DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

  • DNA extraction methods were evaluated to quantify H. pylori in water.

  • The tissue kit was suitable for DNA extraction from treated drinking water.

  • The stool kit was efficient in analyzing samples from the river after heavy rain.

  • The soil kit could extract DNA effectively from the river and stream with no rain.

  • The characteristics of the water sample were important in detecting H. pylori.

Helicobacter pylori has been recognized as one of the main causes of duodenal ulcers and gastritis, and as a risk factor for gastric cancer (Cover & Blaser 1992). The World Health Organization (WHO) has classified it as a class І carcinogen (WHO 1994). Recent studies have focused on the possible waterborne transmission of H. pylori and several studies have reported that H. pylori DNA was detected in treated drinking water as well as surface water and ground water (Moreno et al. 2003; Watson et al. 2004; McDaniels et al. 2005; Voytek et al. 2005; Amirhooshang et al. 2014). As H. pylori in environmental water are present at very low levels and, in some cases, in a viable but non-culturable (VBNC) state under environmentally stressful conditions, they may not be detected by culture-based methods (Horiuchi et al. 2001; Nilsson et al. 2002). Moreover, H. pylori require a long incubation time of 7 days due to their slow growth rate (Degnan et al. 2003). The isolation of H. pylori by culture method is a time-consuming, inefficient process.

Although real-time PCR is widely used for the detection of pathogens, as it is a rapid, sensitive, specific, and quantitative method (Roussel et al. 2005), PCR may generate false-negative results as it is easily affected by inhibitors (Schrader et al. 2012). In water samples, the possibility of PCR inhibition increases when a large volume of water is concentrated in order to detect target microbes present at very low concentration (Layton et al. 2006). Reducing PCR inhibitors is necessary in order to detect H. pylori accurately from water samples using real-time PCR assay.

Several studies have used DNA extraction methods such as the Qiagen DNeasy tissue kit (Janzon et al. 2009), QIAamp DNA stool kit (Janzon et al. 2009), and QIAamp DNA mini kit (Nayak & Rose 2007; Kawaguchi et al. 2009) to extract H. pylori DNA directly from water. In these studies, the efficiency of each method could not be evaluated systematically and little information has been available with regard to the methods. As the results can be affected by various factors including water quality and precipitation, a proper DNA extraction method needs to be used considering the characteristics of the sample. Representative DNA extraction kits were chosen among those commonly reported for H. pylori and other pathogens in water as follows (Jiang et al. 2005; Nayak & Rose 2007; Janzon et al. 2009; Kawaguchi et al. 2009): QIAamp DNA mini kit (Qiagen, USA), QIAamp DNA stool mini kit (Qiagen, USA), and FastDNA SPIN Kit for Soil (Qbiogene (at the times of the experiment), MP biomediclas (current), USA).

In this study, we evaluated three DNA extraction methods for quantifying H. pylori from different water samples, such as river, stream, tap water, and influent of wastewater treatment plants (WWTP), containing various quantities and qualities of inhibitors by water quality and precipitation. We also assessed the effectiveness of immunomagnetic separation (IMS) to reduce the inhibitory effects.

Bacterial strain

Helicobacter pylori (ATCC 43504) was cultured on Columbia agar (Difco, USA) with 5% (v/v) defibrinated horse blood (Oxoid, Spain) at 37 °C for 3–5 days under microaerobic conditions using a CampyGen gas pack (BBL, USA).

Sample collection

21 samples were collected from the six intake stations and four points including Paldang, Kangdong, Chonho, and Jamsil bridges, along Han River in Korea (Figure 1). The water taken from the six intake stations was supplied as the source water for the water treatment plants of Seoul. The water samples were collected from June to July 2009. In order to study the effect of precipitation on PCR inhibitors, the samples were collected into three groups. One group of samples (four samples) was taken in June just after raining with intensity of 25.5 mm/day and another group of samples (four samples) was taken after 140 mm/day of precipitation. The third group of samples (13 samples) was collected during sunny days, i.e., more than 7 days of no precipitation before raining.

