Tehran University of Medical Sciences Journal of Arthropod-Borne Diseases 2322-1984 7 2 2013 12 15 154 163 EN Arulsamy Mary Manonmani Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India. Ashok Kumar Mathivanan Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India. Candassamy Sadanandane Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India. Purushothaman Jambulingam Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India. 2015 10 16 Background: Anopheles culicifacies, a major malarial vector has been recognized as a complex of five sibling species, A, B, C, D and E. These sibling species exhibit varied vectorial capacity, host specificity and susceptibility to malarial parasites/ insecticides. In this study, a PCR assay developed earlier for distinguishing the five individual species was validated on samples of An. culicifacies collected from various parts of India. Methods: The samples were initially screened using the rDNA-ITS2 region based primers which categorised the samples into either A/D group or B/C/E group. A proportion of samples belonging to each group were subjected to the mtDNA-COII PCR assay for identifying individual species. Results: Among the 615 samples analysed by rDNA-ITS2 PCR assay, 303 were found to belong to A/D group and 299 to B/C/E group while 13 turned negative. Among 163 samples belonging to A/D group, only one sample dis- played the profile characteristic of species A and among the 176 samples falling in the B/C/E group, 51 were identi- fied as species B, 14 as species C and 41 as species E respectively by the mtDNA-COII PCR assay. Samples exhib- iting products diagnostic of B/C/E, when subjected to PCR-RFLP assay identified 15 samples as species E. Conclusion: Validation of the mtDNA-COII PCR assay on large number of samples showed that this technique can- not be used universally to distinguish the 5 members of this species complex, as it has been designed based on mi- nor/single base differences observed in the COII region. https://jad.tums.ac.ir/index.php/jad/article/view/231 https://jad.tums.ac.ir/index.php/jad/article/download/231/208