A cost-effective RNA extraction and RT-qPCR approach to detect California serogroup viruses from pooled mosquito samples

Marc Avramov; Vanessa Gallo; Antonia Gross; David R Lapen; Antoinette Ludwig; Catherine I Cullingham

doi:10.1038/s41598-024-52534-1

A cost-effective RNA extraction and RT-qPCR approach to detect California serogroup viruses from pooled mosquito samples

Sci Rep. 2024 Jan 29;14(1):2339. doi: 10.1038/s41598-024-52534-1.

Authors

Marc Avramov^{1

2

3}, Vanessa Gallo⁴, Antonia Gross⁴, David R Lapen⁵, Antoinette Ludwig⁶, Catherine I Cullingham⁴

Affiliations

¹ Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada. marcavramov@cmail.carleton.ca.
² Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, K1A 0C6, Canada. marcavramov@cmail.carleton.ca.
³ National Microbiology Laboratory Branch, Public Health Agency of Canada, 3200 Rue Sicotte, C.P. 5000, St. Hyacinthe, QC, J2S 2M2, Canada. marcavramov@cmail.carleton.ca.
⁴ Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
⁵ Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, K1A 0C6, Canada.
⁶ National Microbiology Laboratory Branch, Public Health Agency of Canada, 3200 Rue Sicotte, C.P. 5000, St. Hyacinthe, QC, J2S 2M2, Canada.

Abstract

Mosquito-borne diseases pose ongoing global health concerns, demanding more cost-efficient methods to detect pathogens to support enhanced surveillance efforts. This study introduces an adapted TRIzol-based high-throughput RNA extraction protocol, tailored for the detection of California serogroup viruses in pooled mosquito samples in a rapid and cost-effective manner. This approach provided consistent RNA yields and sensitive viral detection relative to two commercial extraction kits (QIAGEN RNeasy Mini Kit and MACHEREY-NAGEL NucleoSpin RNA Plus Kit). The incorporation of a user-friendly and non-spiking-based RT-qPCR internal control designed for the 18S rRNA gene in mosquitoes minimizes potential false positives/negatives, improving the fidelity of viral detection outcomes. Effective RNA yields, purity, and successful target amplification across 25 mosquito species and varied pool sizes (1-50 mosquitoes per tube) affirm the reliability of our approach. The extraction method is cost-effective, with an incurred cost of $0.58 CAD per sample, in contrast to the $5.25 CAD cost per sample of the two kits, rendering it promising for mosquito-borne disease surveillance initiatives.

MeSH terms

Animals
Cost-Benefit Analysis
Culicidae*
Encephalitis Virus, California* / genetics
RNA
RNA, Viral / analysis
RNA, Viral / genetics
Reproducibility of Results
Serogroup

Substances

RNA
RNA, Viral