Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing

Abstract

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Microbial Indicators of Fecal Pollution: Recent Progress and Challenges in Assessing Water Quality

“…Ultrafiltration techniques in particular have demonstrated reasonably efficient and consistent recovery for a variety of organisms, water types, and sample volumes, providing a natural complement to multitarget arrays, and increasingly appear to be the default concentration approach for many applications [154,[157][158][159][160][161]. Co-concentration of qPCR inhibitors during ultrafiltration is a concern, but effective inhibition mitigation has been demonstrated by further processing of the concentrate prior to analysis [160,[162][163][164].…”

HEV Occurrence in Waste and Drinking Water Treatment Plants

“…In addition, MgV was spiked and used as process control. HEV primary concentration was performed by a Dead End Hollow Fiber Ultrafiltration (DEUF) using single-use Rexeed-25A dialysis filters (Asahi Kasei Medical Co., Ltd.) with a molecular mass cutoff of 30 kDa, a surface area of 2.5 m 2 , a fiber inner diameter of 185 µm and a priming volume of 137 mL (Borgmästars et al, 2017). A peristaltic pump (model FH100, Thermo Fisher Scientific) was used for all experiments.…”

Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics

“…In validation, it is important to apply relevant samples concerning both sample matrix, cell type and sampling method. For pathogen testing of water, this may include applying clean water as well as water with different amounts of humic substances [44]. Recovery is the most important parameter in relation to sample treatment.…”