Figure 1

Sampling sites. •, intake stations; ♦, Paldang, Kangdong, Chonho, and Jamsil bridges along Han River; ▪, branch streams flowing into Han River; ▴, influent from wastewater treatment plants.

Figure 1

Sampling sites. •, intake stations; ♦, Paldang, Kangdong, Chonho, and Jamsil bridges along Han River; ▪, branch streams flowing into Han River; ▴, influent from wastewater treatment plants.

Close modal

Eleven samples from two of the branch streams flowing into Han River and six samples from the three wastewater treatment plants (WWTP) around Han River basin were collected (Figure 1). These samples were collected from January to September 2011. In addition, two samples were taken from tap located at the extremities of the drinking water distribution system. The samples from the branches and from the WWTP's influent were analyzed to study the effect of different concentration of possible inhibitors. In total, 40 water samples including river, stream, influent, and tap water were used to evaluate the degree of DNA recovery and PCR inhibitors during DNA extraction.

Physiochemical and microbiological analysis of water sample

Total coliforms (TC), fecal coliforms (FC), pH, temperature, conductivity, suspended solids (SS), biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN), total phosphate (TP), and total organic carbon (TOC) were measured for the samples according to the US Standard Methods of Examination of Water and Wastewater (APHA 2005). Heterotrophic plate counts (HPC), TC, E. coli, pH, turbidity, and free residual chlorine (FRC) were measured for the samples from the taps (Ministry of Environment 2011).

DNA extraction

Water samples from taps (500 mL) were filtered through a membrane filter (pore size 0.45 μm; Millipore, USA), and the filter was suspended in 10 mL of sterilized phosphate buffer solution (PBS; pH 7.0). Samples from river, stream, and influent (500 mL) were centrifuged at 3,000 × g for 20 min, and the pellet was washed twice with sterilized PBS. The DNA in the concentrated samples could be extracted using each QIAamp DNA mini kit, QIAamp DNA stool mini kit, and FastDNA SPIN Kit for Soil. The methods with chemical cell lysis, i.e., QIAamp DNA mini kit and QIAamp DNA stool mini kit, used the heat lysis procedure with proteinase K in buffer containing chaotropic salts. The QIAamp DNA stool mini kit had InhibitEX tablets to remove the PCR inhibitors. The FastDNA SPIN Kit for Soil used beads beating in detergents for the physical cell lysis. The three methods used silica membrane-based purification. All DNA extractions were performed according to the manufacturer's instruction with minor adjustment. DNA was stored at −20 °C prior to use for real-time PCR. The results by the three DNA extraction techniques were compared to determine the best technique of getting higher quality DNA from various concentrations of H. pylori DNA. A PCR mixture containing 2 μL of H. pylori DNA equivalent of 104, 103, and 101 cells per reaction was spiked with sample DNA (2–5 μL) extracted by each method. Moreover, to evaluate the recovery of H. pylori DNA from water samples, water samples (n= 5) were spiked with H. pylori suspension of 104 CFU/mL, and DNA was extracted by each method. The concentration was equivalent to 5 × 101 cells per PCR reaction. The copy numbers obtained from the real-time PCR assays for the methods were compared. Each DNA sample was tested in triplicate per method.

QIAamp DNA mini kit: QIAamp DNA mini kit for tissue was used. ATL buffer (180 μL) and proteinase K (20 μL) were added to the water pellet (200 μL), which was then heated at 56 °C for 2 hr.

QIAamp DNA stool mini kit: ASL buffer (1.4 mL) was added to the water pellet (200 μL), which was then heated at 70 °C for 5 min. After vortexing and centrifuge, one InhibitEX tablet (Qiagen) was added to the supernatant to adsorb the inhibitory substances.

Currently, the QIAamp DNA stool mini kit and InhibitEX tablets are no longer manufactured. Instead, the QIAamp Fast DNA Stool Mini Kit with InhibitEX buffer can be used as an alternative kit. The QIAamp DNA Stool Mini Kit requires separate addition of an InhibitEX tablet to remove the PCR inhibitor. However, the QIAamp Fast DNA stool mini kit contains the InhibitEX buffer as liquid format. Also, the alternative kit with InhibitEX buffer can reduce the protocol steps because the procedure to completely suspend the InhibitEX tablet or incubation to allow inhibitors to adsorb to the InhibitEX matrix is no longer required.

