Evaluation of real time PCR for quantitative detection of enterococci in coastal water

Abstract

Introduction: In the past, various methods have been used for the quantification of faecal indicators in water samples. Currently, membrane filtration is widely used for the enumeration of faecal indicators in water samples. However, the turnaround time for membrane filtration is slow, particularly under emergency conditions. Hence, a rapid, reliable, high throughput method is in demand. Real time PCR, also known as the quantitative polymerase chain reaction (qPCR), has been suggested as a tool to replace culture-based methods for water monitoring.

Different faecal indicators such as Total coliforms (TC), Faecal coliforms (FC) and Faecal streptococci (FS) have been used to monitor the water quality. In Hong Kong, E. coli has been used to indicate faecal pollution level since 1987. Recently, Enterococci have been reported as a more stable and reliable faecal indicator in both marine and fresh water. Information on the correlation between E. coli and Enterococci in local context are few.

Therefore, the aim of this study was to evaluate real time PCR for the quantitative detection of Enterococci in coastal water. The relationship between E. coli and Enterococci obtained from water samples collected from the Shing Mun River was also investigated.

Results: In the present studies, a total of 81 water samples were collected from three locations (Tai Wai, Fo Tan and Tai Shui Hang) along the Shing Mun River from September 2014 to February 2015. Apart from the densities of E. coli determined using a culture-based method, the densities of Enterococci in the 81 water samples were simultaneously determined using culture-based and qPCR methods, respectively. The median value of E. coli in Tai Wai, Fo Tan and Tai Shui Hang were 3066, 2200 and 115, respectively. The median value of Enterococci in Tai Wai, Fo Tan and Tai Shui Hang were 690, 46 and 9 CFU/100 ml, respectively. The amounts of Enterococci in the Tai Wai samples were significantly higher than in the Fo Tan and Tai Shui Hang samples, respectively (p<0.001). Moreover, the results showed the overall correlation coefficient between these two faecal indicators was 0.64 (N=81). The results also showed that the amounts of Enterococci measured using qPCR were significantly higher than those measured using culture-based methods (p<0.001). Both methods showed that the amount of Enterococci in Tai Wai samples was significantly higher than that in the Fo Tan and Tai Shui Hang samples, respectively (p<0.005). The overall regression analysis in log-scale showed that these two methods had significant positive linear relationships, with a correlation coefficient (r) of 0.74. The turnaround time for qPCR method is shorter than that for culture-based methods.

Conclusions: The present study showed that real-time PCR has potential to complement traditional culture methods for assessing faecal pollution in coastal waters. However, this molecular-based method has some limitations, such as high start-up cost and an unpredictable qPCR inhibition. Further studies are needed to examine the relationship between the qPCR results and public health risks.