Application of chimeric RNA–DNA oligonucleotides to the detection of pathogenic microorganisms using surface plasmon resonance

Abstract

Chimeric oligonucleotides consisting of 21 bases of ribonucleic acid (RNA) with six bases of deoxyribonucleic acid (DNA) at the 3′-hydroxyl terminus (chimeric RNA–DNA primer) and the recombinant thermostable DNA polymerase derived from Thermus thermophilus (r Tth DNA polymerase) were utilized to efficiently amplify DNA fragments using the conventional polymerase chain reaction (PCR). The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle to form a double-stranded DNA in which one terminus was an RNA : DNA hybrid. Due to the ability of r Tth DNA polymerase to function as both the DNA polymerase and reverse transcriptase, a chimeric RNA–DNA primer was shown to serve as a primer in the conventional PCR procedure. We further demonstrate the advantages of using these PCR products to identify microorganisms using surface plasmon resonance (SPR) technology. This detection system was able to distinguish Shiga toxin-producing Escherichia coli O157 : H7 strains from other bacteria such as Salmonella typhimurium and S. enteritidis.