Water TestingiQ-Check Legionella
Fact Sheet1. SIGNIFICANCE OF Legionella TESTING
Legionellosis is an infection caused by Legionella bacteria; they
are the cause of an acute pneumonia, Legionnaire’s disease anda milder form of pulmonary infection, Pontiac fever. In Europe,
the number of declared cases of Legionnaire’s disease increases
by 25% per year. In the United States, the CDC (Centers forDisease Control and Prevention) estimates the number of casesof Legionellosis at between 10,000 and 20,000 per year. InCanada, there were 332 cases in 2018 which represents 64%
increase compared to 2017. In Quebec an estimate of 200 cases
were reported last year. The actual number of cases, however,is thought to be much higher, as many people with pneumonia
may not be tested for Legionella infection. The Legionella
pneumophila species is responsible for approximately 90% of theclinical cases. Regular control of the presence of Legionella inwater supply systems is the only way of preventing the disease.
One of the most sensitive and specific methods developed forLegionella pneumophila testing is qPCR (quantitative Polymerase
Chain Reaction). To ensure accuracy of data, commerciallyavailable detection methods must comply with specificrequirements and validations recommended by government
agencies and professional organizations that have publishedguidelines not only for the design, but also the commissioning
requirements for cooling towers and water systems.
Bio-Rad’s iQ-Check method has been used for over 10 years tomonitor Legionella in cooling towers and has been demonstrated
to be a very powerful tool for the industry and government
agencies.

2. ISO TECHNICAL SPECIFICATION 12869
ISO (the International Organization for Standardization) is aworldwide federation of national standards bodies (ISO memberbodies). The work of preparing International Standards isnormally carried out through ISO technical committees. ISO/TS
12869 was prepared by Technical Committee ISO/TC 147, Waterquality, Subcommittee SC 4, Microbiological methods. The aimof this standard is to provide proof of performance of a Legionella
PCR system that meets the highest level of quality management,from manufacturing of the product to the results analyses. This
helps build vendor credibility and buyer confidence by providing

the marketplace with the assurance that performance claims arevalid, credible and supported by high-quality, independent
test data.
This technical specification describes a method for the detection
and quantification of Legionella spp. and L. pneumophilausing a quantitative polymerase chain reaction (qPCR).
It specifies general method requirements, performance
evaluation requirements, and quality control requirements
including inclusivity/exclusivity analyses, limit of detection,
limit of quantification of PCR step, calibrating function, link toprimary standard, and efficiency and robustness of extraction
with Aquadien kit. The guidelines, minimum requirements andperformance characteristics are intended to guarantee thatthe results are reliable and reproducible between different
laboratories.
iQ-Check Legionella spp. and iQ-Check Legionella
pneumophila kits are NF VALIDATION certified according
to NF T90-471 and ISO/TS 12896. The MD 15161-2013
Control of Legionella in Mechanical Systems Standard
published by Public Works and Government Standards
Canada (PWGSC) recommends the use of methods thatare in accordance with the ISO TS 12869.
3. iQ-CHECK METHOD
iQ-Check Legionella is an ISO/TS 12869 validated method
based on amplification and detection of genomic sequencesby the real-time PCR method. The Aquadien kit is also an ISO/TS 12969 method that allows an optimal DNA extraction andpurification from bacteria present in water samples for PCRdetection. The principle of the extraction is based on alkaline lysis
of bacteria in presence of thermal shock and DNA purification
using ultrafiltration. The Aquadien W2 wash solution is optimizedfor dirty samples including those containing PCR inhibitors suchas additives and chemicals commonly used for water treatment.
The kits contain ready-to-use reagents required to perform theanalysis of samples: PCR amplification solutions including Taq
DNA Polymerase and internal control, specific fluorescent probes
and primers, negative and positive controls. Additional standards
are provided for quantification.

