J Vet Sci.  2016 Sep;17(3):421-425. 10.4142/jvs.2016.17.3.421.

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

• Kim EM
• Jeon HS
• Kim JJ
• Shin YK
• Lee YJ
• Yeo SG
• Park CK

• KMID: 2413144
• DOI: http://doi.org/10.4142/jvs.2016.17.3.421

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.

Keyword

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

MeSH Terms

Animals
Birds
DNA Contamination
Influenza A virus/*isolation & purification
Influenza in Birds/*diagnosis/virology
Nucleic Acid Amplification Techniques/methods/*veterinary
Reverse Transcription
Sensitivity and Specificity
Uracil-DNA Glycosidase/*chemistry
Uracil-DNA Glycosidase

Figure

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  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).

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  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 108.0 EID50/0.1 mL as the template.

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