The online version of this article (doi:10.1186/1475-2875-11-193) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
MAR, BDB, and KAC contributed to the design of the study. MAR conducted the experiments with assistance from BDB and KAC. MAR and JK conducted the statistical analyses. MAR drafted and edited the manuscript based on critical revisions of all the authors. DMW and KAC provided overall direction and support for the study. All authors read and approved the final manuscript.
Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR).
Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein.
Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification.
Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months.
Authors’ original file for figure 212936_2012_2113_MOESM1_ESM.tiff
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- PCR detection of malaria parasites in desiccated Anopheles mosquitoes is uninhibited by storage time and temperature
Mark A Rider
Brian D Byrd
Dawn M Wesson
Kevin A Caillouet
- BioMed Central
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