Advanced

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

Borgmästars, Emmy; Jazi, Mehrdad Mousavi; Persson, Sofia; Jansson, Linda LU ; Rådström, Peter LU ; Simonsson, Magnus; Hedman, Johannes LU and Eriksson, Ronnie (2017) In Food and Environmental Virology 9(4). p.395-405
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... (More)

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 Cq 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.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Hepatitis A virus, Norovirus, RT-qPCR, RT-qPCR inhibition, Surface water, Ultrafiltration
in
Food and Environmental Virology
volume
9
issue
4
pages
11 pages
publisher
Springer New York
external identifiers
  • scopus:85017444599
  • wos:000414377300003
ISSN
1867-0334
DOI
10.1007/s12560-017-9295-3
language
English
LU publication?
yes
id
3637cd15-9a46-43bb-936e-10add8e67b3b
date added to LUP
2017-11-22 08:34:58
date last changed
2018-01-16 13:26:12
@article{3637cd15-9a46-43bb-936e-10add8e67b3b,
  abstract     = {<p>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<sub>q</sub> 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.</p>},
  author       = {Borgmästars, Emmy and Jazi, Mehrdad Mousavi and Persson, Sofia and Jansson, Linda and Rådström, Peter and Simonsson, Magnus and Hedman, Johannes and Eriksson, Ronnie},
  issn         = {1867-0334},
  keyword      = {Hepatitis A virus,Norovirus,RT-qPCR,RT-qPCR inhibition,Surface water,Ultrafiltration},
  language     = {eng},
  month        = {12},
  number       = {4},
  pages        = {395--405},
  publisher    = {Springer New York},
  series       = {Food and Environmental Virology},
  title        = {Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing},
  url          = {http://dx.doi.org/10.1007/s12560-017-9295-3},
  volume       = {9},
  year         = {2017},
}