Abstract
BackgroundThe aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus.ResultsThe detection limit of LAMP using in vitro RNA transcripts was 3.6×10 copies·μL-1, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples.ConclusionsThe LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test.
Highlights
The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus
Human astroviruses (HAstV) have been shown in several epidemiologic outpatient studies to be an important cause of viral gastroenteritis in infants and young children
Naked-eye observation of LAMP products using hydroxynaphthol blue dye (HNB) The LAMP reaction was incubated in a conventional water bath at 65°C for 90 min, followed by heating at 80°C for 2 min to terminate the reaction
Summary
The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. Human astroviruses (HAstV) have been shown in several epidemiologic outpatient studies to be an important cause of viral gastroenteritis in infants and young children. The incidence of astrovirus infections has been estimated at between 5% and 10% in children with gastroenteritis [2]. Astroviruses are among the most resistant viruses; they show resistance against different physical and chemical agents, they are able to maintain their infectivity at 60°C for 10 min, and they are resistant to treatment at pH 3 [4]. The number of reports on astrovirus detection is relatively low
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