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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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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Figure 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.

Mentions: Next, to determine the analytical sensitivity of RT-LAMP and evaluate the effects of dUTP incorporation and UNG treatment on RT-LAMP, both RT-LAMP and uRT-LAMP assays were performed with 10-fold serially diluted viral RNA extracted from the Korean H5N8 HPAIV (A/broiler duck/Korea/Buan2/2014) at an initial viral titer of 108 median embryo infection dose (EID50)/0.1 mL. The results of RT-LAMP and uRT-LAMP were compared with those of previously reported RRT-PCR using the same viral RNA diluent as the template. The RRT-PCR for the detection of all AIV subtypes was performed using a one-step PrimeScript RT-PCR kit (Takara Bio, Japan) in a real-time PCR instrument (Applied Biosystems, USA) as previously described [6]. The results showed that the detection limit of RT-LAMP was a 107 dilution of the original viral RNA concentration, which is the same as that observed for RRT-PCR. The detection limit of uRT-LAMP (106 dilution) was 10-fold lower than that of RT-LAMP and RRT-PCR (Fig. 2). It is believed that this occurred owing to supplementation with UNG or substitution of dUTP in the reaction mixture as reported by Hsieh et al. [3]. Although the analytical sensitivity of uRT-LAMP showed a slight reduction in response to UNG treatment, it was identified as a valuable screening tool for AIVs because it can effectively prevent potential carry-over contamination. The reduction in detection limit should be improved through further studies.


Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037312&req=5

Figure 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.
Mentions: Next, to determine the analytical sensitivity of RT-LAMP and evaluate the effects of dUTP incorporation and UNG treatment on RT-LAMP, both RT-LAMP and uRT-LAMP assays were performed with 10-fold serially diluted viral RNA extracted from the Korean H5N8 HPAIV (A/broiler duck/Korea/Buan2/2014) at an initial viral titer of 108 median embryo infection dose (EID50)/0.1 mL. The results of RT-LAMP and uRT-LAMP were compared with those of previously reported RRT-PCR using the same viral RNA diluent as the template. The RRT-PCR for the detection of all AIV subtypes was performed using a one-step PrimeScript RT-PCR kit (Takara Bio, Japan) in a real-time PCR instrument (Applied Biosystems, USA) as previously described [6]. The results showed that the detection limit of RT-LAMP was a 107 dilution of the original viral RNA concentration, which is the same as that observed for RRT-PCR. The detection limit of uRT-LAMP (106 dilution) was 10-fold lower than that of RT-LAMP and RRT-PCR (Fig. 2). It is believed that this occurred owing to supplementation with UNG or substitution of dUTP in the reaction mixture as reported by Hsieh et al. [3]. Although the analytical sensitivity of uRT-LAMP showed a slight reduction in response to UNG treatment, it was identified as a valuable screening tool for AIVs because it can effectively prevent potential carry-over contamination. The reduction in detection limit should be improved through further studies.

View Article: PubMed Central - PubMed

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.

No MeSH data available.