4. LIVE/DEAD DIFFERENTIATION WITH FREE-DNA REMOVALSOLUTION (FDRS)One of the primary challenges when performing PCR forenvironmental water testing is the potential presence of free DNA,
which can lead to an overestimation of the level of targeted DNA
sequences. When quantifying Legionella, culture and PCR levels
can differ from each other by up to 2 logs. To improve Legionella
testing in water, a supplemental step using the iQ-Check Free
DNA Removal Solution (FDRS) can be introduced into theAquadien DNA Extraction and Purification Kit. FDRS can be easily

Water samples
Free DNA

integrated in the Aquadien DNA Extraction Kit workflow andprovides a non-carcinogenic and safe way to remove dead cell
DNA or free DNA from environmental water samples prior to PCR
analysis. According to the principle, free DNA is degraded by an
enzymatic treatment of the sample prior to DNA extraction, andinactivated by the Aquadien Extraction Kit R1 solution allowing forthe extraction of DNA from intact and living cells. The inclusionof FDRS in the iQ-Check workflow drastically eliminates theoccurrence of false positives.

Heat to 37°C for 30 min
Live Cells

Free DNA Removal Solution

Sample ready for
DNA extraction with thefree DNA removed

WWW

Fig. 2. The mechanism of action of FDRS

Fig. 3. FDRS protocol

The iQ-Check method is based on the following steps illustrated
below:

Fig. 1. iQ-Check protocol

1. Sampling

2. Filtration

3. DNA Extraction

4. Real-Time PCR Detection
and Quantification5 . Automatic

Data Analysis

Water filtration
10 000

10 000

1 000

1 000

Water
Biofilm
Aerosol CIP10-M

2 | BIO-RAD LABORATORIES, INC.

Concentrate
bacteria by filtering
water

Recover bacteria
from filter and extract
DNA using theAquadien Kit

Add DNA to
PCR microtubes
containing PCR
mix from iQ-Check
Legionella Kits

Start PCR using the
CFX96 Touch™ Deep
Well Real-Time PCR
Detection System

Aquadien protocol

10010010FDRS treatment

102 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

View results in a reportwith full traceabilityusing CFX Manager™
Software, IDE

BIO-RAD LABORATORIES, INC. | 35. iQ-CHECK PERFORMANCE CRITERIA

6. iQ-CHECK VS PORTABLE qPCR TECHNOLOGYPARAMETERFEATURESFEATURES

iQ-Check Legionella

Portable Legionella qPCR

Specificity

Legionella pneumophila and Legionella spp.

TargetMicroorganism

Legionella spp. andLegionella pneumophila

Legionella pneumophila only

Sensitivity

5 GU (genomic units)

Throughput

96 samples

1-3 samples

Genetic Target

Conserved Legionella-specific gene
Extraction Kit

Validated protocols
adapted for all watertypes

No validated protocols

Live/DeadDifferentiation

Yes

No known live/deaddifferentiation

Results

Qualitative andquantitative

Quantitative

Accuracy ofResults

High; Assay performedby trained techniciansat reference labs lowers
the risk of user error

Low; On-site analyses in
non-accredited labs andinexperienced operatorswith lack of PCR skills may
increase the risk of crosscontamination and usererror

Validations

NF Validation according
to T-90 471 and ISO/TS
12869

None

Internal Amplification
ControlYes

Assay Format

Qualitative and quantitative

Reagent shipping & storage

Ambient temperature; +2 to +8°C

Reagent Shelf Life

2 years

Compatible Matrices

Potable and non-potable including complex
water, biofilm, aerosol
L. spp : 35 isolates correctly identified
L.pneumophila: 15 isolates correctlyidentified
L. spp :16 isolates correctly excluded

Exclusivity

L.pneumophila: 25 isolates correctlyexcluded

Time to Results<4 hr

Viability Differentiation

Yes (Free DNA Removal Solution)

Validations

NF T90-471; ISO/TS 12869

19-4031

Inclusivity

For more information on our water testing products visit,
bio-rad.com/water.
Bio-Rad and iQ-Check are trademarks of Bio-Rad Laboratories, Inc. in certain
jurisdictions. All trademarks used herein are the property of their respective owner.