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Smartphone-Based in-Gel Loop-Mediated Isothermal Amplification (gLAMP) System Enables Rapid Coliphage MS2 Quantification in Environmental Waters
journal contribution
posted on 2018-05-08, 00:00 authored by Xiao Huang, Xingyu Lin, Katharina Urmann, Lijie Li, Xing Xie, Sunny Jiang, Michael R. HoffmannModel
coliphages (e.g., ΦX174, MS2, and PRD1) have been widely
used as surrogates to study the fate and transport of pathogenic
viruses in the environment and during wastewater treatment. Two
groups of coliphages (F-specific and somatic) are being explored as
indicators of viral fecal pollution in ambient water. However, the
detection and quantification of coliphages still largely rely on time-consuming
culture-based plaque assays. In this study, we developed an in-gel
loop-mediated isothermal amplification (gLAMP) system enabling coliphage
MS2 quantification within 30 min using standard laboratory devices.
Viral particles (MS2) were immobilized with LAMP reagents in polyethylene
glycol hydrogel, and then viral RNAs were amplified through a LAMP
reaction. Due to the restriction effect of the hydrogel matrix, one
viral particle would only produce one amplicon dot. Therefore, the
sample virus concentrations can be determined based on the number
of fluorescent amplicon dots using a smartphone for imaging. The method
was validated by using artificially spiked and naturally contaminated
water samples. gLAMP results were shown to correlate well with plaque
assay counts (R2 = 0.984, p < 0.05) and achieved similar sensitivity to quantitative reverse-transcription
polymerase chain reaction (RT-qPCR; 1 plaque-forming unit per reaction).
Moreover, gLAMP demonstrated a high level of tolerance against inhibitors
naturally present in wastewater, in which RT-qPCR was completely inhibited.
Besides MS2, gLAMP can also be used for the quantification of other
microbial targets (e.g., Escherichia coli and Salmonella). Considering its simplicity, sensitivity, rapidity,
and versatility, gLAMP holds great potential for microbial water-quality
analysis, especially in resource-limited settings.
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Keywords
amplicon dotculture-based plaque assayswater-quality analysisPRDpathogenic virusesEnvironmental Waters Model coliphagesin-gel loop-mediated. TwoR 2gLAMP resultsSystem Enables Rapid Coliphage MS 2 Quantificationambient waterRNAsample virus concentrationsrestriction effectplaque assay countsresource-limited settingslaboratory devicesLAMP reagents1 plaque-forming unitamplicon dotswastewater treatment . Two groupsLAMP reactionViral particlespolyethylene glycol hydrogelSmartphone-Based in-Gel Loop-Mediated Isothermal Amplification30 minreverse-transcription polymerase chain reactionhydrogel matrixwater samplescoliphage MS 2 quantification
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