Flow Cytometric Sorting of Infected Erythrocytes Demonstrates Reliable Detection of Individual Ring-Stage Plasmodium falciparum Parasites by Plasmodium 18S rRNA Reverse Transcription Polymerase Chain Reaction.
Flow Cytometric Sorting of Infected Erythrocytes Demonstrates Reliable Detection of Individual Ring-Stage Plasmodium falciparum Parasites by Plasmodium 18S rRNA Reverse Transcription Polymerase Chain Reaction.
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感染红细胞的流式细胞仪分选证明了通过疟原虫18S rRNA逆转录聚合酶链反应可靠地检测单个恶性疟原虫环形期寄生虫。
DOI：
10.4269/ajtmh.21-1226
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发表时间：
2022-04-11
影响因子：
3.3
通讯作者：
Murphy, Sean C.
中科院分区：
文献类型：
--
作者：
Matsubara, Jokichi;Chang, Ming;Seilie, Annette M.;Murphy, Sean C.
在临床试验和现场监测研究中，越来越多地使用恶性疟原虫的分子诊断试验来实现对感染的超灵敏检测。以核糖核酸(RNA)为基础的针对18S rRNA的检测特别敏感，据报道，检测限度包括相对大量的血液中单个受感染的红细胞(RBC)。然而，在这种极限浓度下的验证测试受到这种罕见事件的所谓泊松分布的阻碍，这可能导致实验室不准确地将检测极限设置得高于(即，不那么灵敏)于分析实际可以检测到的水平。在此，我们正式展示了疟原虫18S rRNA定量逆转录聚合酶链式反应(qRT-PCR)的分析敏感性。对DNA和RNA双重染色的恶性疟原虫同步培养物进行荧光激活细胞分选(FACS)，然后对加入阴性全血的单个分选细胞进行qRT-PCR。定量逆转录聚合酶链式反应检测到95%以上的单环感染红细胞。正式测量的每个环期恶性疟原虫18S rRNA含量的中位数为9,550个拷贝(四分位数范围为8,130-12,300)。因此，人们可以放心地依靠疟原虫18SRRNA qRT-PCR检测每50微米L血液样本中的一种寄生虫。
Molecular diagnostic tests for Plasmodium falciparum parasites are increasingly used to enable ultrasensitive detection of infection in clinical trials and field surveillance studies. Ribonucleic acid (RNA)-based assays targeting 18S rRNA are particularly sensitive with limits of detection reported to comprise a single infected red blood cell (RBC) in a relatively large volume of blood. However, the validation testing at such limiting concentrations is hampered by the so-called Poisson distribution of such rare events, which can lead laboratorians to inaccurately set the limit of detection higher (i.e., less sensitive) than the assay can actually detect. Here we set out to formally demonstrate the analytical sensitivity of the Plasmodium 18S rRNA quantitative reverse transcription PCR (qRT-PCR). Fluorescence-activated cell sorting (FACS) was used on synchronous P. falciparum cultures doubly stained for DNA and RNA and was followed by qRT-PCR on the individual sorted cells spiked with negative whole blood. Over 95% of individual single-ring infected RBCs were detected by qRT-PCR. The formally measured median 18S rRNA content per individual ring-stage P. falciparum parasite was 9,550 copies (interquartile range 8,130–12,300). Thus, one can confidently rely on Plasmodium 18S rRNA qRT-PCR to detect one parasite per 50-µL blood sample.
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影响因子：
15.8
作者：
Chen I;Clarke SE;Gosling R;Hamainza B;Killeen G;Magill A;O'Meara W;Price RN;Riley EM
通讯作者：
Riley EM
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DuBois, DB
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通讯作者：
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