Establishing method of TaqMan probe real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection system by using 16S rRNA gene as target gene

Abstract

The invention relates to a quick detection and diagnostic method for animal derived chlamydophila (Chlamydophilapsittaci, C.abortus, C.caviae, C.pecorum, C.felis) by applying real-time PCR (Polymerase Chain Reaction), which is one of new molecular biological technologies. The detection system designs a pair of primers and corresponding TaqMan probes based on a conserved zone of 16S rRNA of the animal derived chlamydophila. The system can be used for detecting animal derived chlamydophila genes existing in animal excrement, cotton swab of cloaca and human tracheal eluate with the detection sensitivity higher than that of an ordinary PCR by 10 times. The real-time PCR does not need post-PCR treatment, avoids cross contamination caused by the operation of agarose gel electrophoresis and accordingly enhances detection accuracy and reliability.

Description

Technical field: The invention belongs to the field of molecular biology, and in particular relates to a method for establishing a TaqMan probe real-time fluorescent quantitative PCR detection system using 16S rRNA of animal-derived chlamydia as a target gene. Background technique: Chlamydia is an obligate intracellular parasitic pathogen that differs from other Gram-positive bacteria in its unique bidirectional life cycle, that is, an extracellular survival form—the protosome and an intracellular replicating form—the reticulum , which can be transformed into each other. In 1999, Chlamydiaceae was divided into 2 genera and 9 species. Among them, there are 3 species of Chlamydia, including Chlamydia trachomatis, Chlamydia suis and Chlamydia murine pneumonia; 6 species of Chlamydia, namely Chlamydia abortus, Chlamydia guinea pig, Chlamydia felis, Chlamydia veterinus, Chlamydia pneumoniae Chlamydia and Chlamydia psittaci. These microorganisms are the pathogens of many zoono...

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Problems solved by technology

Pathogen isolation mainly includes chicken embryo inoculation, cell culture and animal regression test. The operation procedures are complicated and time-consuming, and the result judgment is often subjective, which limits the detection

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