A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector
A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector
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DOI：
10.1016/j.ijpara.2004.03.008
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发表时间：
2004-06-01
影响因子：
4
通讯作者：
Ranford-Cartwright, LC
中科院分区：
文献类型：
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作者：
Bell, AS;Ranford-Cartwright, LC
阻断传播的疫苗可防止蚊媒体内疟原虫寄生虫的生长，从而阻止疟疾在社区中传播。确定传播阻断疫苗功效的黄金标准是标准膜喂养测定。该测定需要解剖蚊子并在显微镜下计数蚊子中肠上存在的卵囊，通常在注射后 7-10 天进行。在这里，我们描述了一种实时定量 PCR 测定，该测定快速、目标特异性、稳健，具有灵敏的检测阈值，并且可以在注射前早期使用。比标准膜喂养测定法更适用，并且适用于保存的材料。实时 PCR 检测利用 LightCycler 平台和 SYBR Green I 检测系统来扩增恶性疟原虫 rRNA 基因无性形式的 180 bp。它的定量范围大于四个数量级，检测阈值为 10 种寄生虫。使用具有已知阻断活性的单克隆抗体进行的验证实验表明，实时 PCR 测定可提供与标准膜补料测定相同的结果。此外，PCR 测定可以确定这种单克隆抗体对卵囊内寄生虫发育和子孢子（可传播阶段）产量的影响，从而提供比标准膜喂养测定更相关的传播阻断活性评估。该测定还可用于监测蚊媒内恶性疟原虫寄生虫的孢子发育。 (C) 2004 年澳大利亚寄生虫学协会。由 Elsevier Ltd 出版。保留所有权利。
Transmission-blocking vaccines prevent the development of Plasmodium parasite within the mosquito vector, thereby thwarting the spread of malaria through a community. The gold standard for determining the efficacy of a transmission-blocking vaccine is the standard membrane feeding assay. This assay requires the dissection of mosquitoes and microscopic counting of oocysts present on the mosquito mid-gut, typically at 7-10 days p.i. Here we describe a real-time quantitative PCR assay that is rapid, target-specific and robust, with a sensitive detection threshold and which may be employed earlier p.i. than the standard membrane feeding assay and is applicable to preserved material. The real-time PCR assay utilises the LightCycler platform and SYBR Green I detection system to amplify 180 bp of the asexual form of the Plasmodium falciparum rRNA gene. It has a quantitative range of greater than four orders of magnitude and a detection threshold of 10 parasites. Validation experiments using a monoclonal antibody of known blocking activity revealed the real-time PCR assay to give equivalent results to the standard membrane feeding assay. In addition, the PCR assay can establish the effect of such a monoclonal antibody on the parasites' development within the oocyst and on the sporozoite (the transmissible stage) yield, providing a more pertinent assessment of transmission blocking activity than is possible by the standard membrane feeding assay. This assay may also be employed to monitor the sporogonic development of P. falciparum parasites within the mosquito vector. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.