Preparation RT-PCR method enterococci

The routine determination of the safety of our drinking water is highly important. In practice this involves the use of a culture method for the intestinal bacteria E. coli and enterococci. This method takes more than two days to carry out in practice. The aim is to develop an accepted rapid method for enterococci, as was earlier done for E. coli. This RT-PCR method for E. coli, which produces a result within a few hours, was recently approved by the Ministry of Infrastructure and Water Management.

More sensitive and faster

The detection of E. coli and enterococci is a good measure of the safety of our drinking water. In the context of maintenance work and of emergencies, it is important to be able to quickly control the drinking water for the possible presence of these bacteria. The advantage of the RT-PCR method is that it can provide a definitive answer concerning the safety of the drinking water within half a day. This method applies PCR techniques to RNA molecules. These RNA molecules are present in larger numbers in active microorganisms than they are in dormant microorganisms. The method is therefore more sensitive than molecular methods, which involve analysis at the DNA level. In particular, the DNA-based method does not produce any information about the level of activity of the microorganisms. To determine whether the RT-PCR method is a suitable alternative to the culture method, a series of investigations were carried out, which ultimately led to the legal approval of the   RT-PCR method for E. coli.

RT-PCR detection method for enterococci

Enterococci are a varied group of bacteria, which includes the intestinal bacterium E. coli. There are dozens of species within this group, a number of which have an impact on hygiene and health. For this reason, it is important that the RT-PCR method be capable of detecting those enterococci that are relevant for drinking water. The determine this, a mixture of ten known enterococci was made in the laboratory. This provides the basis for comparing the sensitivities of the new RT-PCR method and the classical culture method.

The use of ten different enterococci requires that the RT-PCR be optimised. In the first step, the RNA is transcribed to DNA to carry out the PCR reaction. The short DNA fragments (primers) are then used; these are unique to different enterococci species. For the ten investigated enterococci, three primers and thus three RT-PCR reactions are required. In order to increase the detection efficiency, a method is therefore being worked on in which these three RT-PCR reactions can be carried out all at once.

Faster determination of safety

This project, within the Joint Research Programme of KWR and the water utilities, falls under the rubric of biological safety, and is connected to the programme’s research on faster verification following work activities. With a view to saving time, a rapid detection method combined with rapid monitoring following work activities are of crucial importance. With the RT-PCR method, the drinking water utilities can carry out a faster safety verification, so that their customers can quickly start using the drinking water again.

Overview of the procedure for the classical culture method (left) and the newly-developed, rapid RT-PCR method (right).