In-Gel Loop Mediated Isothermal Amplification (gLAMP) Enables Coliphage Quantification within 30 Minutes

X. Huang
California Institute of Technology,
United States

Keywords: water quality, microbial pathogens, viral indicator, LAMP, hydrogel, smartphone

Summary:

Human pathogenic enteric viruses in environmental waters are the causative agents of many illness such as hepatitis, gastroenteritis, meningitis, fever, and conjunctivitis. It is generally accepted that monitoring for specific viral pathogen is not in the best interest for public health protection due to methodological limitations. Similar to using bacterial indicators, coliphages (viruses infected E. coli) are being explored as indicators to predict viral contamination of water. In this study, we developed an in-gel Loop Mediated Isothermal Amplification (gLAMP) which enables coliphage quantification within 30 min using standard laboratory devices. Viral particles (MS2) are immobilized with LAMP reagents within polyethylene glycol (PEG) hydrogel. The viral RNA is amplified through in situ isothermal LAMP reaction. The sample viral concentration is determined based on the number of fluorescent amplicon dots at the end of reaction with a fluorescence microscope or a smartphone. gLAMP results showed good correlation to traditional plaque assay and similar sensitivity to RT-qPCR (1 PFU/reaction). Moreover, gLAMP demonstrated significant higher tolerance against inhibitors present in highly contaminated toilet wastewater, in which RT-qPCR was completely inhibited. Considering the simplicity, sensitivity, and rapidity, gLAMP holds great potential for microbial water quality analysis in resource-limited settings.