Eremeeva Marina E, Dasch Gregory A, Silverman David J
School of Medicine, University of Maryland-Baltimore, Baltimore, Maryland 21201-1559, USA.
J Clin Microbiol. 2003 Dec;41(12):5466-72. doi: 10.1128/JCM.41.12.5466-5472.2003.
A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.
开发了一种斑点热立克次体定量PCR检测方法（SQ-PCR），用于检测和计数立氏立克次体及其他密切相关的斑点热群立克次体。该检测方法基于在PCR扩增过程中，SYBR Green染料插入rOmpA基因154 bp片段时的荧光检测。立氏立克次体的rOmpA基因低至5个拷贝即可被检测到。SQ-PCR适用于定量立氏立克次体和斑点热群立克次体的其他10种基因型，但不适用于小蛛立克次体、澳大利亚立克次体、贝利立克次体或斑疹伤寒群立克次体。SQ-PCR的灵敏度与采用离心接种的蚀斑试验相当。SQ-PCR检测方法已成功应用于立克次体储备培养物的鉴定、立克次体在细胞培养中的复制、不同提取方法后立克次体DNA的回收以及感染动物组织、临床样本和蜱中病原体载量的定量。
