Imperial College London
Alternate

Professor George K. Christophides

Faculty of Natural Sciences, Department of Life Sciences

Professor of Infectious Diseases & Immunity
 
 
 
//

Contact

 

+44 (0)20 7594 5342g.christophides

 
 
//

Location

 

6165Sir Alexander Fleming BuildingSouth Kensington Campus

//

Summary

 

Publications

Citation

BibTex format

@article{Habtewold:2015:10.1186/s13071-015-1087-8,
author = {Habtewold, T and Groom, Z and Duchateau, L and Christophides, GK},
doi = {10.1186/s13071-015-1087-8},
journal = {Parasites & Vectors},
title = {Detection of viable Plasmodium ookinetes in the midguts of Anopheles coluzzi using PMA-qrtPCR},
url = {http://dx.doi.org/10.1186/s13071-015-1087-8},
volume = {8},
year = {2015}
}
Download

RIS format (EndNote, RefMan)

TY  - JOUR
AB  - BackgroundMosquito infection with malaria parasites depends on complex interactions between the mosquito immune response, the parasite developmental program and the midgut microbiota. Simultaneous monitoring of the parasite and bacterial dynamics is important when studying these interactions. PCR based methods of genomic DNA (gDNA) have been widely used, but their inability to discriminate between live and dead cells compromises their application. The alternative method of quantification of mRNA mainly reports on cell activity rather than density.MethodQuantitative real-time (qrt) PCR in combination with Propidium Monoazide (PMA) treatment (PMA-qrtPCR) has been previously used for selectively enumerating viable microbial cells. PMA penetrates damaged cell membranes and intercalates in the DNA inhibiting its PCR amplification. Here, we tested the potential of PMA-qrtPCR to discriminate between and quantify live and dead Plasmodium berghei malarial parasites and commensal bacteria in the midgut of Anopheles coluzzii Coetzee & Wilkerson 2013 (formerly An. gambiae M-form).ResultsBy combining microscopic observations with reverse transcriptase PCR (RT-PCR) we reveal that, in addition to gDNA, mRNA from dead parasites also persists inside the mosquito midgut, therefore its quantification cannot accurately reflect live-only parasites at the time of monitoring. In contrast, pre-treating the samples with PMA selectively inhibited qrtPCR amplification of parasite gDNA, with about 15 cycles (Ct-value) difference between PMA-treated and control samples. The limit of detection corresponds to 10 Plasmodium ookinetes. Finally, we show that the PMA-qrtPCR method can be used to quantify bacteria that are present in the mosquito midgut.ConclusionThe PMA-qrtPCR is a suitable method for quantification of viable parasites and bacteria in the midgut of Anopheles mosquitoes. The method will be valuable when studying the molecular interactions between the mosquito, the malaria parasite
AU  - Habtewold,T
AU  - Groom,Z
AU  - Duchateau,L
AU  - Christophides,GK
DO  - 10.1186/s13071-015-1087-8
PY  - 2015///
SN  - 1756-3305
TI  - Detection of viable Plasmodium ookinetes in the midguts of Anopheles coluzzi using PMA-qrtPCR
T2  - Parasites & Vectors
UR  - http://dx.doi.org/10.1186/s13071-015-1087-8
UR  - http://hdl.handle.net/10044/1/27429
VL  - 8
ER  -
Download