FastDNA SPIN Kit for Soil: A water pellet (200 μL) was transferred into the lysing matrix E tube with a mixture of ceramic and silica particles. 978 μL of sodium phosphate buffer and 122 μL of MT buffer were added and the tube was vortexed in FastPrep instrument for 30 s at speed 5.5 (40 s at speed of 6.0 in protocol of current kit).

All other processes were performed according to the manufacturer's instructions. In all figures and data, the QIAamp DNA mini kit is indicated as tissue kit, QIAamp DNA stool mini kit is indicated as stool kit, and FastDNA SPIN Kit for Soil is indicated as soil kit.

Immunomagnetic separation

The immunomagnetic separation (IMS) technique was used to reduce microbial contamination and minimize the effects of any PCR inhibitors present in samples (Watson et al. 2004). IMS was performed prior to DNA extraction with Dynabeads M-280 Sheep anti-Mouse IgG (Dynal Corp., UK) and monoclonal mouse anti- H. pylori immunoglobulin (Fitzgerald Corp., USA). The samples were collected from the WWTP influent (n= 6), stream (n= 7), and river (n= 7) under normal condition (without rain). Concentrated samples were spiked with H. pylori to yield total concentration of 104 cells.

Real-time PCR assay

The primers and probe for the detection of H. pylori were used as described by Horiuchi et al. (2001). The real-time PCR mixture was prepared by combining 500 nM of each primer, 200 nM of probe, 5 μL of DNA template, and 25 μL of iQ super mix (Bio-Rad) to yield a final volume of 50 μL. The PCR conditions consisted of one cycle at 95 °C for 5 min, 50 cycles at 95 °C for 15 s, and 60 °C for 60 s. PCR amplification was carried out in an Icycler (Bio-Rad).

All samples confirmed that H. pylori was not detected by real-time PCR.

Statistical analysis

The results of real-time PCR assay were presented as the gene copies (log values) calculated by standard curve. Analysis of variance (ANOVA) was applied to determine if there was a significant difference (α = 0.05) between the copy numbers obtained by the DNA extraction methods tested (SAS for Windows version 8.0.1).

Water quality parameter

Table 1 summarizes the physiochemical and microbiological properties of the 40 water samples. The concentrations of SS, COD, TP, TN, TOC, and bacteria were relatively higher for river water samples taken just after rain. The samples taken from the branch streams had higher conductivity, BOD, COD, TP, TN and TOC than those from the river regardless of sampling season. The highest TCs were detected in the WWTP's influent samples and no tested bacteria were detected in the tap water samples.

