Quantification of infectious Human mastadenovirus in environmental matrices using PMAxx-qPCR

Abstract

Molecular methodologies providing data on viral concentration and infectivity have been successfully used in environmental virology, supporting quantitative risk assessment studies. The present study aimed to assess human mastadenovirus (HAdV) intact particles using a derivative of propidium monoazide associated with qPCR (PMAxx-qPCR) in aquatic matrices. Initially, different concentrations of PMAxx were evaluated to establish an optimal protocol for treating different naturally contaminated matrices, using 10 min incubation in the dark at 200 rpm at room temperature and 15 min of photoactivation in the PMA-Lite™ LED photolysis device. There was no significant reduction in the quantification of infectious HAdV with increasing concentration of PMAxx used (20 μM, 50 μM, and 100 μM), except for sewage samples. In this matrix, a reduction of 5.01 log of genomic copies (GC)/L was observed from the concentration of 50 μM and revealed 100% HAdV particles with damaged capsids. On the other hand, the mean reduction of 0.51 log in stool samples using the same concentration mentioned above demonstrated 83% of damaged particles eliminated in the stool. Following, 50 μM PMAxx-qPCR protocol revealed a log reduction of 0.91, 0.67, and 1.05 in other samples of raw sewage, brackish, and seawater where HAdV concentration reached 1.47 × 10 4 , 6.81 × 10 2 , and 2.33 × 10 2 GC/L, respectively. Fifty micrometers of PMAxx protocol helped screen intact viruses from different matrices, including sea and brackish water.

Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater

“…These findings demonstrate that the PMAxx dye at 100 μM reduces the detection of heat-inactivated and degraded viruses compared to live viruses, allowing differentiation of capsid integrity by qPCR. Other studies have demonstrated that 50 µM PMAxx is sufficient for detecting human AdV in aquatic matrices [ 60 ] and similarly for NoV on vegetables [ 58 ]. However, the concentration of PMAxx dye treatment required varies depending on the sample type and what compounds maybe present in the sample [ 30 ].…”

Seasonal prevalence of potentially infectious enteric viruses in surface waters below treated wastewater discharge

“…In this study, PMA dye pre-treatment was coupled with qPCR/RT-qPCR, and the application of v-qPCR/v-RT-qPCR eliminated the number of damaged viral particles from the results providing information only about potentially infectious intact viruses. This analytical approach is highly useful for estimating the public health risks posed by the presence of potentially infectious viruses in the environment [35,36]. On the other hand, not all intact virions retain aninfectious capacity in the environment.…”

A Novel RAA Combined Test Strip Method Based on Dual Gene Targets for Pathogenic Vibrio vulnificus in Aquatic Products

“…According to the method of Pedrosa et al [17], the V. vulnificus solution was processed with PMAxx. Briefly, the bacterial solution was mixed with 40 µM PMAxx, incubated in the dark for 5 min and exposed for 15 min on an ice box 20 cm away from a 500 W halogen lamp.…”