Microdensitometric scanning procedure for quantitative assessment of hybridization of rRNA targeting probes in...

In order to develop an objective criteria of grading of positivity of hybridization signals of gene probes targeting rRNA, a microdensitometric scanning procedure was standardised. Ribosomal RNA was extracted from the bacilli harvested from biopsies of leprosy cases across the spectrum and blotted on nitro-cellulose membranes. M. leprae specific rRNA targeting oligonucleotide probes were end-labelled and hybridization was done by the technique standardised and published earlier. The autoradiographs were developed and microdensitometric scanning was done by altering different parameters. Positivity was graded in 5 grades and compared with visual positivity. Microdensitometric scanning procedure and 5 grade system appear to be useful and reproducible. Signals in paucibacillary specimens were in 2+ to 3+ grading range whereas those in multibacillary specimens varied in grades from 2+ to 5+. This approach appears to have potential usefulness for assessing the bacillary load (possibly viable) in the clinical specimens from leprosy cases.