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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

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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.


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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Figure 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).

Mentions: In this study, we developed a carry-over-contamination-free uRT-LAMP for the rapid detection of AIVs. To the best of our knowledge, uRT-LAMP assays have not yet been described for AIVs. Reference AIV strains (subtypes 1–16), two highly pathogenic AIV (HPAIV) subtypes H5N1 and H5N8 (Korean representatives), human influenza B virus (HIBV), and Newcastle disease virus (NDV) were used to evaluate the specificity of the assay (Table 1). Viral RNA was extracted using an RNeasy Mini kit (Qiagen, Germany) according to the manufacturer's instructions. Extracted nucleic acid was stored at -20℃ until further use. RT-LAMP assay for AIV detection was performed using AIV matrix gene-specific primer sets as previously described [5]. The amplification reaction was performed at 58℃ for 40 min, followed by heating at 80℃ for 5 min to terminate the reaction. After RT-LAMP, the positive results were visually confirmed by a colorimetric change from purple to sky blue in the reaction tubes without an additional detection process (Fig. 1). Because of the high mutation rate of AIV genes, it is difficult to design a multi-set of RT-LAMP primers to detect all subtypes of AIVs [1013]. Six primer sets (F3, B3, LF, LB, FIP, and BIP) for the proposed RT-LAMP that specifically target eight different regions highly conserved among all subtypes of AIVs were carefully designed by analyzing most of the AIV matrix gene deposited in the Influenza Sequence Database during 2012–2014 [15]. The RT-LAMP assay using these primers specifically detected all subtypes of AIVs tested, but not HIBV and NDV (Table 1), indicating the assay was highly accurate and specific for all subtypes of AIVs.


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

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

Figure 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).
Mentions: In this study, we developed a carry-over-contamination-free uRT-LAMP for the rapid detection of AIVs. To the best of our knowledge, uRT-LAMP assays have not yet been described for AIVs. Reference AIV strains (subtypes 1–16), two highly pathogenic AIV (HPAIV) subtypes H5N1 and H5N8 (Korean representatives), human influenza B virus (HIBV), and Newcastle disease virus (NDV) were used to evaluate the specificity of the assay (Table 1). Viral RNA was extracted using an RNeasy Mini kit (Qiagen, Germany) according to the manufacturer's instructions. Extracted nucleic acid was stored at -20℃ until further use. RT-LAMP assay for AIV detection was performed using AIV matrix gene-specific primer sets as previously described [5]. The amplification reaction was performed at 58℃ for 40 min, followed by heating at 80℃ for 5 min to terminate the reaction. After RT-LAMP, the positive results were visually confirmed by a colorimetric change from purple to sky blue in the reaction tubes without an additional detection process (Fig. 1). Because of the high mutation rate of AIV genes, it is difficult to design a multi-set of RT-LAMP primers to detect all subtypes of AIVs [1013]. Six primer sets (F3, B3, LF, LB, FIP, and BIP) for the proposed RT-LAMP that specifically target eight different regions highly conserved among all subtypes of AIVs were carefully designed by analyzing most of the AIV matrix gene deposited in the Influenza Sequence Database during 2012–2014 [15]. The RT-LAMP assay using these primers specifically detected all subtypes of AIVs tested, but not HIBV and NDV (Table 1), indicating the assay was highly accurate and specific for all subtypes of AIVs.

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.