Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers.
Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers.
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DOI：
10.1186/1475-2875-8-86
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发表时间：
2009-04-29
期刊：
影响因子：
3
通讯作者：
Ariey F
中科院分区：
文献类型：
--
作者：
Steenkeste N;Incardona S;Chy S;Duval L;Ekala MT;Lim P;Hewitt S;Sochantha T;Socheat D;Rogier C;Mercereau-Puijalon O;Fandeur T;Ariey F
目前在热带地区的许多国家部署了几项战略，以加强疟疾控制，实现消灭疟疾。为了衡量任何干预措施的影响，需要适当检测疟疾。大多数情况下，决策仍然依赖于显微镜诊断。但是需要能够处理大量样品的灵敏诊断工具。分子检测方法提供了更高的灵敏度，以及自动化和升级的灵活性。发展了两种新的分子方法：dot 18 S，一种基于18 S rRNA基因的疟原虫特异性巢式PCR，然后使用种特异性探针进行种的斑点印迹检测;以及CYTB，一种基于细胞色素B基因的疟原虫特异性巢式PCR，然后使用SNP分析进行种的检测。将结果与显微镜检查和使用种特异性引物的基于“标准”18 S rRNA基因的巢式PCR获得的结果进行比较。337份样本确诊。与显微镜相比，这三种分子方法更敏感，大大增加了疟原虫感染的估计患病率，包括三日疟原虫和卵形疟原虫。发现混合感染率很高，约三分之一的村民感染了一种以上的疟疾寄生虫。Dot 18 S和CYTB的敏感性超过了“标准”巢式PCR方法，CYTB是最敏感的。因此，与检测疟原虫的“标准”巢式PCR方法相比，dot 18 S和CYTB的敏感性分别为95.3%和97.3%。一致检测疟原虫属。三种分子生物学方法的阳性率为83%。相互矛盾的结果大多与混合感染中三日疟原虫和卵形疟原虫的检测有关，这是由于在低水平寄生虫血症中的“全或无”检测效应。使用分子方法发现了大量无症状感染者。Dot 18 S和CYTB，本文报道的新方法是高度灵敏的，允许寄生虫DNA提取以及数百个样品的属和种特异性诊断，并且适合于高通量放大以用于更大的样品尺寸。这些方法提供了关于疟疾流行和流行病学的新信息，并适合于主动疟疾检测。本文讨论了这种敏感的疟疾诊断工具的有用性，特别是在低流行地区的根除计划正在进行中。
Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded. Two new molecular methods were developed: dot18S, a Plasmodium-specific nested PCR based on the 18S rRNA gene followed by dot-blot detection of species by using species-specific probes and CYTB, a Plasmodium-specific nested PCR based on cytochrome b gene followed by species detection using SNP analysis. The results were compared to those obtained with microscopic examination and the "standard" 18S rRNA gene based nested PCR using species specific primers. 337 samples were diagnosed. Compared to the microscopy the three molecular methods were more sensitive, greatly increasing the estimated prevalence of Plasmodium infection, including P. malariae and P. ovale. A high rate of mixed infections was uncovered with about one third of the villagers infected with more than one malaria parasite species. Dot18S and CYTB sensitivity outranged the "standard" nested PCR method, CYTB being the most sensitive. As a consequence, compared to the "standard" nested PCR method for the detection of Plasmodium spp., the sensitivity of dot18S and CYTB was respectively 95.3% and 97.3%. Consistent detection of Plasmodium spp. by the three molecular methods was obtained for 83% of tested isolates. Contradictory results were mostly related to detection of Plasmodium malariae and Plasmodium ovale in mixed infections, due to an "all-or-none" detection effect at low-level parasitaemia. A large reservoir of asymptomatic infections was uncovered using the molecular methods. Dot18S and CYTB, the new methods reported herein are highly sensitive, allow parasite DNA extraction as well as genus- and species-specific diagnosis of several hundreds of samples, and are amenable to high-throughput scaling up for larger sample sizes. Such methods provide novel information on malaria prevalence and epidemiology and are suited for active malaria detection. The usefulness of such sensitive malaria diagnosis tools, especially in low endemic areas where eradication plans are now on-going, is discussed in this paper.
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影响因子：
3
作者：
Duval, Linda;Robert, Vincent;Ariey, Frederic
通讯作者：
Ariey, Frederic
影响因子：
9.4
作者：
Kawamoto, F;Miyake, H;Wataya, W
通讯作者：
Wataya, W
DOI：
10.4269/ajtmh.1999.60.1019
发表时间：
1999-06-01
影响因子：
3.3
作者：
Price, R;Nosten, F;White, NJ
通讯作者：
White, NJ
DOI：
10.4269/ajtmh.1998.59.582
发表时间：
1998-10-01
影响因子：
3.3
作者：
Babiker, HA;Abdel-Muhsin, ABA;Walliker, D
通讯作者：
Walliker, D
DOI：
10.4269/ajtmh.2006.75.588
发表时间：
2006-10-01
影响因子：
3.3
作者：
Kasehagen, Laurin J.;Mueller, Ivo;Zimmerman, Peter A.
通讯作者：
Zimmerman, Peter A.