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首页> 外文期刊>Journal of Clinical Microbiology >Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays
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Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

机译：通过基于18S rRNA基因亚基和物种特异性的实时PCR检测人类血液中的四种疟原虫

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There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the “gold standard” in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.
机译：有报告说，移民和旅行者中的疟疾病例增加。尽管血液涂片的显微镜检查仍然是诊断的“金标准”，但这种方法灵敏度不足，需要大量的专业知识。为了改善诊断，开发了一种实时多重PCR。设计了一套针对疟原虫属18S rRNA基因高度保守区域的通用引物。引物组内部足够多态，可以为 P设计四个物种特异性探针。恶心， P。 vivax ， P。恶意软件和 P。椭圆形。使用物种特异性探针的实时PCR检测到一个质粒拷贝的 P。恶心， P。 vivax ， P。疟疾和 P。椭圆形。使用18S筛选探针通过实时PCR对所有物种实现了相同的灵敏度。研究了九十七份血液样本。他们对其中的66名（60名患者）进行了显微镜检查和实时PCR结果进行了比较，并发现粗丝虫病检出率为86％。用临床，分子和测序数据重新评估不一致的结果，以解决这些问题。解决了18S筛选PCR和显微镜检查之间的所有九种不一致性，该方法支持分子方法，两种方法均阳性的31个样本中，针对物种特异性PCR的物种一级的九种不一致性中的八个也得到了解决。测试了其他31个血液样本，以监测7例患者的抗疟疾治疗情况。通过实时PCR测量的寄生虫数量在七名患者中有六名迅速下降，与显微镜下确定的寄生虫病平行。这表明定量PCR在监测接受抗疟疾治疗的患者中的作用。

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