RT‑qPCR Method for Four‑Hour Negative Reporting of E. coli in Water

Figure 1: Correlation between plate counts and PCR estimates for E. coli in spiked samples.

 

The new PCR method from ALS combines two E. coli markers with proven sensitivity and specificity, supported by extensive research. EC16S, first described by Huijsdens et al. (2002), offers broad inclusivity, while EC23S857 – validated under U.S. EPA Draft Method C (Sivaganesan et al., 2019) and widely adopted for regulatory applications – delivers superior sensitivity and quantitative performance.

Together, these markers provide complementary strengths, forming the basis of a dual-target strategy that helps reduce false negatives and build confidence in results. This approach is integrated into a PCR workflow aligned with international best-practice principles for molecular water testing, helping ensure reliable detection across diverse water matrices. 

Sensitivity and specificity 

PCR results demonstrated strong concordance with plate culture, with sensitivity and specificity both exceeding 95% (Table 2), meeting internationally recognised performance criteria for qPCR. 

The method achieved 100% negative predictive value, offering exceptional confidence in negative results.

Estimated counts based on PCR correlated closely with plate culture counts in spiked samples, with Pearson correlation coefficients >0.95 (Figure 1). This confirms that the method can not only detect E. coli contamination reliably but also provide meaningful estimates of colony forming units (CFU).

While PCR specificity was slightly below 100% (Table 2), culture-based methods detect only cells capable of growth under laboratory conditions. They may miss viable but non-culturable (VBNC) cells or trace contamination, both of which PCR can identify thanks to its lower detection limit.

Although PCR can theoretically amplify DNA from non-viable cells, the strong correlation between PCR and culture counts across spiked samples indicates that interference from extraneous DNA is negligible and does not compromise quantitation. These findings underscore PCR’s reliability as a rapid, sensitive alternative to culture for potable water monitoring.

Reliability in potable water

Performance was assessed exclusively in potable water samples, with trace amounts of effluent used only as a spike source to achieve target E. coli concentrations. Results confirmed consistent amplification efficiency and reproducibility, with replicate analyses showing <5% coefficient of variation (CV).

No significant matrix interference was observed.

* PCR detects VBNC cells and offers a lower detection limit, enabling identification of contamination that culture may miss. While PCR can theoretically amplify DNA from non-viable cells, performance metrics meet internationally recognised benchmarks for qPCR.

TP = true positive; FN = false negative; FP = false positive; TN = true negative

Potable water samples are collected in sterile bottles dosed with sodium thiosulfate, stored at 5 ± 3 °C, and processed within 26 hours. A 1 L sample is concentrated by vacuum filtration through a 0.45 µm membrane using aseptic technique.

The membrane is transferred to a bead tube for nucleic acid extraction. Each batch includes a process blank and positive to help verify workflow performance.

Nucleic acid purification and PCRTotal nucleic acid is extracted using the MagMAX™ CORE kit with bead-beating on an automated platform. Extracted eluate is used in a duplex RT-qPCR assay with an internal amplification control (IAC) included to monitor inhibition. Each PCR batch includes standards and a no-template control to help ensure quality. 

Result analysis and reporting

Copies per reaction are converted to estimated CFU per 100 mL using an experimentally verified conversion factor and the effective volume.

Results are reported as detected/not detected, alongside quantitative estimates. The method achieves sensitivity consistent with ADWG requirements (1 cfu/100 mL) as outlined by NHMRC and NMMRC (2011).

Compliance alignment

The method was developed to meet ADWG requirements for E. coli detection, helping ensure sensitivity consistent with potable water safety standards.

The workflow and verification process are structured to support NATA accreditation for potable water testing, reinforcing ALS’ commitment to accredited, high-quality analytical services.

Key compliance features include:

• Dual-marker design for enhanced inclusivity and sensitivity
• Rigorous QA measures including multi-level process controls and internal controls for robust confidence in results.
• NATA accreditation pending
• Quantitative reporting with strong correlation to cfu using experimentally verified conversion factors

Special requirements 

Method request 

When submitting samples, clearly indicate method code MM336 on the chain of custody (CoC). Include any priority turnaround or out-of-hours requirements to ensure correct processing.

Sample submission

Samples are to be received by the ALS Scoresby lab within 20 hours of collection.

For same-day reporting, samples must be received by the ALS Scoresby lab by 6 pm.

ALS can supply a 2L sterile bottle for sample collection.

Parallel analysis

This PCR method is performed in parallel with the standard plate culture method (MM698).

Clients should collect sufficient sample volume to allow both analyses to be completed without compromise.

Out-of-hours reporting

For urgent or time-sensitive results, ALS offers reporting outside standard business hours. To avoid delays, clients must nominate a dedicated contact person on the CoC who will be available to receive data and respond promptly. Out-of-hours rates apply.

Given the critical nature of this method, we recommend contacting ALS’ Client Services team by phone immediately after sample dispatch, so appropriate measures can be put in place and all arrangements confirmed.