Development and evaluation of a rapid nucleic acid amplification method to detect influenza A and B viruses in human respiratory specimens

Isothermal nucleic acid amplification methods can potentially shorten the amount of time required to diagnose influenza. We developed and evaluated a novel isothermal nucleic acid amplification method, RT-SIBA to rapidly detect and differentiate between influenza A and B viruses in a single reaction tube. The performance of the RT-SIBA Influenza assay was compared with two established RT-PCR methods. The sensitivities of the RT-SIBA, RealStar RT-PCR, and CDC RT-PCR assays for the detection of influenza A and B viruses in the clinical specimens were 98.8%, 100%, and 89.3%, respectively. All three assays demonstrated a specificity of 100%. The average time to positive result was significantly shorter with the RT-SIBA Influenza assay (90 min). The method can be run using battery-operated, portable devices with a small footprint and therefore has potential applications in both laboratory and near-patient settings. (C) 2018 Elsevier Inc. All rights reserved.Peer reviewe

Detection of human rhinoviruses by reverse transcription strand invasion based amplification method (RT-SIBA)

Background: Rhinovirus (RV), a major cause of respiratory infection in humans, imposes an enormous economic burden due to the direct and indirect costs associated with the illness. Accurate and timely diagnosis is crucial for deciding the appropriate clinical approach and minimizing unnecessary prescription of antibiotics. Diagnosis of RV is extremely challenging due to genetic and serological variability among its numerous types and their similarity to enteroviruses. Objective: We sought to develop a rapid nucleic acid test that can be used for the detection of Rhinovirus within both laboratory and near patient settings. Study design: We developed and evaluated a novel isothermal nucleic acid amplification method called Reverse Transcription Strand Invasion-Based Amplification (RT-SIBA) to rapidly detect Rhinovirus from clinical specimens. Result: The method, RT-SIBA, detected RV in clinical specimens with high analytical sensitivity (96%) and specificity (100%). The time to positive result was significantly shorter for the RV RT-SIBA assay than for a reference RV nucleic acid amplification method (RT-qPCR). Conclusion: The rapid detection time of the RV SIBA assay, as well as its compatibility with portable instruments, will facilitate prompt diagnosis of infection and thereby improve patient care.Peer reviewe

Strand Invasion Based Amplification (SIBA®): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte

<div><p>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 <i>Salmonella.</i></p></div

SIBA primers are unable to amplify target DNA independently of the invasion oligonucleotide (IO).

<p>(A) Real-time monitoring of amplification using SYBR Green I, (B) melting curve analysis ((-dF (fluorescence)/dT (temperature) versus temperature), and (C) non-denaturing electrophoresis of the corresponding reaction products. Lane 1, BioRad EZ Load 20 bp Molecular Ruler (20–1000 bp); lane 2, primers + IO + template (10⁷ copies); lane 3, primers + IO + template (10⁵ copies); lane 4, primers + IO + water; lane 5, primers + template (10⁷ copies); lane 6, primers + template (10⁵ copies); lane 7, IO + template (10⁷ copies); lane 8, primers + non-homologous IO + template (10⁷ copies); lane 9, primers + IO + non-homologous template (10⁷ copies); lane 10, 200 nM primers in the absence of SIBA reaction reagents; lane 11, 200 nM IO in the absence of SIBA reaction reagents; lane 12, 200 nM primers and 200 nM IO in the absence of SIBA reagents. Lanes 10–12 served as controls for monitoring the presence of oligonucleotides in the reaction products. These were diluted in TBE buffer and run alongside the SIBA reaction products. SB-F21 and SB-R21 are the forward and reverse primers, respectively. The IO used was SB-IO. The homologous target DNA used was SB-template. nhom  =  non-homologous to the target template (SB nhom template) or non-homologous IO (SB nhom IO).</p

Sensitivity of SIBA extension to point mutations.

<p>The SIBA reaction was performed with either a fully homologous target template (SB-template) or with templates containing 1–4 base point mutation(s). The results are expressed as the delay in the threshold detection time (dt), i.e., the average Δdt =  average dt of a template containing point mutation(s) minus the average dt of the fully homologous target template (SB-template). ND denotes no detectable amplification of a template. SB-F21 and SB-R21 were the upstream and downstream primers, respectively. The invasion oligonucleotide (IO) used was SB-IO.</p><p>Sensitivity of SIBA extension to point mutations.</p

Artifactual amplification is abolished by using an invasion oligonucleotide (IO) with a 2′-O-methyl RNA modification.

<p>(A) Configuration of the IO molecules used. (B) Real-time monitoring of SIBA reactions with SYBR Green I using different IOs: (i) IO with a 2′-O-methyl RNA modification and fully homologous to the target duplex, SB-IO; (ii) IO fully homologous to the target duplex, where the 2′-O-methyl RNA modification was replaced with natural DNA nucleotides, SB-IO DNA; (iii) IO with a 2′-O-methyl RNA modification that is not homologous to the target duplex, SB-IO DIFF-METH; and (iv) IO with the 2′-O-methyl RNA modification deleted, SB-IO NON-METH. SB-F21 and SB-R21 were the forward and reverse primers, respectively. The reactions were either performed using 106 target template molecules (SB-template) or in the absence of template.</p