Dirus Complex Species Identification PCR (DiCSIP) improves identification ofAnopheles diruscomplex from Greater Mekong Subregion
Authors: Saeung Manop , Pengon Jutharat , Pethrak Chatpong , Thaiudomsap Sarunya , Lhaosudto Suthat , Saeung Atiporn , Manguin Sylvie , Chareonviriyaphap Theeraphap , Jupatanakul Natapong 
Published: October 2023

AbstractBackgroundTheAnopheles diruscomplex plays a significant role as malaria vectors in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deployment of effective vector control measures. This becomes increasingly crucial as the GMS advances towards malaria elimination while facing with the emergence of zoonotic simian malaria transmission. However, the original molecular identification assay, Dirus Allele Specific PCR (AS-PCR) targeting the ITS2 region have pronounced non-specific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers for accurate identification of species within theAnopheles diruscomplex.Methodology/Principal findingsDespite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications, particularly forAnopheles dirus,Anopheles scanloni, andAnopheles nemophilous. Subsequently,in silicoanalyses pinpointed problematic primers with high GC content and multiple off-target binding sites. Through a series ofin silicoanalyses and laboratory validation, a new set of primers for Dirus Complex Species Identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity for accurate identification of five complex member species found in the GMS. Validation with laboratory and fieldAn. diruscomplex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentifyAn. dirusas other species when used with different thermocyclers.Conclusion/SignificanceThe DiCSIP assay offers a significant improvement inAn. diruscomplex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission, and prevent the re-introduction of malaria in the GMS.Author SummarySeveral species ofAnophelesmosquitoes belong to species complexes due to their indistinguishable morphology from closely related sibling species. However, members of the same species complex may exhibit varied vectorial capability, i.e. the ability to spread human pathogens, in this case malaria parasites, ranging from dominant vector to non-vector. The Dirus AS-PCR molecular assay to identify species within theAn. diruscomplex, a significant malaria vector species in the GMS, can produce inconsistent PCR results leading to misidentification. Furthermore, our analysis ofAn. dirusITS2 sequences in the NCBI database indicated misidentification between these sibling species suggesting the need for a new set of primers to improve reproducibility and sensitivity in identifying members of theAn. diruscomplex. This study presents thorough analyses of the existing primers that cause difficulties in correct amplification and a development of a new set of primers for Dirus Complex Species Identification PCR (DiCSIP) assay. The DiCSIP assay offers several advantages over the original Dirus AS-PCR. It provides a higher specificity, sensitivity and wider operational range allowing for the use of the DNAzol direct reagent to process mosquito samples without the need for DNA extraction, saving both time and cost in sample processing. Our study provides a valuable molecular tool for entomological surveys, which is crucial for effective vector control measures in the GMS.