Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage.

Abstract:

AIMS:Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. METHODS AND RESULTS:Noroviruses in environmental water samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3'-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. CONCLUSIONS:Sewage was found to contain up to 1.8 x 10(6) norovirus cDNA copies per 100 ml and effluent contained up to 1.7 x 10(6) copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. SIGNIFICANCE AND IMPACT OF THE STUDY:The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods.

journal_name

Lett Appl Microbiol

authors

Laverick MA,Wyn-Jones AP,Carter MJ

doi

10.1111/j.1472-765X.2004.01534.x

subject

Has Abstract

pub_date

2004-01-01 00:00:00

pages

127-36

issue

2

eissn

0266-8254

issn

1472-765X

pii

LAM1534

journal_volume

39

pub_type

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