Integrated use of biomarkers and bioaccumulation data in Zebra mussel (Dreissena polymorpha) for site-specific quality assessment.

One of the useful biological tools for environmental management is the measurement of biomarkers whose changes are related to the exposure to chemicals or environmental stress. Since these responses might vary with different contaminants or depending on the pollutant concentration reached in the organism, the support of bioaccumulation data is needed to prevent false conclusions. In this study, several persistent organic pollutants -- 23 polychlorinated biphenyl (PCB) congeners, 11 polycyclic aromatic hydrocarbons (PAHs), six dichlorodiphenyltricholroethane (DDT) relatives, hexachlorobenzene (HCB), chlorpyrifos and its oxidized metabolite -- and some herbicides (lindane and the isomers alpha, beta, delta; terbutilazine; alachlor; metolachlor) were measured in the soft tissues of the freshwater mollusc Zebra mussel (Dreissena polymorpha) from 25 sampling sites in the Italian portions of the sub-alpine great lakes along with the measure of ethoxyresorufin dealkylation (EROD) and acetylcholinesterase (AChE) activity. The linkage between bioaccumulation and biomarker data allowed us to create site-specific environmental quality indexes towards man-made chemicals. This classification highlighted three different degrees of xenobiotic contamination of the Italian sub-alpine great lakes: a high water quality in Lake Lugano with negligible pollutant levels and no effects on enzyme activities, an homogeneous poor quality for Lakes Garda, Iseo and Como, and the presence of some xenobiotic point-sources in Lake Maggiore, whose ecological status could be jeopardized, also due to the heavy DDT contamination revealed since 1996.     

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.     

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.     

Zebra mussel (Dreissena polymorpha) seasonal colonization patterns in a sub-tropical floodplain river

Zebra mussel (Dreissena polymorpha) seasonal colonization patterns, growth and habitat preferences were determined in a sub-tropical floodplain river at the southern edge of its distribution in North America during 1995–96 (Atchafalaya River Basin, Louisiana). Zebra mussel movement into subtropical areas represents a major frontier for this species worldwide. The onset of adult zebra mussel colonies occurred when the minimum daily temperature dropped below 31 °C and dissolved oxygen levels rose above 6.5 mg l^-1 in the fall. By mid-winter, mussel populations were established at lateral distances >10 km from the main river channel. Mussel growth occurred throughout the winter with an increase in growth in April and May. Adult mortality occurred during May–August as dissolved oxygen levels declined and minimum daily temperature warmed above 29 °C in the floodplain and 32.5 °C in riverine sites. Limiting factors responsible for the seasonal pattern include temperature and dissolved oxygen tolerances experienced during summer months in the ARB. Summer water conditions apparently preclude establishment of resident zebra mussel populations in the Atchafalaya floodplain. Naturally occurring seasonal patterns in temperature and dissolved oxygen in floodplain rivers may have implications for the expansion of this exotic mollusk in warmwater systems with source colonies restricted to mainstem rivers and seasonal sinks in floodplain regions. This revised version was published online in July 2006 with corrections to the Cover Date.     

The filtration rate of the zebra mussel Dreissena polymorpha used for water quality assessment in Dutch rivers

The effects of contaminated river water on the filtration rate of zebra mussels from a clean reference site were studied. After a 48-h exposure period to filtered water from the rivers Rhine, Meuse and Amstel (The Netherlands), the filtration rate was measured. It was demonstrated that water from contaminated locations inhibited the filtration rate. Inhibition was higher during low water levels in the rivers Rhine and Meuse than during high water levels, suggesting that contaminants are diluted during high water levels. It is concluded that the shortterm filtration assay with D. polymorpha can be used for assessing water quality.     

