title: Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation.
abstract: Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection.
authors.affiliation: DVGW-Technologiezentrum Wasser (TZW), Department Microbiology and Molecular Biology, Karlsruher Str. 84, Karlsruhe, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Ho
authors.affiliation: Chair of Analytical Chemistry and Institute of Hydrochemistry, Technical University of Munich, Marchioninistr. 17, 81377 Munich, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Seidel
authors.affiliation: Chair of Analytical Chemistry and Institute of Hydrochemistry, Technical University of Munich, Marchioninistr. 17, 81377 Munich, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Niessner
authors.affiliation: DVGW-Technologiezentrum Wasser (TZW), Department Technology and Economics, Karlsruher Str. 84, Karlsruhe, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Eggers
authors.affiliation: DVGW-Technologiezentrum Wasser (TZW), Department Microbiology and Molecular Biology, Karlsruher Str. 84, Karlsruhe, Germany. Electronic address: andreas.tiehm@tzw.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tiehm
mesh_terms: D001386:Azides; D010584:Bacteriophage phi X 174; D001435:Bacteriophages; D004203:Disinfection; D007364:Intercalating Agents; D011419:Propidium; D014466:Ultraviolet Rays
chemical_list: D001386:Azides; D007364:Intercalating Agents; D011419:Propidium
keywords: Amplicon size; LA-qPCR; Low pressure UV; PCR primers; Plaque assay; Propidium monoazide (PMA)