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. 2020 Sep 9;13(1):460.
doi: 10.1186/s13071-020-04327-4.

High sensitivity of one-step real-time reverse transcription quantitative PCR to detect low virus titers in large mosquito pools

Zhaoyang Tang  1   2 , Hanano Yamada  1 , Carina Kraupa  1 , Sumejja Canic  1 , Núria Busquets  3 , Sandra Talavera  3 , Davy Jiolle  4 , Marc J B Vreysen  1 , Jérémy Bouyer  1 , Adly M M Abd-Alla  5
Affiliations

High sensitivity of one-step real-time reverse transcription quantitative PCR to detect low virus titers in large mosquito pools

Zhaoyang Tang et al. Parasit Vectors. .

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 control 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 mosquito 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 exceeding 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: Arbovirus; Chikungunya virus (CHIKV); Flavivirus; Pool size; RT-qPCR; Usutu virus (USUV); West Nile virus (WNV); Zika virus (ZIKV).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Amplification and standard curves of serial dilution of plasmid containing the sequence targeted by the primers and probes for the RT-qPCR for CHIKV, WNV, USUV and ZIKV detection. The correlation between the relative florescent unit (RFU) and the quantification cycle (Cq) on the left and between the virus log 10 copy number and the Cq on the right. Measurements were taken in triplicates. The regression equations and correlation coefficients (R) are given for each plot
Fig. 2
Fig. 2
Correlation between the log10 virus titer in the heads and bodies of infected mosquitoes
Fig. 3
Fig. 3
Screening of mosquito-borne virus in the head of infected mosquitoes. The horizontal red bar indicates the cut-off of Cq value 36. Negative control (NC) of USUV and ZIKV did not show a Cq value
Fig. 4
Fig. 4
Correlation between the log10 copy number per reaction and the quantification cycle threshold (Cq) to determine the mosquito-borne virus detection limit based on the cut-off value of the Cq of 36
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