Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S.cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 × 10⁵ to 3.8 CFU/ml in sweet wine and from 5 × 10⁶ to 50CFU/ml in red wine.     

Enumeration of Salmonellae in Table Eggs,Pasteurized Egg Products,and Egg-Containing Dishes by Using Quantitative Real-Time PCR

Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (C_q) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.     

Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10³ CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.     

Viability PCR,a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.Legionella organisms are ubiquitous bacteria found in many types of water sources in the environment. Their growth is especially favored in human-made warm water systems, including cooling towers, hot tubs, showerheads, and spas (3, 14, 15, 38). Legionella bacteria replicate as intracellular parasites of amoebae and persist in the environment as free-living microbes or in biofilms. In aerosol form, they enter the lungs and can cause an acute form of pneumonia known as Legionnaires'' disease or a milder form of pulmonary infection called Pontiac fever. The species Legionella pneumophila is responsible for the vast majority of the most severe form of this atypical pneumonia (52, 70). Legionellosis outbreaks are associated with high mortality rates (15 to 20%) (15, 16, 38, 46), which can reach up to 50% for people with weakened immune systems (immunocompromised patients) (69). Legionella surveillance programs include regular monitoring of environmental water samples (9, 13, 66). It is generally acknowledged that Legionella represents a health risk to humans when cell densities are greater than 10⁴ to 10⁵ CFU per liter of water, and epidemiological data show that outbreaks of legionellosis occur at these concentrations (36, 47).The evaluation of the risk associated with Legionella has traditionally been performed using culture-based methods (1, 24). Culture is essential for identifying and typing Legionella strains during epidemics. However, Legionella culture requires long incubation times (up to 10 days) before results can be scored. This problem makes culture unsuitable for preventive actions and rapid response in emergency situations. Moreover, under certain conditions (i.e., low-nutrient environments, oxidative or osmotic stress, etc.), Legionella cells can lose the ability to be cultured, although they are still viable (7, 17, 20, 22, 39, 45, 67). These viable but nonculturable (VBNC) Legionella cells may still represent a public health hazard because they can regain their ability to grow in new, more favorable conditions (12, 19, 23, 61).Molecular approaches, such as quantitative real-time PCR (qPCR), are faster and can mitigate the main drawbacks of culture-based methods. qPCR is an alternative tool that offers rapid, sensitive, and specific detection of Legionella bacteria in environmental water samples (4, 5, 12, 26, 65, 68). PCR results can be obtained in hours instead of days, and VBNC Legionella cells can also be detected (12, 26). However, the major disadvantage of qPCR lies in its inability to evaluate viability due to the persistence of DNA in cells after death (27, 34). The monitoring of Legionella contamination levels by conventional qPCR may thus result in an overestimation of the risk of infection because false-positive results can be scored. However, the real risk from Legionella is limited to the live fraction of the total Legionella population. Only live or viable Legionella cells are able to replicate in pulmonary macrophages and cause severe pneumonia (14, 15). The development of more rapid, culture-independent methods capable of discriminating between live and dead cells is of major interest for measuring Legionella infection risks and preventing legionellosis. The nucleic acid-binding dye ethidium monoazide bromide (EMA), used in combination with qPCR, is an attractive alternative for selectively detecting and enumerating viable bacteria. EMA is particularly useful because it selectively penetrates cells with damaged membranes and covalently binds to DNA after photoactivation (21, 53). DNA-bound EMA molecules prevent PCR amplification and thereby lead to a strong signal reduction during qPCR. DNA from viable cells with intact cell membranes prevents EMA molecules from entering the cell and therefore can be amplified and quantified (56). Nocker et al. (41, 42) suggested that the signal reduction was due to a selective loss of genomic DNA from dead cells (rendered insoluble after cross-linkage) during the DNA extraction procedure rather than to PCR inhibition. However, Soejima et al. (59, 60) recently reported that treatment with EMA followed by visible light irradiation directly cleaves the chromosomal DNA of dead bacteria.In this study we optimized the EMA-staining procedure in conjunction with qPCR with pure cultures of L. pneumophila. We analyzed the potential for the EMA-qPCR method to discriminate Legionella cells with compromised or intact cell membranes. We optimized this EMA-qPCR technique, viability PCR, hereafter named v-PCR, and used it to quantify viable Legionella cells in environmental water samples. We compared our results with those obtained by conventional qPCR and culture methods. In addition, we evaluated the ability of v-PCR to monitor the efficacy of different disinfection strategies.     

Rapid Quantitative Detection of Listeria monocytogenes in Meat Products by Real-Time PCR

We describe a quick and simple method for the quantitative detection of Listeria monocytogenes in meat products. This method is based on filtration, Chelex-100-based DNA purification, and real-time PCR. It can detect as few as 100 CFU/g and quantify as few as 1,000 CFU/g, with excellent accuracy compared to that of the plate count method. Therefore, it is a promising alternative for the detection of L. monocytogenes in meat products.     

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10⁻² to 1.4 × 10⁻² pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.     

