Water Analysis and Technical Consulting

Bacteria of the genus Legionella are opportunistic pathogens, commonly spread in aquatic environments: springs, rivers, lakes, thermal waters. From these environments they can spread to and in artificial works, such as: drinking water pipes; building water systems; fountains; swimming pools; tanks etc. in which the bacterium can develop.

Traditional

Rentacs carry out environmental sampling of drinking, boiler and cooling tower waters, as well as filters and air conditioning infrastructure, additionally medical and industrial water, certified by the ACCREDIA laboratory, with quantitative counting of the UFC (Cologne Forming Units) and research of the type (Serum group).

Samples comply with the guidelines and legislation in force.

Rentacs carry out sampling for Legionella research and quantification with Real Time PCR technique, certified by the ACCREDIA laboratory, with quantitative counting of UFC (Colony Forming Units) in less than 24 hours.

PCR Method

Traditional methods for the detection of Legionella and Legionella pneumophila in water samples have the disadvantage of having long incubation periods. The research of the same with the PCR method allows Rentacs to obtain results in a short time and with greater sensitivity.

These methods allow for the detection or quantification of Legionella and Legionella pneumophila in all water samples in a few hours, in which the following steps take place:

1. Sample filtration
2. DNA extraction
3. PCR reaction for the detection of Legionella and Legionella pneumophila

The PCR test is based on the amplification and detection of genomic sequences of a target DNA that acts as a target, in this case that of Legionella. During the test, primers (i.e., nucleic acid strands that serve as a trigger point for DNA replication) bind to a target region of the bacterium and the Taq polymerase uses these primers and deoxy nucleotide triphosphates (dNTPs) to extend the DNA, creating exact copies of them (called amplicons). Subsequently, an oligonucleotide probe hybridizes to these amplicons, and, by emitting fluorescence thanks to a fluorophore in its possession, is allowed to “see” the presence of the bacterium’s DNA. When the target DNA is present in the sample, the fluorescence intensity increases proportionally to the increase in the amount of the amplified products and is measured by the optical module inside the thermal cycler during the hybridization step. The software, combined with the instrument, displays the fluorescence intensity in real time based on the number of amplification cycles. For each reaction, the software determines a Cq, i.e. the cycle from which the fluorescence significantly increases above the background noise. The value of Cq is related to the logarithm of the number of initial copies of the target sequence.

The reading of the Cq values allows time to detect the presence of the DNA of the Legionella bacterium and this is representative of the presence of the DNA in the original water sample. The quantification is possible thanks to the interpolation of the results obtained with a curve created specifically using standards containing various quantities of bacteria. The kits for Legionella and Legionella pneumophila are certified NF Validation according to the validation protocol based on the NF T90-471 standard (June 2015). The scope of the validation has been extended to the ISO / TS 12869 method (November 2012): Water quality – Detection and quantification of Legionella spp. and / or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR).