Smartphone-Based in-Gel Loop-Mediated Isothermal Amplification (gLAMP) System Enables Rapid Coliphage MS2 Quantification in Environmental Waters.

Xiao Huang,Xingyu Lin,Lijie Li,Xing Xie,Sunny Jiang,Katharina Urmann,Michael R Hoffmann

doi:10.1021/acs.est.8b00241

Abstract

Model 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.

Highlights

Human pathogenic enteric viruses found in domestic wastewater have been identified as important causative agents responsible for a wide range of infections in humans.[1]
Inspired by earlier work on in situ PCR,[27] immobilization of microbes in hydrogels,[28] PCR amplification in polyacrylamide gels,[29] and multiple-displacement amplification (MDA) amplification in polyethylene glycol (PEG) hydrogels,[16] we have developed a smartphone-based in-gel loop-mediated isothermal amplification (LAMP)
We found that the quenching of unincorporated amplification signal reporters (QUASR) primers did not reduce gel LAMP (gLAMP) amplification efficiency, a higher concentration of quencher primer (2× of the complementary probe primer) was needed to maintain a clean gel background at the end of the gLAMP reaction

Summary

Introduction

Human pathogenic enteric viruses (e.g., adenovirus, enterovirus, and norovirus) found in domestic wastewater have been identified as important causative agents responsible for a wide range of infections in humans.[1]. The dot sizes represented the largest size that the amplicons could develop within the given reaction time, while the size variability in a single gel may result from variable initial template conformation, the degree of template denaturation or from local inhomogeneities in the hydrogel structure (due to the free dangling ends, self-looping, or entanglements of macromers).

Results

Conclusion