Routine cell line characterization procedures are not adequate for cha
racterizing the cell lines of insect origin. Ribosomal RNA (rRNA) gene
sequences and their comparisons have been used successfully for delin
eating 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 fragme
nt of the cell lines yields a homoduplex with the larvae of known spec
ies, 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 belong
ing to two different species. (C) 1997 Academic Press.