Legionella Triplex qPCR

Legionella qPCR

Legionella Triplex qPCR




Quantitative polymerase chain reaction (qPCR), also known as Real-Time qPCR, is a rapid molecular method that can be used to detect Legionella in potable and non-potable water samples.  At SPL, we have been using qPCR since 2014 to detect Legionella pneumophila with serogroup 1 determination and have expanded our offering to now include detection of all Legionella species. This is achieved by simultaneously amplifying 3 target DNA sequences unique to Legionella, L. pneumophila and L. pneumophila serogroup 1 then detecting the resulting fluorescent signal for each individually. Results are provided as presence/absence for Legionella with indication if L. pneumophila is present and if serogroup 1 is detected.

Testing is performed in 2 to 4 days as compared to 7 to 10 days for culture. The procedure follows International Standard Organization (ISO) standard 12869-2019 with minor modifications. Standard qPCR cannot discriminate the genome of live vs dead/impaired organisms and may also detect Legionella that are not culturable. This detection of all DNA is a benefit as a negative result indicates that no Legionella (live or dead) is present.

When species identification beyond L. pneumophila serogroup 1 is important, we recommend pairing qPCR with culture.  The qPCR test can rapidly identify the presence of Legionella, L. pneumophila, and L. pneumophila serogroup 1 while culture can provide the identification of additional species and serotypes (L. pneumophila).  Culture will also enable isolate retention for later comparisons such as required in clinical case investigations.

Test Method

  • Water samples are concentrated by filtration.
  • DNA is extracted using the Qiagen DNeasy Power Water Kit.
  • Multiplex quantitative polymerase chain reaction (qPCR) for detection of Legionella species, L. pneumophila and L. pneumophila, serogroup 1.
  • Qualitative (+/-) result for all Legionella assays.

Turn Around Time
2–4 days

Test Code

Sample Transport
See Sampling and Shipping page for instructions.


qPCR Application Guide – Experimental Overview, Protocol, Troubleshooting. 3rd Edition. 2011-2012. Integrated DNA Technologies.

Qiagen DNeasy PowerWater DNA Isolation Kit Instruction Manual.

Applied Biosystems. Quantstudio 6 and 7 Flex Real-Time PCR System Software. 2013. Booklets 2 and 5.

Donohue MJ, O’Connell K, Vesper SJ, Mistry JH, King D, Kostich M, Pfaller S. Widespread molecule detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States. Environ Sci Technol. 2014 Mar 18; 48(6):3145-52.

ISO Standard 12869: 2019. Water Quality – Detection and quantification of Legionella spp. by concentration and genic amplification by quantitative polymerase chain reaction (qPCR)

Accreditations and Proficiency
See Accreditations and Proficiency page for information on our accreditations and Legionella testing proficiency programs.