Table 1

Physiochemical and microbiological properties of the samples

ParametersSample type (40*)
River (21)
Stream (11)WWTP influent (6)Tap (2)
With rain (8)Without rain (13)
TC** (CFU/100 mL) 3.1 × 104 (2.2 × 103–3.3 × 105) 3.0 × 102 (4.2 × 101–3.4 × 1037.4 × 103 (3.9 × 102–1.0 × 1051.4 × 107 (7.9 × 106–1.8 × 107– 
FC** (CFU/100 mL) 4.1 × 103 (8.5 × 102–4.9 × 1046.6 × 101 (5.0 × 100–8.4 × 1021.4 × 103 (6.0 × 101–1.0 × 104– – 
Temperature (°C) 20.1 (19.0–20.7) 22.2 (19.1–25.3) 17.1 (2.1–25.4) 20.8 (15.0–24.0) – 
pH 7.4 (6.9–8.0) 7.8 (7.2–8.9) 7.8 (7.2–9.0) 7.2 (7.0–7.3) 7.0 (7.1–6.9) 
SS (mg/L) 18.8 (7.6–56.4) 5.9 (2.4–8.4) 11.0 (2.0–21.6) 84.2 (50.0–114.0) – 
BOD (mg/L) 1.8 (1.2–2.5) 1.8 (1.2–2.6) 4.9 (1.4–9.7) – – 
COD (mg/L) 2.9 (2.4–3.3) 2.4 (1.9–3.0) 5.9 (3.0–9.5) – – 
Conductivity (μs/cm) 132 (107–155) 159 (139–216) 373 (195–619) – – 
T-P (mg/L) 0.191 (0.033–0.526) 0.036 (0.020–0.056) 0.404 (0.012–0.811) – – 
T-N (mg/L) 2.324 (1.662–3.340) 1.630 (1.476–1.873) 8.236 (3.071–15.209) – – 
TOC (mg/L) 2.63 (1.92–3.55) 2.11 (1.79–2.67) 4.15 (1.95–5.99) – – 
HPC (CFU/mL) – – – – 
TC/E. coli (/100 mL) – – – – Absence 
Turbidity (NTU§    0.06 
FRC (mg/L) – – – – 0.21 (0.19–0.23) 
ParametersSample type (40*)
River (21)
Stream (11)WWTP influent (6)Tap (2)
With rain (8)Without rain (13)
TC** (CFU/100 mL) 3.1 × 104 (2.2 × 103–3.3 × 105) 3.0 × 102 (4.2 × 101–3.4 × 1037.4 × 103 (3.9 × 102–1.0 × 1051.4 × 107 (7.9 × 106–1.8 × 107– 
FC** (CFU/100 mL) 4.1 × 103 (8.5 × 102–4.9 × 1046.6 × 101 (5.0 × 100–8.4 × 1021.4 × 103 (6.0 × 101–1.0 × 104– – 
Temperature (°C) 20.1 (19.0–20.7) 22.2 (19.1–25.3) 17.1 (2.1–25.4) 20.8 (15.0–24.0) – 
pH 7.4 (6.9–8.0) 7.8 (7.2–8.9) 7.8 (7.2–9.0) 7.2 (7.0–7.3) 7.0 (7.1–6.9) 
SS (mg/L) 18.8 (7.6–56.4) 5.9 (2.4–8.4) 11.0 (2.0–21.6) 84.2 (50.0–114.0) – 
BOD (mg/L) 1.8 (1.2–2.5) 1.8 (1.2–2.6) 4.9 (1.4–9.7) – – 
COD (mg/L) 2.9 (2.4–3.3) 2.4 (1.9–3.0) 5.9 (3.0–9.5) – – 
Conductivity (μs/cm) 132 (107–155) 159 (139–216) 373 (195–619) – – 
T-P (mg/L) 0.191 (0.033–0.526) 0.036 (0.020–0.056) 0.404 (0.012–0.811) – – 
T-N (mg/L) 2.324 (1.662–3.340) 1.630 (1.476–1.873) 8.236 (3.071–15.209) – – 
TOC (mg/L) 2.63 (1.92–3.55) 2.11 (1.79–2.67) 4.15 (1.95–5.99) – – 
HPC (CFU/mL) – – – – 
TC/E. coli (/100 mL) – – – – Absence 
Turbidity (NTU§    0.06 
FRC (mg/L) – – – – 0.21 (0.19–0.23) 

*n, Sample number.

**Geometric mean.

CFU, colony forming unit.

Range.

§NTU, nephelometric turbidity unit.

DNA recovery by DNA extraction methods

The recovery of H. pylori DNA was evaluated by extracting DNA using the three methods from the concentrates of five samples, i.e., two samples from tap, two from the river (one for rainy day and another for sunny day) and one from stream, spiked with H. pylori suspensions of 104 CFU/mL. The results are shown in Figure 2. For the tap water samples and H. pylori suspension in PBS, the greatest recovery of H. pylori DNA was obtained from the tissue kit, but no H. pylori DNA was recovered from the soil kit and stool kit. In the samples from the river and stream, the soil kit and stool kit extracted more H. pylori DNA than the tissue kit. While the soil kit produced consistent recovery for the samples from the river (without rain) and stream, the stool kit resulted in higher recovery for the river samples with rain.

