Detection of Helicobacter pylori in sewage and water using a new quantitative PCR method with SYBR green.
OBJECTIVE
To demonstrate the application of a new quantitative polymerase chain reaction (qPCR) technique for the determination of Helicobacter pylori concentrations in water, and to use this method to investigate the occurrence of the bacteria in sewage. The other aim was to study the survival capacity and detectability of the bacteria in artificially contaminated groundwater at different temperatures of 4 and 15 degrees C.


RESULTS
The detection of H. pylori in water was aided by PCR using specific primers designed for the amplification of a fragment within the major vacuolating cytotoxin gene. Conventional culture was compared with conventional PCR and the new real-time (RT) qPCR approach for the quantification of the bacterium. Helicobacter pylori remained culturable for 120 h at 4 degrees C as opposed to only 24 h at 15 degrees C. RT qPCR demonstrated a 100-fold greater sensitivity for the detection of H. pylori DNA in comparison with conventional PCR. Scanning electron microscopic (SEM) observation showed that the normal spiral form changed to a coccoid form after 24 and 72 h at 15 and 4 degrees C, respectively. Helicobacter pylori was found at 2-28 cells ml(-1) in sewage, of the 23 sewage samples - 84% were positive for H. pylori species-specific vacuolating cyctotoxin gene (vacA) by RT qPCR, but were negative by conventional PCR.


CONCLUSIONS
The RT qPCR assay provided a specific, sensitive and rapid method for the quantitative detection of H. pylori in sewage. This molecular method would be valuable in studying the prevalence of H. pylori as required by the United States Environmental Protection Agency Contaminant Candidate List, particularly in nondisinfected ground waters, in sewage as a source of contamination, and for addressing the possible presence of viable but nonculturable of H. pylori.


CONCLUSIONS
The quantitative detection of H. pylori by rapid and less-expensive methods than the TaqMan Assay using SYBR green could be an important tool to monitor infection in community by measuring the concentrations in sewage and to meet the new regulatory and risk-based frameworks for water supplies.
Nayak
A K
AK
Michigan State University, Department of Fisheries and Wildlife, East Lansing, MI 48824, USA.
Rose
J B
JB
D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D004784:Environmental Monitoring / Q000379:methods; D005456:Fluorescent Dyes; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D050296:Microbial Viability; D008855:Microscopy, Electron, Scanning; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D012722:Sewage; D013696:Temperature; D014871:Water Microbiology
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012722:Sewage; C098022:SYBR Green I