High sensitivity of one-step real-time reverse transcription quantitative PCR to detect low virus titers in large mosqui
- PDF / 2,476,058 Bytes
- 13 Pages / 595.276 x 790.866 pts Page_size
- 18 Downloads / 186 Views
Parasites & Vectors Open Access
RESEARCH
High sensitivity of one‑step real‑time reverse transcription quantitative PCR to detect low virus titers in large mosquito pools Zhaoyang Tang1,2, Hanano Yamada1, Carina Kraupa1, Sumejja Canic1, Núria Busquets3, Sandra Talavera3, Davy Jiolle4, Marc J. B. Vreysen1, Jérémy Bouyer1 and Adly M. M. Abd‑Alla1*
Abstract Background: Mosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases primarily relies on the management of the vector. Traditional control methods are insufficient to control mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other con‑ trol tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males with the aim to induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as the released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mos‑ quito samples, its detection limit in mosquito samples has yet to be defined. Methods: We evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile virus (WNV) and Zika virus (ZIKV). Results: CHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1 and 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed the prediction of the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection. Conclusions: Mosquito-borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceed‑ ing 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have a SIT component by reducing the time and the manpower needed to conduct this quality control process. Keywords: Flavivirus, Arbovirus, Chikungunya virus (CHIKV), Usutu virus (USUV), West Nile virus (WNV), Zika virus (ZIKV), Pool size, RT-qPCR *Correspondence: a.m.m.abd‑[email protected] 1 Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna International Centre, P.O. Box 100, 1400 Vienna, Austria Full list of author information is
Data Loading...
