Interlaboratory accuracy and precision among results of three sewage-associated marker genes in urban environmental estuarine waters and freshwater streams

Abstract

The application of quantitative polymerase chain reaction (qPCR) based microbial source tracking (MST) marker genes are increasingly being used to identify contaminating sources and inform management decisions. In this study, we assessed interlaboratory agreement on duplicate environmental water samples collected from estuarine and freshwater locations, by comparing results of qPCR based testing for Bacteroides HF183, crAssphage CPQ_056, and pepper mild mottle virus (PMMoV). The overall agreements (co-detection and non-co-detection) between CSIRO Land and Water (CLW) laboratory and Sydney Water (SW) laboratory for the HF183, crAssphage CPQ_056 and PMMoV marker genes for duplicate water samples were 74, 75 and 74%, respectively. Cohene's kappa (k) revealed fair to moderate agreements and acceptable relative percent difference (RPD) values of <15% for duplicate samples. The pooled mean abundances of HF183, CPQ_056, and PMMoV in measurable samples at the CLW laboratory were 5.19 ± 0.93, 5.12 ± 0.82, and 4.42 ± 0.65 log₁₀ copies/L, respectively. However, the pooled mean abundances were significantly lower at the SW laboratory, HF183 (4.58 ± 0.84 log₁₀ copies/L), crAssphage CPQ_056 (4.20 ± 0.63 log₁₀ copies/L), and PMMoV (3.89 ± 0.41 log₁₀ copies/L). At individual sample level, most of the paired samples had <1 log₁₀ difference. Significant positive Spearman rank correlations were obtained between two laboratories for the HF183 (R_s = 0.65; p < 0.05), CPQ_056 (R_s = 0.79; p < 0.05), and PMMoV (R_s = 0.54; p < 0.05) marker genes. Several factors such as standards, qPCR platforms, PCR inhibitors, nucleic acid extraction efficiency and low levels of targets in some samples may have contributed to the observed discrepancies. Results presented in this study highlight the importance of standardized protocol, laboratory equipment (such as digital PCR), sample processing strategies and appropriate quality controls that may need implementation to further improve accuracy and precision of results between laboratories.

Keywords: CrAssphage; HF183; Interlaboratory validation; Method comparison; Microbial source tracking; PMMoV; Sewage contamination.