Figure 2

Recovery of H. pylori DNA extracted by three methods according to sample types.

Figure 2

Recovery of H. pylori DNA extracted by three methods according to sample types.

Close modal

DNA quality by DNA extraction methods

DNA was extracted from 40 water samples using the three DNA extraction methods. The real-time PCR mixture containing 2 μL of H. pylori DNA corresponding to 104 cells per reaction was spiked with 2 μL of sample DNA to check for any PCR inhibitor remaining in the extracted DNA. The results are shown in Figure 3. The average copy numbers of 40 samples containing DNA extracted by the tissue kit were >3-log lower than those of positive-only controls despite being mixed with H. pylori DNA as positive control. In particular, the copy numbers could not be obtained in 20 samples taken from river on rainy days, stream, and influent. Most samples containing DNA extracted by the soil kit showed copy numbers similar to those of the positive controls, but the copy numbers of four samples were different from those of the positive controls. The samples were collected from the river after heavy precipitation of over 140 mm/day and the volumes of the final concentrated samples were over 0.2 mL. All PCRs containing DNA samples extracted by the stool kit showed consistent copy numbers and that were similar to those of positive controls. The copy numbers for DNA extracted by the tissue kit were significantly different from those by the stool kit and the soil kit (P < 0.0001). The two techniques (i.e., stool kit and soil kit) were selected based on the results for additional tests to evaluate the efficiency of the two methods in detail. For 20 samples including four samples collected from the river on rainy days (25.5 mm/day) and eight samples on sunny days, and eight samples from the stream in days without rain, a PCR mixture containing 2 μL of a pure H. pylori DNA corresponding to 103 cells per reaction was spiked with 2–5 μL of DNA extracted by the stool kit and the soil kit. As illustrated in Figure 4, the copy numbers were consistent regardless of the added volume of extracted DNA samples in real-time PCRs containing DNA samples extracted by the stool kit and H. pylori DNA as positive control. Conversely, the copy numbers of five samples among DNA samples extracted by the soil kit fluctuated with increasing amount of extracted DNA sample. The four samples were collected from the river after heavy precipitation of 25.5 mm/day and one sample was collected from the branch streams flowing into Han River, the sample was analyzed to have the highest suspended solids (21.6 mg/L) among the stream samples. The copy numbers of these samples were significantly different from those of the positive controls (P < 0.0001). To determine whether a variation of copy numbers was present in the PCRs containing H. pylori DNA of low concentration and DNA extracted by the stool kit and the soil kit, a PCR mixture containing 2 μL of pure H. pylori DNA corresponding to 101 cells per reaction was spiked with 2–5 μL of sample DNA for ten water samples. The copy numbers obtained with an increase of DNA samples extracted by the stool kit were similar to those of the positive controls similar (P > 0.05). The copy numbers of samples containing DNA extracted by the soil kit and H. pylori DNA could not be obtained. The stool kit resulted in the best-quality DNA from the water samples for real-time PCR assay.

Figure 3

Comparison of copy numbers for DNA extracts obtained by three methods.

Figure 3

Comparison of copy numbers for DNA extracts obtained by three methods.

Close modal
Figure 4

Variation of copy numbers with increasing amounts of DNA samples.

Figure 4

Variation of copy numbers with increasing amounts of DNA samples.

Close modal

Efficiency of IMS

The efficiency of DNA extraction after IMS and DNA extraction without IMS was evaluated (Figure 5). DNA was extracted using the stool kit. The copy numbers obtained by the real-time PCR assay of H. pylori DNA as the positive control were similar for the two methods (P > 0.05). The copy numbers of the river samples without rainfall were similar (P > 0.05) regardless of IMS application (Figure 5). However, the copy numbers of influent and stream samples were higher (P < 0.0001) in terms of DNA extraction after IMS than without IMS (Figure 5).

Figure 5

Variation of copy numbers according to the application or non-application of IMS.

Figure 5

Variation of copy numbers according to the application or non-application of IMS.