Development of a Real-Time qPCR Method for Detection and Enumeration of Mycobacterium spp. in Surface Water

A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).Water exposure (15) is one source of human infection caused by nontuberculous mycobacteria (NTM). Nevertheless, the isolation and enumeration of NTM from water is difficult because other microorganisms overgrow NTM colonies (22). Consequently, the development of an alternative detection and enumeration method is essential for monitoring NTM sources in the environment.Two real-time quantitative PCR (qPCR) methods for NTM measurement have been described (7, 29). The primer pair used in the first real-time qPCR method (7) targets 16S rRNA and was previously used to track mycobacterial growth in industrial water samples by conventional PCR (31). It was presented as a sensitive test for members of the Mycobacterium genus because it detected 34 species of mycobacteria (19, 25). However, the primer specificity was only measured by conventional PCR against DNA of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus (31) or by in silico analysis (7). The second real-time qPCR method, targeting the hsp65 gene (29), was also sensitive (detection of 34 out of 37 Mycobacterium spp. tested). Although the primers showed high specificity (no detection of 16 different nonmycobacterial species) by conventional PCR (21), their specificity combined with the qPCR probe was only tested against Candida albicans DNA (29).We sought to develop a reliable real-time qPCR method to detect Mycobacterium spp. in water samples. The development involved in silico primer screening followed by a specificity study by conventional PCR. Furthermore, the efficiency (Ef), correlation coefficient (r²), limit of quantification (LOQ), specificity (Sp), and sensitivity (Ss) of this new method targeting 16S rRNA were compared with those of the two previously described methods (7, 29).     

Quantitative Detection of Schistosoma japonicum Cercariae in Water by Real-Time PCR

In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R² = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas.     

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.Methanogens are integral to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent greenhouse gases, during organic matter degradation in anaerobic soils and sediment (8). Methanogens are widespread in anaerobic environments, including tundra (36), freshwater lake and wetland sediments (9, 12), estuarine and marine sediments (2), acidic peatlands (4, 14), rice field soil (10, 16), animal guts (41), landfills (30), and anaerobic digesters treating animal manure (1), food processing wastewater (27), and municipal wastewater and solid waste (37, 57). Methane produced in anaerobic digesters may be captured and used for energy production, thus offsetting some or all of the cost of operation and reducing the global warming potential of methane release to the atmosphere.Methanogens are difficult to study through culture-based methods, and therefore many researchers have instead used culture-independent techniques to study methanogen populations. The 16S rRNA gene is the most widely used target for gene surveys, and a number of primers and probes have been developed to target methanogen groups (9, 11, 31, 36, 38, 40, 46, 48, 57). To eliminate potential problems with nonspecific amplification, some researchers have developed primers for the gene sequence of the α-subunit of the methyl coenzyme M reductase (mcrA) (17, 30, 49). The Mcr is exclusive to the methanogens with the exception of the methane-oxidizing Archaea (18) and shows mostly congruent phylogeny to the 16S rRNA gene, allowing mcrA analysis to be used in conjunction with, or independently of, that of the 16S rRNA gene (3, 30, 49). A number of researchers have examined methanogen communities with mcrA and have found uncultured clades quite different in sequence from cultured methanogen representatives (9, 10, 12, 14, 17, 22, 28, 47).Previous studies described methanogen communities by quantitation of different clades through the use of rRNA-targeted or rRNA gene-targeted probes with techniques such as dot blot hybridization (1, 27, 37, 38, 48) and fluorescent in situ hybridization (11, 40, 44, 57). Real-time quantitative PCR (qPCR) is an alternate technique capable of determining the copy number of a particular gene present in the DNA extracted from an environmental sample. Only a few studies have used qPCR to quantitatively examine different clades within methanogen communities, and most of these studies have exclusively targeted the 16S rRNA gene (19, 41, 42, 54-56). Far fewer researchers have used qPCR to quantify methanogen clades by targeting the mcrA (21, 34, 45), and these studies were limited to only a few phylogenetic groups.In this paper we present a methodology for determining methanogen gene copy numbers through the use of qPCR targeting the mcrA. Methanogens were quantified in total using methanogen-specific primers in SYBR green assays and also as members of nine different phylogenetic groups using TaqMan probes targeting specific subsets of methanogens.     

Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.     

Differential Real-Time PCR Assay for Enumeration of Lactic Acid Bacteria in Wine

Oenococcus oeni is often employed to perform the malolactic fermentation in wine production, while nonoenococcal lactic acid bacteria often contribute to wine spoilage. Two real-time PCR assays were developed to enumerate the total, and nonoenococcal, lactic acid bacterial populations in wine. Used together, these assays can assess the spoilage risk of juice or wine from lactic acid bacteria.     

TaqMan MGB Probe Fluorescence Real-Time Quantitative PCR for Rapid Detection of Chinese Sacbrood Virus

Sacbrood virus (SBV) is a picorna-like virus that affects honey bees (Apis mellifera) and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV) in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB) probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.     