Assessment of the genotoxic potential of benzo(a)pyrene and pp'-dichlorodiphenyldichloroethylene in Zebra mussel (Dreissena polymorpha)

The single-cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test were carried out with haemocytes of Zebra mussel (Dreissena polymorpha) specimens to evaluate the potential genotoxicity of benzo(a)pyrene (BaP) and pp'-dichlorodiphenyldichloroethylene (pp'-DDE, a metabolite of pp'-DDT). Mussels were exposed to three different concentrations (0.1 microg/L, 2 microg/L, 10 microg/L) of each chemical in water during 168 h (SCGE assay) and 96 h (MN test) of exposure under laboratory conditions. These levels correspond to nominal molar concentrations of 0.4 nM, 7.9 nM and 40 nM for BaP and 0.3 nM, 6.2 nM and 31 nM for pp'-DDE, respectively. Concurrently, the levels of toxicants were measured in soft tissues of the mussels by gas-chromatographic analyses, to evaluate their temporal trends and the dose/response relationships. Significant increases of the ratio between the comet length and the diameter of the comet head (LDR) and of micronucleus frequencies in comparison with baseline levels were observed not only for all concentrations of BaP, but also for pp'-DDE (except 0.3 nM). The concentration above which DNA damage starts to be significantly increased was 0.8 nmol/g lipids for BaP and 1.6 nmol/g lipids for pp'-DDE, respectively. The results of these experiments show a clear genotoxic effect on this non-target organism not only for the well-known genotoxicant BaP, but also for the final metabolite of pp'-DDT at soft-tissue concentrations that have been found in several aquatic ecosystems worldwide.     

Horizontal and Vertical Distribution of the Zebra Mussel (Dreissena polymorpha,Pallas) Larvae in the Modrac Reservoir,Bosnia and Herzegovina

In the Modrac Reservoir, loaded with coal separation suspended material, rapid development of the zebra mussel (Dreissena polymorpha) took place from at least 1987. It is probably the first distinguished “infection” for this part of Balkan Peninsula, caused by this organism. Horizontal and vertical distribution of this mussel, at 15 different reservoir profiles, had been investigated.     

Using Massive Parallel Sequencing for the Development,Validation, and Application of Population Genetics Markers in the Invasive Bivalve Zebra Mussel (Dreissena polymorpha)

The zebra mussel (Dreissena polymorpha, Pallas, 1771) is one of the most invasive species of freshwater bivalves, due to a combination of biological and anthropogenic factors. Once this species has been introduced to a new area, individuals form dense aggregations that are very difficult to remove, leading to many adverse socioeconomic and ecological consequences. In this study, we identified, tested, and validated a new set of polymorphic microsatellite loci (also known as SSRs, Single Sequence Repeats) using a Massive Parallel Sequencing (MPS) platform. After several pruning steps, 93 SSRs could potentially be amplified. Out of these SSRs, 14 were polymorphic, producing a polymorphic yield of 15.05%. These 14 polymorphic microsatellites were fully validated in a first approximation of the genetic population structure of D. polymorpha in the Iberian Peninsula. Based on this polymorphic yield, we propose a criterion for establishing the number of SSRs that require validation in similar species, depending on the final use of the markers. These results could be used to optimize MPS approaches in the development of microsatellites as genetic markers, which would reduce the cost of this process.     

AChE在水稻抗性诱导的褐飞虱凋亡中肠细胞中的定位及表达

【目的】褐飞虱Nilaparvata lugens是水稻的重要害虫之一。本研究旨在了解水稻品种抗性诱导的褐飞虱中肠细胞凋亡与细胞中乙酰胆碱酯酶(acetylcholinesterase,ACh E)的关系。【方法】从4龄Ⅰ型褐飞虱若虫中肠组织分离原代细胞,用不同抗性的水稻幼苗汁液处理第一代继代细胞,利用TUNEL染色法检测细胞的凋亡情况,再分别利用免疫组织化学和荧光定量PCR技术对ACh E进行亚细胞定位和检测其表达水平的变化。【结果】在对照组(未处理)细胞中,检测不到代表凋亡细胞核的绿色荧光,而免疫组化后阳性反应颜色很浅,表明细胞中存在ACh E的本底水平表达。感虫水稻品种TN1幼苗汁液处理细胞中,细胞的凋亡率为8%,抗性水稻B5幼苗汁液处理的细胞中有65%的细胞发生凋亡;而抗性水稻TKM-6幼苗汁液处理的细胞中则有85%的细胞发生凋亡。免疫组化的检测结果表明,所有凋亡的细胞中都存在ACh E的积累,且主要分布在细胞质中;ACh E在凋亡后期并不迁入凋亡小体中,而是集中在细胞核附近。荧光定量PCR检测表明,TKM-6,B5和TN1幼苗汁液处理的细胞中ACh E的表达量分别为对照细胞的29.9,18.4和8倍,该结果与细胞水平上免疫组化的检测结果一致。【结论】本研究结果证实了水稻品种抗性与其诱导的中肠细胞凋亡率呈正相关,且凋亡细胞的细胞质中存在ACh E的积累。这些发现为揭示ACh E在褐飞虱与水稻的互作中的功能、促进抗性水稻新品种的选育及开发新的褐飞虱防治措施提供了一定的参考信息。     

