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. 2026 Jul 22;25:371. doi: 10.1186/s12936-026-06065-x
Box 1. Current mosquito sampling and genotyping strategies
Depending on location, a monitoring program that samples mosquitoes monthly can collect anywhere from a few mosquitoes per village per week in the dry season to several hundred or thousands per village per week at the peak of the rainy season(s) [18, 19]. Common adult sampling techniques include pyrethroid spray catches (PSCs), indoor aspirations, and/or the use of traps [20]. Common species are usually identified taxonomically, and target species are stored either in 75–80% ethanol or dried with silica gel ahead of molecular analyses used to characterize morphologically identical members of the An. gambiae or An. funestus species complexes. Once in the laboratory, DNA is extracted from samples using either cheap extractions based on either buffer digestion and ethanol precipitation with differential centrifugation, or a more expensive affinity column-based extraction. The former approach is used for processing large volumes at lower cost using simple PCR with fragment visualization on agarose gels [21, 22], while the latter approach is used when downstream analyses require purer DNA, such as multiplexed or nested PCR, qPCR, and sequencing-based analyses, or when the target DNA is rare compared to mosquito genomic DNA (e.g., mosquito pathogen detection) [23, 24]
Most African institutions leading and contributing to mosquito surveillance programs, and/or participating in the baseline studies informing gene drive mosquito release programs, have existing workflows for individual mosquito sample processing based around simple PCR-based approaches for species diagnostics, and sometimes bloodmeal analyses, whilst Plasmodium detection can be performed either by nested PCR, Taqman qPCR, or ELISA. Hence, in the context of gene drive field trial monitoring, assays for the genetic construct should fit into existing workstreams based on PCR and make use of existing equipment that does not require novel or complex supply chains.
For simple genetic construct detection assays, PCR can provide a presence/absence signal if primers are designed to bind to sequences only found on the transgene. Through adding a primer specific to the wild-type sequence, the resulting three-primer multiplexed PCR assays can be used to discriminate between heterozygotes and homozygotes [25]. Care must be taken to design primers that consider local sequence polymorphisms, as mutations in priming regions are the most common issue affecting the specificity of PCR-based approaches used on field-collected samples [22, 26]. When mosquito samples are abundant, or for large-scale monitoring, genetic construct detection assays that interrogate pools of mosquitoes may be cost-effective. However, detecting rare genetically modified mosquitoes in large pools may exceed the sensitivity of PCR, which cannot reliably detect targets below 1:100. For this, other molecular assays such as loop-mediated isothermal amplification (LAMP) may be better suited. Like PCR, LAMP amplifies specific DNA regions, but operates at a constant 60–65 °C temperature, eliminating the need for a thermocycler and enabling faster turnaround, as LAMP products can be detected using fluorescence with intercalating dyes, or colorimetric methods [27, 28]. LAMP requires strict standard operating procedures (SOPs), dedicated instruments, and workspace specifications, as its high sensitivity makes it prone to contamination. While LAMP assays can be multiplexed, it is more challenging than with PCR since each target requires at least four primers [29, 30]
As molecular technologies, such as DNA biosensors and other rapid nucleic acid detection platforms [31, 32], advance and institutions in malaria-endemic countries gain better access to funding and equipment, new cost-effective genotyping options for monitoring genetic constructs may become available; however, high-tech solutions are not always the most appropriate, as they require dedicated facilities, established supply chains and servicing, and trained personnel