Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes

Abstract

Background

Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens.

Methodology/Principal findings

Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack.

Conclusions/Significance

Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.

Author summary

Molecular xenomonitoring has demonstrated significant potential as a non-invasive means of providing reliable surveillance for the presence of lymphatic filariasis (LF)-causing parasites. Given the continuing successes of global mass drug administration efforts, the need for such non-invasive surveillance techniques is expanding. However, considering the significant infrastructural demands which such surveillance requires, the development of simplified surveillance methodologies will be fundamental to future programmatic implementation efforts. Accordingly, we have developed a novel, simplified diagnostic platform for point-of-collection-based detection of the LF-causing parasite, Brugia malayi in pools of mosquitoes. By coupling a rapid and inexpensive DNA extraction methodology with a field-friendly amplification platform and test strip-based detection assay, this backpack PCR system eliminates the need for expensive instrumentation and laboratory-based infrastructure. Furthermore, adaptation of the platform described here will allow for the straightforward and rapid development of a parallel assay for the detection of Wuchereria bancrofti, facilitating the increased use of xenomonitoring and enabling mosquito surveillance efforts in regions lacking sophisticated laboratory infrastructure.