PCR methods for legal parameters

In determining the (hygienic) quality of drinking water, use is made of E. coli, intestinal enterococci and bacteria from the coli group (coliforms) as indicator organisms. Culture methods are the standard technique used to establish the presence of these indicators. However, there is a strong need for alternative methods that can reliably assess the water quality more rapidly. Within a series of projects in the Joint Research Programme of KWR and the water utilities (BTO), RT-PCR methods have been developed and validated for the detection of E. coli and intestinal enterococci. The present project will 1) investigate what the possibilities are for the development of an RT-PCR method to determine the presence of bacteria from the coli group or an alternative organism; 2) study the conditions under which RT-PCR and culture can produce different results; and 3) organise an international workshop to elucidate the activities taking place internationally that aim at the application and acceptance of molecular-biological analytical techniques within the water and drinking water sector.

Molecular PCR methods for rapid and reliable insight into the hygienic quality of drinking water

Rapid and reliable analytical methods for drinking water are essential for safeguarding the water’s hygienic quality. Drinking water laboratories can use these methods in situations in which there is a need for quick and clear information about the water’s safety. This is particularly important in the context of emergencies or of work on the distribution network. Rapid analyses limit possible health risks and can provide quick insight into the effect of necessary measures following emergencies. They thus limit the nuisance for consumers.

Introduction of molecular-biological analyses with (RT-)PCR ‘step by step’

RT-PCR methods for the rapid detection of E. coli and intestinal enterococci were developed and validated in earlier BTO projects.  These methods have since been accepted  as a rapid alternative to the legally prescribed reference method, and may be applied by drinking water laboratories. Besides these two parameters, the detection of bacteria from the coli group is also legally prescribed. For this, however, there is as yet no rapid method available.

The value added of the ‘bacteria from the coli group’ parameter is still the subject of much discussion. This is why the present project will first evaluate the extent to which this parameter is a valuable supplement in determining the hygienic reliability of drinking water, or whether there are alternatives that produce clearer information. Steps will then be taken towards developing a rapid method to determine the presence of the coli group bacteria or of an alternative.

RT-PCR and culture method differences

A second priority for the drinking water utilities concerns the fact that the current RT-PCR methods for E. coli and intestinal enterococci occasionally produce results that differ from those of the culture method. Previous research showed that UV and chlorine (dioxide) can induce disinfection processes in the treatment, and that inactivated indicator bacteria are still detectable with RT-PCR but no longer with culture (BTO 2019.005). This means that RT-PCR can be used to demonstrate the absence of indicator organisms. However, in demonstrating the presence of indicator organisms with RT-PCR, it is important that the information concerning the applied disinfection methods be taken into account during the interpretation of the analyses results.

But indicator bacteria are also more often detected with RT-PCR than with culture in situations where no disinfection is applied. This is not surprising, given the different nature of the culture and RT-PCR methods, but it is of great importance in order to properly interpret these differences. The objective of this part of the research is to look at which factors can cause differences between the culture and RT-PCR method for E. coli and intestinal enterococci, and to research whether the optimisation of the RT-PCR method can minimise the number of discrepancies between PCR and culture.

The Netherlands is a leader in the EU in the development and application of RT-PCR to safeguard drinking water quality, but work is also being done in other Member States on rapid alternatives for the indicator bacteria. These developments can be mutually reinforcing, both substantively and with regard to the approval process of alternative methods. For this reason, this project’s third priority is the organisation of a workshop during the ‘Health Related Water Microbiology’ congress ‘WaterMicro2025’.

Steps towards better and faster analytical methods to determine the hygienic quality of drinking water

The project outcomes will lead to an extension of the possibilities for the use of RT-PCR methods in the assessment of water quality. Steps will be taken towards expanding the analytical possibilities (methods for bacteria from the coli group, or an alternative parameter), and more insight will be gained into the causes of the differences between culture and RT-PCR.

One part of the outcomes will be the additional move taken along the route towards PCR methods becoming an accepted and validated alternative to the current culture methods. This applies to the European coordination, and also to the first phase of the method development for an alternative to the culture method for bacteria from the coli group. If the first phase produces good results, further validation, jointly with the drinking water laboratories and drinking water utilities, will be necessary for legal approval.