Bell Constance A, Patel Robin
Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Diagn Microbiol Infect Dis. 2005 Dec;53(4):301-6. doi: 10.1016/j.diagmicrobio.2005.06.019. Epub 2005 Nov 2.
A rapid real-time polymerase chain reaction (PCR) assay capable of the simultaneous detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii was developed using the LightCyclertrade mark instrument (Roche Applied Sciences, Indianapolis, IN). The assay targets the operon groEL of the heat shock protein. Base pair mismatches in amplified DNA in regions of detection probe hybridization allowed organism differentiation by melting curve analysis. The analytical sensitivity was at least 10 copies per reaction. DNA extracts from 59 specimens previously confirmed positive for A. phagocytophilum (n = 37), E. chaffeensis (n = 19), or E. ewingii (n = 3) were used to evaluate the assay. All of the specimens positive for 1 of the 3 organisms by conventional PCR were likewise positive by the LightCycler method. Sensitivity and specificity were at least 100% compared with conventional PCR. This assay provides a rapid method for the detection and differentiation of the causative agents of human ehrlichiosis in the United States.
使用LightCycler商标仪器（罗氏应用科学公司，印第安纳波利斯，印第安纳州）开发了一种能够同时检测和区分嗜吞噬细胞无形体、查菲埃立克体和尤因埃立克体的快速实时聚合酶链反应（PCR）检测方法。该检测方法针对热休克蛋白的操纵子groEL。检测探针杂交区域中扩增DNA的碱基对错配允许通过熔解曲线分析进行生物体区分。分析灵敏度为每个反应至少10个拷贝。使用先前经确认对嗜吞噬细胞无形体（n = 37）、查菲埃立克体（n = 19）或尤因埃立克体（n = 3）呈阳性的59个标本的DNA提取物来评估该检测方法。通过常规PCR对这3种生物体中的1种呈阳性的所有标本通过LightCycler方法同样呈阳性。与常规PCR相比，灵敏度和特异性至少为100%。该检测方法为在美国检测和区分人类埃立克体病的病原体提供了一种快速方法。