Close modal

The differences in PCR inhibitors, microorganisms, and physical characteristics of water samples make it difficult to extract DNA from water samples (Jofre & Blanch 2010). When the concentrations of the target microorganisms, such as H. pylori, in the samples were relatively low, and DNA was directly extracted from the samples, the sensitivity and efficiency of the DNA extraction method were very important for successful PCR, especially real-time PCR (Horiuchi et al. 2001; Layton et al. 2006; Jofre & Blanch 2010). Although the PCR inhibitors can be reduced by dilution of samples or extracted DNA, dilution can lead to decreased sensitivity (Schrader et al. 2012). The addition of substances to reduce PCR inhibitors was not always effective (Schrader et al. 2012).

Therefore, three different DNA extraction methods used in chemical or physical methods for cell lysis were tested to remove PCR inhibitors and detect H. pylori from water effectively. In the chemical methods, such as the tissue kit and stool kit, detergents used for the disruption of cells may inhibit the enzymes applied in nucleic acid amplification (Weyant et al. 1990). The application of physical methods, such as the soil kit, may cut the nucleic acids and form chimeric PCR products (Liesack et al. 1991). Each method may affect the amplification of target microbes. As a result of this study, the tissue kit was not sufficient to remove PCR inhibitors, generating false results in water samples with inhibitory compounds. The tissue kit was preferred for extracting DNA from treated water such as drinking water with little inhibitory compounds. The soil kit was highly efficient in recovering H. pylori DNA and removing PCR inhibitors in environmental waters except for the samples collected with heavy rain. As storm water carries fecal matter and other pollutants from the watershed (Pednekar et al. 2005; Bhat et al. 2007), when the samples were collected in heavy rainfall, the concentrate must be separated into multiple subsamples in order to extract DNA using the soil kit. The stool kit could remove PCR inhibitors effectively from all water samples tested regardless of their characteristics. InhibitEX tablets used for the kit might be efficient in adsorbing inhibitory compounds from the water samples. However, the recovery efficiency of the kit was high only for water samples with poor water quality. Therefore, the stool kit can be said to be suitable for DNA extraction from stream or river water after heavy rainfall.

It would be more efficient to apply IMS prior to extracting DNA than the extraction of DNA without IMS from samples taken from influent and streams. Further study with more water samples is recommended to determine the efficiency of IMS.

This study suggests that the characteristics of water samples by the source and rainfall are one of the major factors for determining the efficiency of DNA extraction, and that a single DNA extraction method is not always optimal for the detection of target pathogens. DNA extraction by the soil kit with physical cell lysis such as bead beating had fewer PCR inhibitors than that by the tissue kit. However, the stool kit with inhibitor removal, the addition of InhibitEX tablets to adsorb the inhibitory substance, could result in the best removal of PCR inhibitors compared with the soil kit. Nonetheless, the soil kit allows the higher recovery of H. pylori from river and stream waters than the stool kit with inhibitor removal. The recovery of H. pylori in treated water was highest using the tissue kit without inhibitor removal. Higher inhibitor removal could not always guarantee higher DNA recovery, especially in samples with low concentrations of target DNA.

Choosing the appropriate DNA extraction method considering the characteristics of water sample is critically important for quantifying H. pylori and reducing PCR inhibitors in environmental water samples. The approach can be applied to the detection of other microorganisms in water as well as H. pylori.

All relevant data are included in the paper or its Supplementary Information.

