Development of a Highly Sensitive Genus-Specific Quantitative Reverse Transcriptase Real-Time PCR Assay for Detection and Quantitation of Plasmodium by Amplifying RNA and DNA of the 18S rRNA Genes
Development of a Highly Sensitive Genus-Specific Quantitative Reverse Transcriptase Real-Time PCR Assay for Detection and Quantitation of Plasmodium by Amplifying RNA and DNA of the 18S rRNA Genes
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DOI：
10.1128/jcm.00276-11
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发表时间：
2011-08-01
影响因子：
9.4
通讯作者：
Ockenhouse, Christian F.
中科院分区：
文献类型：
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作者：
Kamau, Edwin;Tolbert, LaDonna S.;Ockenhouse, Christian F.
建立了一种检测疟原虫属特异性的高灵敏定量逆转录酶实时聚合酶链式反应(qRT-PCR)方法。用培养的同步环期3D7寄生虫，该方法可扩增18SRRNA基因的总核酸，检出限为0.002只寄生虫/亩L。在对临床标本进行分析时，检出寄生虫密度低至0.000362只/亩L。对临床样本的分析表明，从总核酸中检测18S rRNA基因比仅从DNA中检测18S rRNA基因的分析灵敏度提高了1个对数单位以上。对镜检未检出寄生虫的临床标本进行qRT-PCR分析，90%(117/130)的标本检测出疟原虫核酸。使用总核酸和DNA的qRT-PCR方法对临床样本进行定量，并与显微镜进行比较。当单独使用DNA时，寄生虫密度与显微镜检查的相关性明显高于总核酸。我们的结论是，定量逆转录聚合酶链式反应分析总核酸是检测早期疟疾患者和/或亚显微感染患者低寄生虫水平的一种合适的方法，并可极大地帮助疟疾诊断、干预试验和疟疾控制和消除工作。
A highly sensitive genus-specific quantitative reverse transcriptase real-time PCR (qRT-PCR) assay for detection of Plasmodium has been developed. The assay amplifies total nucleic acids (RNA and DNA) of the 18S rRNA genes with a limit of detection of 0.002 parasite/mu l using cultured synchronized ring stage 3D7 parasites. Parasite densities as low as 0.000362 parasite/mu l were detected when analyzing clinical samples. Analysis of clinical samples showed that detection of 18S rRNA genes from total nucleic acids increased the analytical sensitivity of the assay by more than 1 log unit compared to DNA only. When clinical samples with no parasites present by microscopy were analyzed by qRT-PCR, 90% (117 of 130) were positive for the presence of Plasmodium nucleic acids. Quantification of clinical samples by qRT-PCR using total nucleic acid versus DNA was compared to microscopy. There was a significantly greater correlation of parasite density to microscopy when DNA alone was used than with total nucleic acid. We conclude that analysis of total nucleic acids by qRT-PCR is a suitable assay for detection of low parasite levels in patients with early-stage malaria and/or submicroscopic infections and could greatly benefit malaria diagnosis, intervention trials, and malaria control and elimination efforts.