Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

Abstract

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

Keywords: avian influenza virus; contamination; loop-mediated isothermal amplification; uracil-DNA glycosylase.

Fig. 1. Prevention of false-positive reaction by carry-over contamination of pre-amplified deoxyuridine triphosphate (dUTP)-incorporated reverse transcription loop-mediated isothermal amplification (RT-LAMP) products. In the uracil-DNA glycosylase (UNG)-untreated RT-LAMP, amplification-positive color change from negative purple to positive sky blue and LAMP-specific ladder-like electrophoresis pattern were observed in reaction tubes 1–6, but in the UNG-treated UNG-RT-LAMP this change was only observed in reaction tube 1–4 (B). Lane NC, negative control; Lane M, 100-bp DNA marker; Lane 1–7, results of RT-LAMP or UNG-RT-LAMP contaminated with 10-fold diluted pre-amplified dUTP-incorporated RT-LAMP products (from 10 picograms to 10 attograms per reaction).

Fig. 2. Detection limit of the RT-LAMP (A), UNG-RT-LAMP (B), and real-time reverse transcription polymerase chain reaction (C). Lane NC, negative control; Lane M, 100-bp DNA marker; Lane 1–8, results of amplification with 10-fold diluted viral RNA extracted from the Korean H5N8 HPAIV (A/broiler duck/Korea/Buan2/2014), with an initial viral titer of 10^8.0 EID₅₀/0.1 mL as the template.