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

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.

SUBMITTER: Huang X

PROVIDER: S-EPMC5990930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ImportanceA critical need exists in low-income and middle-income countries for low-cost, low-tech, yet highly reliable and scalable testing for SARS-CoV-2 virus that is robust against circulating variants.ObjectiveTo assess whether a smartphone-based assay is suitable for SARS-CoV-2 and influenza virus testing without requiring specialized equipment, accessory devices, or custom reagents.Design, setting, and participantsThis cohort study enrolled 2 subgroups of participants (symptomatic and asymptomatic) at Santa Barbara Cottage Hospital. The symptomatic group consisted of 20 recruited patients who tested positive for SARS-CoV-2 with symptoms; 30 asymptomatic patients were recruited from the same community, through negative admission screening tests for SARS-CoV-2. The smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) was first optimized for analysis of human saliva samples spiked with either SARS-CoV-2 or influenza A or B virus; these results then were compared with those obtained by side-by-side analysis of spiked samples using the Centers for Disease Control and Prevention (CDC) criterion-standard reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. Next, both assays were used to test for SARS-CoV-2 and influenza viruses present in blinded clinical saliva samples obtained from 50 hospitalized patients. Statistical analysis was performed from May to June 2021.ExposuresTesting for SARS-CoV-2 and influenza A and B viruses.Main outcomes and measuresSARS-CoV-2 and influenza infection status and quantitative viral load were determined.ResultsAmong the 50 eligible participants with no prior SARS-CoV-2 infection included in the study, 29 were men. The mean age was 57 years (range, 21 to 93 years). SmaRT-LAMP exhibited 100% concordance (50 of 50 patient samples) with the CDC criterion-standard diagnostic for SARS-CoV-2 sensitivity (20 of 20 positive and 30 of 30 negative) and for quantitative detection of viral load. This platform also met the CDC criterion standard for detection of clinically similar influenza A and B viruses in spiked saliva samples (n = 20), and in saliva samples from hospitalized patients (50 of 50 negative). The smartphone-based LAMP assay was rapid (25 minutes), sensitive (1000 copies/mL), low-cost (<$7/test), and scalable (96 samples/phone).Conclusions and relevanceIn this cohort study of saliva samples from patients, the smartphone-based LAMP assay detected SARS-CoV-2 infection and exhibited concordance with RT-qPCR tests. These findings suggest that this tool could be adapted in response to novel CoV-2 variants and other pathogens with pandemic potential including influenza and may be useful in settings with limited resources.

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

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