Characterization of Insect Cell Lines: Heteroduplex Analysis Employing a Mitochondrial 16S Ribosomal RNA Gene Fragment

Routine cell line characterization procedures are not adequate for characterizing the cell lines of insect origin. Ribosomal RNA (rRNA) gene sequences and their comparisons have been used successfully for delineating species and phylogenetic analysis. Using similar principles, we have standardized a protocol for the confirmation of species identity of insect cell lines. The procedure includes PCR amplification of the mitochondrial 16S rRNA gene fragment from the cell line and larvae of known insect species and heteroduplex analysis to detect the sequence variation in the PCR-amplified rRNA gene fragments. If the PCR fragment of the cell lines yields a homoduplex with the larvae of known species, then the cell line is conspecific with the larvae. If the larvae and cell line are of two different species, then the analysis exhibits multiple bands of heteroduplexes. The technique also allows detection of cross-contamination of culture having two insect cell lines belonging to two different species.