A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.
A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.
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DOI：
10.1371/journal.pone.0165506
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Ayong L
中科院分区：
文献类型：
--
作者：
Kemleu S;Guelig D;Eboumbou Moukoko C;Essangui E;Diesburg S;Mouliom A;Melingui B;Manga J;Donkeu C;Epote A;Texier G;LaBarre P;Burton R;Ayong L
高度敏感和现场部署的分子诊断工具是迫切需要的，以检测亚显微镜，但传播水平的疟疾寄生虫流行在世界各地的疟疾流行的国家。开发了逆转录环介导等温扩增（RT-LAMP）试验，并与厚血涂片显微镜检查、基于抗原的快速诊断试验（RDT）和针对相同RT-LAMP转录本的内部RT-PCR进行了比较。优化的检测方法可检测到低至0.25 ng总寄生虫RNA的恶性疟原虫感染，当直接对感染的全血裂解物进行检测时，检测限为0.08个寄生虫/ μL，或当使用RNA提取物时，检测限为~0.0008个寄生虫/ μL。早在RT-LAMP开始后8分钟就观察到测定阳性，并且在大多数情况下，反应在20分钟前完成。对132例疑似疟疾病例进行的临床评价显示，使用提取的RNA进行RT-LAMP的阳性率为90%，使用全血裂解物时为85%。恶性疟原虫特异性RDT的阳性率为70%，RT-PCR的阳性率为83%，厚血涂片镜检的阳性率为74%（平均寄生虫密度= 36，986个寄生虫/ μL）。所开发的RT-LAMP和比较测试之间的一致性率大于75%，最低的是光学显微镜（78%，McNemar检验：P = 0.0002），最高的是RT-PCR（87%，McNemar检验：P = 0.0523）。与参考RT-PCR相比，全血RT-LAMP的检测灵敏度为90%，使用相应RNA提取物的RT-LAMP的检测灵敏度为96%。无电加热器进行了进一步的开发和评估，与电池供电的等温扩增机在资源有限的环境中使用的开发测试。总之，这些数据突出了在分子诊断中靶向高丰度RNA转录物的益处，以及所开发的RT-LAMP测定在低至高寄生虫密度环境中疟疾诊断中的潜在有用性。
Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings.
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影响因子：
9.8
作者：
Bozdech, Zbynek;Llinas, Manuel;Pulliam, Brian Lee;Wong, Edith D;Zhu, Jingchun;DeRisi, Joseph L
通讯作者：
DeRisi, Joseph L
DOI：
10.4269/ajtmh.2012.12-0095
发表时间：
2013-02-01
影响因子：
3.3
作者：
Baltzell, Kimberly A.;Shakely, Deter;Greenhouse, Bryan
通讯作者：
Greenhouse, Bryan
DOI：
10.1101/pdb.prot4104
发表时间：
2006-06-01
期刊：
CSH protocols
影响因子：
--
作者：
Connolly, Michael A;Clausen, Peter A;Lazar, James G
通讯作者：
Lazar, James G
影响因子：
3
作者：
Britton S;Cheng Q;McCarthy JS
通讯作者：
McCarthy JS
影响因子：
9.4
作者：
Kamau, Edwin;Tolbert, LaDonna S.;Ockenhouse, Christian F.
通讯作者：
Ockenhouse, Christian F.