Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae
Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae
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DOI：
10.1128/jcm.41.12.5466-5472.2003
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发表时间：
2003-12-01
影响因子：
9.4
通讯作者：
Silverman, DJ
中科院分区：
文献类型：
--
作者：
Eremeeva, ME;Dasch, GA;Silverman, DJ
建立了斑点热立克次体定量PCR检测方法（SQ-PCR），用于斑点热群立克次体及其它密切相关的立克次体的检测和计数。该测定基于荧光检测rOmpA的154-bp片段中的SYBR绿色染料嵌入。在PCR扩增过程中，rOmpA基因只有5个拷贝。可以检测到立克次氏体。SQ-PCR适用于R.立克次体和其它10种斑点热群立克次体基因型，而R.阿卡里河australis，R.贝氏体或斑疹伤寒群立克次体。SQ-PCR的灵敏度与使用离心接种的空斑试验的灵敏度相当。SQ-PCR试验成功地应用于立克次体储备培养物的表征，立克次体在细胞培养物中的复制，立克次体DNA的回收，以及感染动物组织、临床样本和蜱中立克次体载量的定量。
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.