Amirhooshang
A.
,
Ramin
A.
,
Ehsan
A.
,
Mansour
R.
&
Shahram
B.
2014
High frequency of Helicobacter pylori DNA in drinking water in Kermanshah, Iran, during June–November 2012
.
Journal of Water and Health
12
,
504
512
.
APHA
2005
Standard Methods for the Examination of Water and Wastewater
, 21st edn.
American Public Health Association
,
Washington, DC
,
USA
.
Bhat
S.
,
Hatfield
K.
,
Jacobs
J. M.
,
Lowrance
R.
&
Williams
R.
2007
Surface runoff contribution of nitrogen during storm events in a forested watershed
.
Biogeochemistry
85
,
253
262
.
Cover
T.
&
Blaser
M.
1992
Helicobacter pylori and gastroduodenal disease
.
Annual Review of Medicine
42
,
135
145
.
Degnan
A. J.
,
Sonzogni
W. C.
&
Standridge
J. H.
2003
Development of plating medium for selection of Helicobacter pylori from water samples
.
Applied Environmental Microbiology
69
,
2914
2918
.
Horiuchi
T.
,
Ohkusa
T.
,
Watanabe
M.
,
Kobayashi
D.
,
Miwa
H.
&
Eishi
Y.
2001
Helicobacter pylori DNA in drinking water in Japan
.
Microbiology and Immunology
45
,
515
519
.
Janzon
A.
,
Sjoling
A.
,
Lothigius
A.
,
Ahmed
D.
,
Qadri
F.
&
Svennerholm
A. M.
2009
Failure to detect Helicobacter pylori DNA in drinking and environmental water in Dhaka, Bangladesh, using highly sensitive real-time PCR assays
.
Applied Environmental Microbiology
75
,
3039
3044
.
Kawaguchi
K.
,
Matsuo
J.
,
Osaki
T.
,
Kamiya
S.
&
Yamaguchi
H.
2009
Prevalence of Helicobacter and Acanthamoeba in natural environment
.
Letters in Applied Microbiology
48
,
465
491
.
Layton
A.
,
McKay
L.
,
Williams
D.
,
Garrett
V.
,
Gentry
R.
&
Sayler
G.
2006
Development of bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water
.
Applied Environmental Microbiology
72
,
4214
4224
.
Ministry of Environment
2011
Korean Standard Method for the Examination of Drinking Water
.
Ministry of Environment, Republic of Korea
.
Moreno
Y.
,
Ferrus
M. A.
,
Alonso
J. L.
,
Jimenez
A.
&
Hernandez
J.
2003
Use of fluorescent in situ hybridization to evidence the presence of Helicobacter pylori in water
.
Water Research
37
,
2251
2256
.
Nilsson
H. O.
,
Bloom
J.
,
Al-Soud
W. A.
,
Ljungh
A.
,
Andersen
L. P.
&
Wadstrom
T.
2002
Effect of cold starvation, acid stress, and nutrients on metabolic activity of Helicobacter pylori
.
Applied Environmental Microbiology
68
,
11
19
.
Roussel
Y.
,
Wilks
M.
,
Harris
A.
,
Mein
C.
&
Tabaqchali
S.
2005
Evaluation DNA extraction methods from mouse stomachs for the quantification of H. pylori by real-time PCR
.
Journal of Microbiological Methods
62
,
71
81
.
Schrader
C.
,
Schielke
A.
,
Ellerbroek
L.
&
Johne
R.
2012
PCR inhibitors-occurrence, properties and removal
.
Journal of Applied Microbiology
113
,
1014
1026
.
Voytek
M. A.
,
Ashen
J. B.
,
Fogarty
L. R.
,
Kirshtein
J. D.
&
Landa
E. R.
2005
Detection of Helicobacter pylori and fecal indicator bacteria in five North American rivers
.
Journal of Water and Health
3
,
405
422
.
Watson
C. L.
,
Owen
R. J.
,
Said
B.
,
Lai
S.
,
Lee
J. V.
,
Surman-Lee
S.
&
Nichols
G.
2004
Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England
.
Journal of Applied Microbiology
97
,
690
698
.
Weyant
R. S.
,
Edmonds
P.
&
Swaminathan
B.
1990
Effect of ionic and non-ionic detergents on the Taq polymerase
.
Biotechniques
9
,
308
309
.
World Health Organization (WHO)
1994
World Health Organization's evaluation of carcinogenic risk to humans. Schistosome, liver flukes and Helicobacter pylori
.
IARC Monographs
61
,
45
119
.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY-NC-ND 4.0), which permits copying and redistribution for non-commercial purposes with no derivatives, provided the original work is properly cited (http://creativecommons.org/licenses/by-nc-nd/4.0/).