Application of modified water quality indices as indicators to assess the spatial and temporal trends of water quality in the Dongjiang River

The Dongjiang River plays an important role in southern China, as a source for irrigation and potable water of Hongkong and the other parts of the Pearl River Delta (PRD). The water quality index (WQI) was calculated to assess the spatial and temporal variability and identify the classification of water quality in the river. In order to simplify the procedure and reduce the analytical costs of the water quality evaluation, a modified WQI (defined as WQI_min) was introduced based on Principal Component Analysis (PCA) and correlations analyses of the water parameters detected in dry and wet seasons during 2011–2012. Compared with the previous index, similar spatial changing trend and classification of the water quality were obtained by WQI_min, which was composed of pH, temperature, total suspended solid, NH₄⁺-N, and NO₃⁻-N. The results showed an excellent water quality in the tributary site near the reservoir, a good water quality in the upstream of the river, and medium water quality in the downstream of the river, which suggested that the urban wastewater originated from increasing population size and industry development in the downstream mainly led to the deterioration of water quality along the river. Moreover, WQI_min could more adequately reflect the seasonal changes of water quality which was slightly worse in dry season than wet season. Our results also suggest that continuous monitoring should be conducted to prevent pollution from industry and anthropogenic activities.     

Distribution and aspects of population dynamics of the zebra mussel,Dreissena polymorpha (Pallas, 1771), in the lake IJsselmeer area (The Netherlands)

Summary The distribution, settling and growth of the zebra mussel, Dreissena polymorpha Pallas, in Lake IJsselmeer and Lake Markermeer in The Netherlands were studied from 1980 to 1985. In these lakes D. polymorpha is the most important food source for wintering diving ducks. The study was part of an investigation into the carrying capacity of the lakes for these birds.     

Seasonal changes in the respiratory electron transport system (ETS) and respiration of the zebra mussel,Dreissena polymorpha in Saginaw Bay,Lake Huron

Electron transport system activity (ETS) and respiration rates (R) of the zebra mussel, Dreissena polymorpha, were determined monthly from April to November over 2 years at two sites in Saginaw Bay, Lake Huron. The sites were located in the inner and outer bay and contrasted in food quantity and quality. ETS ranged from 2 to 40 g O₂ mg DW^–1 h^–1 over the study period. Both ETS and respiration were strongly related to temperature, and maximum values were found between June and August. ETS also peaked in June/July when assays were conducted at a constant temperature (25 °C), indicating other factors besides temperature affected metabolic activity. R:ETS ratios decreased with increased temperature at the inner bay site, but trends were minimal at the outer bay site. In late summer, blooms of the cyanophyte Microcystis occurred in the inner bay, likely depressing filtration rates, and leading to lower respiration rates relative to ETS. ETS activity was consistently higher in the outer bay and was likely a result of higher food quality. Despite these spatial differences, annual mean R:ETS ratios varied only from 0.04 to 0.09 at the two sites over the 2-year period. Based on these values, ETS may be useful as an indicator of long-term metabolic activity in annual energy budgets of D. polymorpha. However, food conditions differentially affect respiration relative to ETS, and variability in this ratio must be considered when interested in shorter time scales.     

