The positive control fails
The positive control should have a Ct greater than 0. Twenty microliters of the positive control should be added to a reaction. Repeat. If positive control continues to fail, reach out to LuminUltra for a replacement.
The negative control fails (Ct>0)
The negative control was detected. The LuminUltraLegionella pneumophila assay is designed to be very sensitive and cross-contamination of a sample can cause small amounts of DNA to be present in the negative sample. Try re-running the assay in a cleaner location and keep all qPCR reagents separate from the extraction process. Wipe down the area with 10% bleach to destroy errant DNA template.
The internal control isn’t visible
The internal amplification control has a Ct usually between 30 and 40. Sometimes the IAC can fail if the target DNA is high or if inhibitors are present in the extracted sample. Repeat the sample by diluting in half (10ul sample, 10ul water) to overcome inhibition by dilution.
An incorrect Q16 protocol was run.
The Legionella pneumophila amplification reaction is robust and should amplify in a wide range of cycling conditions.
Will DNA from recently deceased bacteria be measured? Does this matter?
Our technology uses a filtration-based method to preferentially capture living organisms, while allowing interferences, microbial debris and aqueous phase components to pass through. With that being said, it might also capture some recently deceased, still intact organisms that will be lysed and measured along with the living organisms. This is not a bad thing. Our clients that are using Legionella qPCR assays are trying to mitigate risk in their systems. Even if they detect some small amount of recently deceased organisms, that means that they recently had pathogenic Legionella in their system, which means Legionella has become established in their system. A false positive is better than a non-detect or a false negative.
How are results reported for Legionella qPCR testing?
ISO 12869, “Water quality –Detection and quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR)” states that results should be reported as GU (genome units) per litre (GU/L).
Does 1 GU/mL = 1 CFU/mL when comparing qPCR to culture results?
Unfortunately, not, and there is no universal conversion factor that can be used due to the inherent technological differences between the two methods. While generally 1 CFU is indicative of one starting organism, and Legionella pneumophila bacterium contains one GU on average, this does not mean that the two measurements are directly comparable, even though they try to measure the same thing: potentially virulent individual Legionella bacteria. Culture methods that seek to enumerate Legionella consistently underrepresent the actual number of bacteria present, are highly dependent on the individual laboratory and have worse limits of detection such that there is a significant false negative rate. It is more than likely possible to determine a correlation between CFU/mL and GU/mL but it will be specific to one system that uses one laboratory for culture testing. After performing this comparison work, thresholds based on GU/mL or GU/L can be used for risk mitigation.