Detection of Treponema pallidum by a sensitive reverse transcriptase PCR
Detection of Treponema pallidum by a sensitive reverse transcriptase PCR
复制标题
DOI：
10.1128/jcm.35.6.1348-1352.1997
复制
发表时间：
1997-06-01
影响因子：
9.4
通讯作者：
Lukehart, SA
中科院分区：
文献类型：
--
作者：
CenturionLara, A;Castro, C;Lukehart, SA
梅毒是通过血清学检测或病原体梅毒螺旋体的鉴定来诊断的。历史上，通过暗视野显微镜、用多克隆或单克隆抗体免疫染色或兔接种试验（RIT）在临床标本中检测到该细菌。RIT被认为是非常敏感和特异的，尽管它仅在研究环境中可用，并且由于获得结果所需的时间长度而没有临床用途。已经开发了几种PCR方法用于检测T.我们建立了一种特异、高灵敏度的逆转录酶PCR（RT-PCR）方法，该方法针对T.苍白球该RT-PCR可通过Southern分析检测单个微生物，当整个微生物被稀释并加入10（-2）至10（-3）T时。当RNA等价物用于制备cDNA时，该试验证明可检测10（-2）T。脑脊液中苍白球RNA等价物，20种不同的T.从脑脊液、房水、血液和下疳中分离的苍白球被证明可以通过该试验检测。这种有效和灵敏的技术可能比现有的方法更有用，用于检测临床样品中非常低数量的微生物。
Syphilis is diagnosed by serologic testing or by identification of the causative agent, Treponema pallidum. The bacterium has historically been detected in clinical specimens by dark-field microscopy, immunostaining with polyclonal or monoclonal antibodies, or the rabbit inoculation test (RIT), RIT is considered to be very sensitive and specific, although it is available only in research settings and is not clinically useful due to the length of time required to obtain a result, In recent years, several PCR methods have been developed for the detection of T. pallidum, but none of these has shown a clear advantage in sensitivity over RIT, We have developed a specific and highly sensitive reverse transcriptase PCR (RT-PCR) that targets a 366 bp region of the 16S rRNA of T. pallidum. This RT-PCR can detect a single organism by Southern analysis when whole organisms are diluted and 10(-2) to 10(-3) T. pallidum organisms when RNA equivalents are used to make cDNA. The test was demonstrated to detect 10(-2) T. pallidum RNA equivalents in cerebrospinal fluid, Twenty different strains of T. pallidum, isolated from cerebrospinal fluids, aqueous humor, blood, and chancres, were shown to be detectable by this test, This efficient and sensitive technique could be more useful than existing methods for detecting very low numbers of organisms in clinical samples.