Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

Mark J Hoser,Scott W Morrical,Hannu K Mansukoski,Kevin E Eboigbodin,Krzysztof Pyrc

doi:10.1371/journal.pone.0112656

Abstract

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

Highlights

The polymerase chain reaction (PCR) revolutionized the field of molecular diagnostics and biological research by allowing specific genes or nucleotide sequences present in sample material to be amplified to detectable levels within approximately 2 hours
We describe a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA), which is inherently resistant to nonspecific amplification and, sensitive for a single target molecule (Fig. 1)
The methods consists of a modified invasion oligonucleotide (IO) which is a substrate for the recombinase and two terminal primers, a forward and a reverse primer with lengths that are too short to be a substrate for the recombinase

Summary

Introduction

The polymerase chain reaction (PCR) revolutionized the field of molecular diagnostics and biological research by allowing specific genes or nucleotide sequences present in sample material to be amplified to detectable levels within approximately 2 hours. Non-specific amplification results either from binding of the primers to non-target nucleic acids or from direct copying of one primer onto another, a phenomenon known as primer dimers [4]. In contrast to PCR, the rate of artifactual amplification in isothermal technologies is not aligned with that of the target region throughout the thermal cycle. This can result in non-target-specific products being more rapidly amplified than the target and subsequently overwhelming the reaction, with a further reduction in sensitivity [5,6]

Methods

Results

Conclusion