A multiplex qPCR followed by high resolution melting analysis for the detection of blood-feeding sources in Culex sp. (Diptera: Culicidae) mosquitoes

Culex species, such as Culex quinquefasciatus and Culex nigripalpus
display a range of feeding habits and act as vectors for pathogens that
can cause diseases in both humans and animals. Understanding their feeding
habits is pivotal for enhancing disease prevention strategies. The present
study introduces the application of two multiplex real-time PCR (qPCR)
followed by High-Resolution Melting (HRM) as a cost-effective and
time-efficient alternative. This investigation involved the development of
two multiplex qPCR-HRM: assay 1 detects human, dog, and chicken, while
assay 2 detects cat, cattle, and horse in Culex sp. engorged female
mosquitoes. The qPCR-HRM reactions showed a detection limit of one copy of
genomic DNA when performed as single and multiplex qPCR-HRM. The reaction
efficiencies were 97.96% for human, 100.60% for dog, 99.03% for chicken,
99.92% for feline, 99.18% for cattle, and 97.68% for horse. The qPCR-HRM
method, employing multiplex 1 and 2, was applied to field-collected
mosquitoes and demonstrated the ability to detect DNA from multiple blood
sources within a single sample. By analyzing both multiplexes, it was
possible to identify up to five distinct blood sources in Cx.
quinquefasciatus and Cx. nigripalpus, and up to two sources in Culex
coronatus. Sequencing corroborated the qPCR-HRM results, confirming the
presence of DNA from one to four different blood sources with 100%
accuracy. The development of these molecular methods may contribute for
identification of blood-feeding patterns in mosquitoes. It contributes to
studies on the dissemination and transmission of pathogens among various
animals and humans, thereby bridging the gap between epidemiology and
vector monitoring.