Details

Autor(en) / Beteiligte

• Ho, Johannes
• Seidel, Michael
• Niessner, Reinhard
• Eggers, Jutta
• Tiehm, Andreas

Titel

Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation

Ist Teil von

• Water research (Oxford), 2016-10, Vol.103, p.141-148

Ort / Verlag

England: Elsevier Ltd

Erscheinungsjahr

2016

Quelle

Elsevier ScienceDirect Journals

Beschreibungen/Notizen

• 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. [Display omitted] •PCR signal reduction after UV treatment increases with increasing amplicon size.•Genome size qPCR shows similar results to the plaque assay.•PMA has no effect on UV treated phiX174 bacteriophages.•Log reduction of qPCR is correlated to [applied UV dose x amplicon size].•The normalized PCR effect constant is suitable for calculating plaque assay results.