One-Step Multiplex Polymerase Chain Reaction Assay for the Detection of Major Disease-Transmitting Mosquito Vectors in India.

Mosquitoes are important vectors that transmit viral, protozoan, and helminthic diseases across the world. Climate change and unplanned urbanization are accelerating the spread of these diseases. Controlling vector-borne diseases can be performed most effectively through vector control. Inadequate knowledge of vector bionomics is an impediment and can lead to inappropriate vector control efforts. However, the conventional methods of vector identification are based on morphological differences, demand a significant amount of time and specific skills, and are often misleading. An efficient and affordable solution is needed to quickly and accurately identify pooled samples from vast geographical territories. To ensure the correct identification of distorted or pooled samples in India, a set of definitive steps is required, including the construction of unique primers and the standardization of a one-step assay based on the second internal transcribed spacer gene of the ribosomal DNA. We have successfully developed and confirmed a highly efficient one-step multiplex reverse transcriptase polymerase chain reaction assay for the accurate identification of major mosquito vectors, especially in the cases of both the adult and larval forms of Anopheles sp., Aedes sp., and Culex sp. Hence, the specificity, universality, and uniqueness of these primers could serve as a critical tool for the rapid one-step and one-reaction identification of mosquitoes to control mosquito-borne disease outbreaks and public health emergencies.