Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi
Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi
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DOI：
10.4269/ajtmh.2004.70.351
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发表时间：
2004-04-01
影响因子：
3.3
通讯作者：
Richards, AL
中科院分区：
文献类型：
--
作者：
Jiang, J;Chan, TC;Richards, AL
两个特定的和敏感的聚合酶链反应（PCR）检测和定量恙虫病东方体，恙虫病的代理人，使用的47-kD的外膜蛋白抗原/高温要求A基因的一部分作为目标。用一对47 kD的引物从26株O.评估了恙虫病，但它没有产生扩增子时，17立克次体和18个不太相关的细菌核酸提取物进行了测试。使用相同的引物和31个碱基对的荧光探针，证明了实时PCR检测试剂的相似特异性。该方法灵敏、定量，可用于测定O.恙虫病核酸使用含有来自Kato株的完整47-kD基因的质粒作为标准。O.实验感染东方体后，从小鼠和猴的感染组织中提取的恙虫病DNA显示，小鼠血液中有27- 5，552个拷贝/穆尔，小鼠肝/脾匀浆中有14，448 - 86，012个拷贝/穆尔，猴血液中有3-21个拷贝/穆尔。
Two specific and sensitive polymerase chain reaction (PCR) assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen/ high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27-5,552 copies/muL of mouse blood, 14,448-86,012 copies/muL of mouse liver/spleen homogenate, and 3-21 copies/muL of monkey blood.