Unexpected detection of DNA by nucleic acid sequence-based amplification technique

Abstract

Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective.