Validation and in-field testing of a new on-site qPCR system for quantification of Legionella pneumophila according to ISO/TS 12869:2012 in HVAC cooling towers.

Legionella pneumophila, found in engineered water systems such as HVAC cooling towers, poses a significant public health risk. Culture, though routinely used to quantify L. pneumophila, has several disadvantages including long turnaround time, low sensitivity, and inter-laboratory variability. In this study, we validated the performance of an on-site quantitative polymerase chain reaction (qPCR) detection system for L. pneumophila in accordance with International Standards Organization Technical Specification 12869:2012. We evaluated specificity, limit of detection and quantification, and calibration curve linearity. Additionally, we evaluated whole system recovery and robustness using samples taken from taps and evaporative cooling towers. We then compared the system's performance against laboratory culture and laboratory qPCR across 53 cooling towers in a 12-week in-field study. We found that concordance between on-site qPCR and culture was both laboratory- and site/sample-dependent. Comparison of laboratory qPCR with on-site qPCR revealed that laboratory results were highly variable and showed little concordance. Some discordance may be explained by time delay between sample collection and testing ('shipping effect') which may lead to inaccurate reporting. Overall, our study highlights the value of on-site qPCR detection of L. pneumophila, demonstrates that laboratories are prone to misreporting results due to shipping effects, and reveals significant discordance between laboratory qPCR and culture.

Evaluation and validation of reference gene stability during Marek's disease virus (MDV) infection.

Quantitative RT-PCR (qRT-PCR) is widely used in the study of relative gene expression in general, and has been used in the field of Marek's disease (MD) research to measure transcriptional responses to infection and/or vaccination. Studies in the past have either employed cellular ß-actin (BACT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference genes, although the stability of their expression in the context of Marek's disease virus (MDV) infection has never been investigated. In the present study, we compared the stability of five reference genes (BACT, 28S RNA, 18S RNA, GAPDH, Peptidyl-prolyl-isomerase B [PPIB], a.k.a. cyclophilin B) as standard internal controls in chicken embryo fibroblast (CEFs) cultures infected with either MD vaccine or oncogenic MDV1 viruses. We further extend these analyses to reference gene stability in spleen lymphomas induced by infection of commercial broiler chickens with a very virulent plus MDV1 (vv+ TK-2a virus). Two excel based algorithms, (Bestkeeper and Normfinder) were employed to compare reference gene stability. Bestkeeper and Normfinder analysis of reference gene stability in virus- and mock-infected cells, showed that 28S RNA and PPIB displayed higher stability in CEF infections with either oncogenic or vaccine viruses. In addition, both Bestkeeper and Normfinder determined 28S RNA and PPIB to be the most stably-expressed reference genes in vivo in vv+ TK-2a-induced spleen lymphomas. Furthermore, Bestkeeper and Normfinder analyses both determined BACT to be the least stable reference gene during MDV infection of CEF with oncogenic viruses, vaccine viruses, as well as in vv+ TK-2a-induced spleen lymphomas.