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使用SYBR绿I和LightCycler仪器通过实时聚合酶链反应快速检测百日咳博德特氏菌

Rapid detection of Bordetella pertussis by real-time PCR using SYBR green I and a LightCycler instrument.

作者信息

Poddar S K

机构信息

Pediatric Pharmacology Research Unit, University of California at San Diego, La Jolla 92093-0808, USA.

出版信息

J Clin Lab Anal. 2004;18(5):265-70. doi: 10.1002/jcla.20035.

DOI:10.1002/jcla.20035

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6807937/

Abstract

A polymerase chain reaction (PCR) assay in real-time for detection of B. pertussis using SYBR green I as the reporter fluorophore and LightCycler instrument (a thermocycler coupled to a fluorescence detection device) was established and evaluated. The amplified amplicon using series diluted control prototype strain (ATCC strain #9797) of B. pertussis was analyzed for the fluorescent melting profile, and melting temperature (Tm) was determined. When examined, amplicons using a representative set of clinical isolates of B. pertussis were found to have the same Tm value (86 +/- 0.5 degrees C, the specificity parameter of detection) as the control prototype strain as expected. Amplified product was also analyzed and detected by agarose gel electrophoresis. The detection limit by fluorescent profile and Tm analysis was 10-fold better than that detected by agarose gel analysis.

摘要

建立并评估了一种实时聚合酶链反应（PCR）检测方法，该方法使用SYBR green I作为报告荧光团，LightCycler仪器（一种与荧光检测装置相连的热循环仪）来检测百日咳博德特氏菌。对使用系列稀释的百日咳博德特氏菌对照原型菌株（ATCC菌株#9797）扩增得到的扩增子进行荧光熔解曲线分析，并确定熔解温度（Tm）。检测时发现，使用一组代表性的百日咳博德特氏菌临床分离株扩增得到的扩增子，其Tm值（86±0.5℃，检测的特异性参数）与对照原型菌株预期的相同。扩增产物也通过琼脂糖凝胶电泳进行分析和检测。荧光曲线和Tm分析的检测限比琼脂糖凝胶分析的检测限高10倍。