TY - JOUR
T1 - A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector
AU - Bell, A. S.
AU - Ranford-Cartwright, L. C.
N1 - Funding Information:
We would like to thank Dr Will Roeffen for the gift of mAb 85RF45.1, Fiona McMonagle, Liz Peat and Keith Scott for their maintenance of the mosquito colony, Marian Bruce for Plasmodium vivax and Plasmodium malariae DNA, Luke Baton for aid with parasite cultures and mosquito dissections, and Dr M. Peterkin and staff at the Glasgow and West of Scotland Blood Transfusion Service, for the provision of blood and serum. Acknowledgement is made for the financial support of the European Commission (contract no. QLK2 CT 1999 00753).
PY - 2004/6
Y1 - 2004/6
N2 - 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.
AB - 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.
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U2 - 10.1016/j.ijpara.2004.03.008
DO - 10.1016/j.ijpara.2004.03.008
M3 - Article
C2 - 15157762
AN - SCOPUS:2442513988
SN - 0020-7519
VL - 34
SP - 795
EP - 802
JO - International Journal for Parasitology
JF - International Journal for Parasitology
IS - 7
ER -
