Development of a novel reverse transcriptase polymerase chain reaction to determine the Gram reaction and viability of bacteria in clinical specimens.
To develop a novel RNA based assay to determine the Gram reaction and viability of bacteria in clinical specimens. Reverse transcriptase PCR (RT-PCR) targeting 16SrRNA region was optimized using Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 by using two novel sets of primers. Sixty clinical specimens consisting of 31 intraocular specimens (19 vitreous fluids and 12 aqueous humor), 11 peripheral blood specimens and 18 other clinical specimens were subjected to standard microbiological culture and RT-PCR to determine the Gram reaction and viability of bacteria. The amplified products were subjected to DNA sequencing to identify the bacterium. The sensitivity of RT-PCR was 0.4fg and the primers amplified bacterial cDNA. RT-PCR detected the presence of bacteria in 60 clinical specimens indicating the presence of viable bacteria. Concordant results were obtained with both primer sets. Seventy five bacterium comprising 52 single (69.3%) and 23 mixed bacteria (30.6%), both Gram positive and Gram negative were detected. These results correlated with the bacterial identity by PCR based DNA sequencing. RT-PCR is a reliable tool to identify the presence of viable bacteria and to precisely determine Gram reaction.