Fast and robust single PCR for Plasmodium sporozoite detection in mosquitoes using the cytochrome oxidase I gene

• Published in: Malaria Journal
• Main Authors: Diego F. Echeverry, Nicholas A. Deason, Victoria Makuru, Jenna Davidson, Honglin Xiao, Julie Niedbalski, Xiaoyu Yu, Jennifer C. Stevenson, Hugo Bugoro, Allan Aparaimo, Hedrick Reuben, Robert Cooper, Thomas R. Burkot, Tanya L. Russell, Frank H. Collins, Neil F. Lobo
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2017
• Subjects: Malaria; Plasmodium; Diagnosis; Sporozoite; Anopheles; 18s-rRNA; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1186/s12936-017-1881-1; https://doaj.org/article/64d2d9009af647d8aa6c5431dacd6c02

Abstract Background Molecular tools for detecting malaria-infected mosquitoes with improved practicality, sensitivity and specificity, and high-throughput are required. A common PCR technique used to detect mosquitoes infected with Plasmodium spp. is a nested PCR assay based on the 18s-rRNA gene. However, this technique has several technical limitations, is laborious and time consuming. Methods In this study, a PCR-based on the Plasmodium cytochrome oxidase I (COX-I) gene was compared with the 18s-rRNA nested PCR using serial dilutions (330–0.0012 pg) of DNA from Plasmodium vivax, Plasmodium falciparum and Plasmodium knowlesi and with DNA from 48 positive and negative Kenyan mosquitoes (previously detected by using both ELISA and PCR). This assay for Plasmodium spp. DNA detection using the fast COX-I PCR assay was then performed individually on 2122 field collected mosquitoes (from the Solomon Islands) in which DNA was extracted from head and thorax. Results The fast COX-I PCR assay took 1 h to run and consistently detected as low as to 0.043 pg of parasite DNA (equivalent to two parasites) in a single PCR, while analyses with the 18s-rRNA nested PCR required 4 h to complete with a consistent detection threshold of 1.5 pg of DNA. Both assays produced concordant results when applied to the 48 Kenyan control samples with known Plasmodium spp. infection status. The fast COX-I PCR identified 23/2122 Plasmodium-infected mosquitoes from the Solomon Islands. Conclusions This new COX-I PCR adapted for a single PCR reaction is a faster, simpler, cheaper, more sensitive technique amenable to high-throughput analyses for Plasmodium DNA detection in mosquitoes and is comparable to the 18s-rRNA nested PCR. The improved sensitivity seen with the fast COX-I PCR will improve the accuracy of mosquito infection rate determination.