Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method.
The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.
LCPME-UMR 7564 CNRS-UHP, Equipe Microbiologie et Physique, Faculté de Pharmacie, 5 rue A. Lebrun, Nancy cedex, BP 403, 54 001 France. Isabelle.Bertrand@pharma.uhp-nancy.fr
Isabelle
I
Bertrand
Christophe
C
Gantzer
Thierry
T
Chesnot
Janine
J
Schwartzbrod
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D005455:Fluorescent Antibody Technique; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012722:Sewage / Q000469:parasitology*; D014867:Water / Q000469:parasitology*
D017931:DNA Primers; D016054:DNA, Protozoan; D012722:Sewage; D014867:Water