The use of biomarkers of oxidative stress in zebra mussel <Emphasis Type="Italic">Dreissena polymorpha</Emphasis> (Pallas, 1771) for chronic anthropogenic pollution assessment of the Rybinsk Reservoir

Biomarkers of oxidative stress such as catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR) activity, and malondialdehyde and reduced glutathione content, as well as heavy metal concentrations (HM: Pb, V, Cr, Mn, Co, Ni, Cu, Zn, and Cd), were studied in Dreissena polymorpha tissues. Mussels were collected on three sites located on the Rybinskoe Reservoir different in levels of anthropogenic pressure: the most polluted sites were 1 and 2 and site 3 was relatively clean. Mussels from sites 1 and 2 had higher concentrations of HM (Pb, V, Cr, Mn, Ni, Cu, and Mn) and their response to the pollutant action was manifested in increased processes of lipid peroxidation (LPO), the activation of CAT, and elevated level of GHS.     

Preferences of invasive Ponto-Caspian and native European gammarids for zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>, Bivalvia) shell habitat

The migratory behavior and swimming patterns of anadromous upstream migratory fish have been poorly described in the Shibetsu River in eastern Hokkaido, Japan. In this 2004 study, we used electromyogram (EMG) transmitters and depth/ temperature (DT) loggers to compare the upstream migratory behavior of adult male chum salmon (Oncorhynchus keta) and pink salmon (O. gorbuscha) in the canalized and reconstructed segments of the Shibetsu River, where a part of canalized section was preliminary reconstructed meander to restore a more natural section. The EMG transmitter and DT logger were externally attached to the left side of the body, below the front edge of the dorsal fin. Fish of both species often migrated along the riverbanks and near the bottom of the water column, sometimes engaged in holding behavior, which was defined as cessation of swimming during their upstream migration for 5 minutes. Modal swimming depth calculated by DT loggers for chum salmon (0.2–0.4 m) was shallower than pink salmon (0.6–0.8 m). Further, modal swimming speeds measured by calibrated EMG for chum salmon (0.2–0.4 BL s⁻¹) were slower than pink salmon (1.2–1.4 BL s⁻¹). Pink salmon swam faster as well as in relatively deeper than chum salmon, suggesting that they expend more energy than chum salmon in the reconstructed segment. Based on these results, it seemed likely that the upstream migration behavior of chum and pink salmon was different with species-specific strategies.     

A modified drinking water quality index (DWQI) for assessing drinking source water quality in rural communities of Khuzestan Province,Iran

We reconsidered the Canadian Council of Ministers of the Environment Water Quality Index (CCME WQI) to achieve an efficient drinking water quality index (DWQI) for assessment of drinking source water quality in rural communities of Khuzestan Province, Iran in 2009–2013. In contribution with a panel of water quality experts, the CCME WQI was mainly modified by four changes: (1 and 2) assigning weight factors for input parameters and index factors, (3) modifying excursion concept for carcinogens and bioaccumulative pollutants and (4) removing effect of unequal measurements of input parameters. The DWQI characterizes the drinking source water quality through comparing the measured values of input parameters with relevant benchmarks. The DWQI score (from 0 to 100) classifies the water quality in five categories as poor (0–54.9), marginal (55.0–69.9), fair (70.0–84.9), good (85.0–94.9) and excellent (95.0–100). Based on the DWQI, the temporal changes of the rural drinking source water quality were not significant; while the spatial variations of the water quality were considerable across the province, so the DWQI scores in the northern counties were higher than that in the southern ones. At the county level, the highest and lowest average scores of the DWQI (±standard deviation: SD) were observed in Izeh and Shadegan to be 90 ± 5 and 69 ± 10, respectively. Based on the DWQI, proportions of the drinking water sources with the excellent, good, fair, marginal and poor qualities were determined to be 6.7, 59.1, 26.2, 7.8 and 0.1%, respectively. Turbidity and Ryznar Index (RI) were introduced respectively as the health-based and esthetic parameters with the most violations (22.7 and 63.2%, respectively). The results of the case study and sensitivity analysis indicated that the DWQI is a simple, flexible, stable and reliable index and could be used as an effective tool to characterize drinking source water quality.     

