and sensitively detect the target sequences by a two-step amplification process under simple reaction conditions, is simple to operate, does not need any expensive instrument or reagent, and has the advantages of no technical requirements for operating personnel, low detection cost and short detection time. The method can avoid cross contamination which can possibly occur due to agarose electrophoresis, thereby enhancing the detection accuracy and reliability.

Description

technical field [0001] The invention relates to the field of animal molecular biology test methods and test reagents, in particular to a primer, a kit and a method for multiple real-time fluorescent quantitative PCR detection of animal chlamydia Taqman-MGB probes. 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 ( Chlamydophilafelis ), Li...

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 聂福平王昱李应国杨俊肖进文袁曾壮王国民李贤良
Owner 重庆海关技术中心
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