Rapid identification method for legionella pneumophila

Abstract

The invention provides a rapid identification method for legionella pneumophila. The rapid identification method comprises the following steps: adopting a pair of legionella pneumophila specific primers to amplify a region on a 16S rRNA (ribosomal Robonucleic Acid) gene; designing a pair of hybridization probes in an amplified segment aiming at specific sequences of the legionella pneumophila specific primers; anchoring a probe 3'-end marked FAM fluorescent gene and quenching a probe 5'-end marked BHQ1 (Black Hole Quencher 1); carrying out phosphorylation on the 3' end; analyzing the legionella pneumophila in a sample by real-time fluorescence PCR (Polymerase Chain Reaction) melting curve analysis. The detection method mainly comprises the following three steps: extracting a sample DNA (Deoxyribose Nucleic Acid), carrying out real-time fluorescence PCR and analyzing a melting curve. The method is simple and rapid to operate, is intuitive in result, is high in specificity and good in stability, and can be widely applied to rapid identification of the legionella pneumophila in clinical and environmental water samples.

Description

technical field [0001] The invention belongs to the field of medicine, in particular to a method for identifying Legionella pneumophila by using a fluorescent PCR melting curve. Background technique [0002] Legionella, a facultative intracellular parasite, is an important pathogen causing legionellosis in humans. In recent years, Legionnaires' disease has outbreaks and epidemics all over the world, and my country has listed it as one of 14 new infectious diseases. According to foreign reports, 10% of nosocomial pneumonia infections of unknown origin are caused by Legionella. At present, there are 52 species of Legionella and more than 70 serotypes. However, more than 80% of Legionella infections are clinically caused by Legionella pneumophila. Therefore, the identification of Legionella pneumophila is of great value for the diagnosis of Legionnaires' disease. [0003] At present, the detection and identification methods of Legionella mainly include separation and cultur...

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Problems solved by technology

However, the above detection methods all have deficiencies, such as direct isolation and culture, the time required is as long as 3 to 10 days, the positive rate is low, the medium configuration is complicated, and the price is expensive; urine antigen detection is only effective for Legionella pneumophila serotype 1 Specificity; Although molecular typing technology has greatly improved the sensitivity and specificity of Legionella detection, it still has its own shortcomings, such as the traditional PCR detection is cumbersome, time-consuming, and easy to contaminate; based on fluorescence resonance energy The hybridization probe labeling technology based on the transfer principle is expensive and complicated to operate; DNA sequencing is the most accurate identification method for Legionella species, but it takes a long time and is difficult to analyze, and cannot meet the rapid clinical requirements. Other molecular detection methods have various problems. kind of deficiency

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