Abstract
Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, ‘Strand Invasion Based Amplification’ (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7491-y) contains supplementary material, which is available to authorized users.
Highlights
Influenza A and B are among the most common causes of acute human respiratory illness and are associated with high morbidity and mortality rates in infants, the elderly, and immunocompromised individuals (Mallia and Johnston 2007; Simonsen et al 2000)
We demonstrated the ability of SIBA technology to detect RNA-pathogens, reverse transcription SIBA (RT-SIBA), for the rapid detection of influenza A and B with high analytical sensitivity and specificity
We demonstrate that RT-SIBA can be applied for the rapid detection of pathogens that have RNA, rather than DNA, as their genetic material, as is the case for the majority of viruses
Summary
Influenza A and B are among the most common causes of acute human respiratory illness and are associated with high morbidity and mortality rates in infants, the elderly, and immunocompromised individuals (Mallia and Johnston 2007; Simonsen et al 2000). Patients with influenza infection may be treated with antiviral drugs; these treatments are only effective when started within the first 2 days of the illness (Stiver 2003). Timely and accurate diagnosis of influenza plays an important role in targeting antiviral treatment and, has a significant role in the prevention of the misuse of antibiotics, since the symptoms of influenza are often similar to those of other respiratory illnesses caused by bacterial infections (Low 2008). Nucleic acid amplification tests (NAATs) are becoming the method of choice for routine diagnosis of influenza and other respiratory viruses within clinical laboratory settings. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays have been developed for the diagnosis of influenza and remain the most common platform used in NAAT. RT-PCR requires the use of heavy and sophisticated
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