Investigating the effects of point source and nonpoint source pollution on the water quality of the East River (Dongjiang) in South China

Understanding the physical processes of point source (PS) and nonpoint source (NPS) pollution is critical to evaluate river water quality and identify major pollutant sources in a watershed. In this study, we used the physically-based hydrological/water quality model, Soil and Water Assessment Tool, to investigate the influence of PS and NPS pollution on the water quality of the East River (Dongjiang in Chinese) in southern China. Our results indicate that NPS pollution was the dominant contribution (>94%) to nutrient loads except for mineral phosphorus (50%). A comprehensive Water Quality Index (WQI) computed using eight key water quality variables demonstrates that water quality is better upstream than downstream despite the higher level of ammonium nitrogen found in upstream waters. Also, the temporal (seasonal) and spatial distributions of nutrient loads clearly indicate the critical time period (from late dry season to early wet season) and pollution source areas within the basin (middle and downstream agricultural lands), which resource managers can use to accomplish substantial reduction of NPS pollutant loadings. Overall, this study helps our understanding of the relationship between human activities and pollutant loads and further contributes to decision support for local watershed managers to protect water quality in this region. In particular, the methods presented such as integrating WQI with watershed modeling and identifying the critical time period and pollutions source areas can be valuable for other researchers worldwide.     

Evaluations of a flat-panel based compact daily quality assurance device for proton pencil beam scanning (PBS) system

PurposeTo evaluate the flat-panel detector quenching effect and clinical usability of a flat-panel based compact QA device for PBS daily constancy measurements.Materials & MethodThe QA device, named Sphinx Compact, is composed of a 20x20 cm² flat-panel imager mounted on a portable frame with removable plastic modules for constancy checks of proton energy (100 MeV, 150 MeV, 200 MeV), Spread-Out-Bragg-Peak (SOBP) profile, and machine output. The potential quenching effect of the flat-panel detector was evaluated. Daily PBS QA tests of X-ray/proton isocenter coincidence, the constancy of proton spot position and sigma as well as the energy of pristine proton beam, and the flatness of SOBP proton beam through the 'transformed' profile were performed and analyzed. Furthermore, the sensitivity of detecting energy changes of pristine proton beam was also evaluated.ResultsThe quenching effect was observed at depths near the pristine peak regions. The flat-panel measured range of the distal 80% is within 0.9 mm to the defined ranges of the delivered proton beams. X-ray/proton isocenter coincidence tests demonstrated maximum mismatch of 0.3 mm between the two isocenters. The device can detect 0.1 mm change of spot position and 0.1 MeV energy changes of pristine proton beams. The measured transformed SOBP beam profile through the wedge module rendered as flat.ConclusionsEven though the flat-panel detector exhibited quenching effect at the Bragg peak region, the proton range can still be accurately measured. The device can fulfill the requirements of the daily QA tests recommended by the AAPM TG224 Report.     

利用RIL群体进行小麦品质性状及其与产量性状的相关分析

利用小麦重组自交系(RIL)群体,对小麦主要品质性状、及其与产量的关系进行了分析。结果表明,RIL群体品质、产量性状都表现了较大的变异幅度;蛋白质各性状内、淀粉各性状内相关性多数显著,但二者之间相关性多不显著,这说明在小麦品种改良中,优良的蛋白质性状和优良的淀粉性状可以兼得;稳定时间、沉降值等重要的加工品质性状和淀粉性状与产量相关不显著,说明加工品质可以与产量很好地协调起来;适当降低千粒重,增加穗数,有利于强筋的形成和粘度的提高;矮杆、半矮杆性状能够与良好的加工品质、高蛋白质含量、高GMP含量协调起来。     

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R² ⩾ 0.999) and faecal samples (R² ⩾ 0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective amplifications by quantitative PCR would be one way to combine the advantages of the two technologies.