Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes

TL;DR

This study compares RNA extraction methods for detecting Chikungunya virus in Aedes aegypti mosquitoes, finding that some kits perform better with more mosquitoes per sample.

Contribution

The study evaluates and compares four RNA extraction protocols for Chikungunya virus detection in mosquitoes, identifying optimal methods for surveillance.

Findings

QIAamp® and PureLink® with TRIzol® kits showed greater sensitivity for RNA extraction.

Higher numbers of mosquitoes per pool correlate with lower Ct values and higher viral load.

QIAamp® provided the best RNA purity and concentration with lower Ct values.

Abstract

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus’s diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.

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