枣疯病植原体实时荧光定量PCR检测方法的研究

目的:建立枣疯病植原体拷贝数检测实时荧光定量PCR方法,为枣疯病植原体定量检测提供技术支持。方法:构建质粒标准品,设计实时荧光PCR探针引物,优化体系,建立标准曲线,并进行重复性验证。结果:制备了枣疯病植原体标准质粒,建立了稳定的质粒标准品检测体系(R2=0.998,检测限10拷贝,定量限100拷贝)。结论:实时荧光定量PCR检测方法重复性好,可用于枣疯病植原体的拷贝数检测,为枣疯病植原体检验检测和病害防治提供了技术支持。     

An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve

The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r² = 0.72, slope = 1.07, p < 0.001). Therefore, this improved qPCR method should be a powerful tool for the accurate quantification of H. akashiwo cysts in sediment samples.     

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log₁₀. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log₁₀. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log₁₀ lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log₁₀. Conversely, sensitivity was only 0.30 log₁₀ better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.     

Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.     

Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.     

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.     

Quantitative Real-Time PCR Assays for Sensitive Detection of Canada Goose-Specific Fecal Pollution in Water Sources

Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.The Canada goose (Branta canadensis) is a prevalent waterfowl species in North America. The population density of Canada geese has doubled during the past 15 years, and the population was estimated to be close to 3 million in 2007 (4). Canada geese often congregate within urban settings, likely due to available water sources, predator-free grasslands, and readily available food supplied by humans (6). They are suspected to contribute to pollution of aquatic environments due to the large amounts of fecal matter that can be transported into the water. This can create a public health threat if the fecal droppings contain pathogenic microorganisms (6, 7, 9, 10, 12, 13, 19). Therefore, tracking transient fecal pollution of water due to fecal inputs from waterfowl, such as Canada geese, is of importance for protecting public health.PCR detection of host-specific 16S rRNA gene sequences from Bacteroidales of fecal origin has been described as a promising microbial source-tracking (MST) approach due to its rapidity and high specificity (2, 3). Recently, Lu et al. (15) characterized the fecal microbial community from Canada geese by constructing a 16S rRNA gene sequence database using primers designed to amplify all bacterial 16S rRNA gene sequences. The authors reported that the majority of the 16S rRNA gene sequences obtained were related to Clostridia or Bacilli and to a lesser degree Bacteroidetes, which represent possible targets for host-specific source-tracking assays.The main objective of this study was to identify novel Bacteroidales 16S rRNA gene sequences that are specific to Canada goose feces and design primers and TaqMan fluorescent probes for sensitive and specific quantification of Canada goose fecal contamination in water sources.Primers 32F and 708R from Bernhard and Field (2) were used to construct a Bacteroidales-specific 16S rRNA gene clone library from Canada goose fecal samples (n = 15) collected from grass lawns surrounding Wascana Lake (Regina, SK, Canada) in May 2009 (for a detailed protocol, see File S1 in the supplemental material). Two hundred eighty-eight clones were randomly selected and subjected to DNA sequencing (at the Plant Biotechnology Institute DNA Technologies Unit, Saskatoon, SK, Canada). Representative sequences of each operational taxonomic unit (OTU) were recovered using an approach similar to that described by Mieszkin et al. (16). Sequences that were less than 93% similar to 16S rRNA gene sequences from nontarget host species in GenBank were used in multiple alignments to identify regions of DNA sequence that were putatively goose specific. Subsequently, two TaqMan fluorescent probe sets (targeting markers designated CGOF1-Bac and CGOF2-Bac) were designed using the RealTimeDesign software provided by Biosearch Technologies (http://www.biosearchtech.com/). The newly designed primer and probe set for the CGOF1-Bac assay included CG1F (5′-GTAGGCCGTGTTTTAAGTCAGC-3′) and CG1R (5′-AGTTCCGCCTGCCTTGTCTA-3′) and a TaqMan probe (5′-6-carboxyfluorescein [FAM]-CCGTGCCGTTATACTGAGACACTTGAG-Black Hole Quencher 1 [BHQ-1]-3′), and the CGOF2-Bac assay had primers CG2F (5′-ACTCAGGGATAGCCTTTCGA-3′) and CG2R (5′-ACCGATGAATCTTTCTTTGTCTCC-3′) and a TaqMan probe (5′-FAM-AATACCTGATGCCTTTGTTTCCCTGCA-BHQ-1-3′). Oligonucleotide specificities for the Canada goose-associated Bacteroides 16S rRNA primers were verified through in silico analysis using BLASTN (1) and the probe match program of the Ribosomal Database Project (release 10) (5). Host specificity was further confirmed using DNA extracts from 6 raw human sewage samples from various geographical locations in Saskatchewan and 386 fecal samples originating from 17 different animal species in Saskatchewan, including samples from Canada geese (n = 101) (Table ​(Table1).1). An existing nested PCR assay for detecting Canada goose feces (15) (targeting genetic marker CG-Prev f5) (see Table S1 in the supplemental material) was also tested for specificity using the individual fecal and raw sewage samples (Table ​(Table1).1). All fecal DNA extracts were obtained from 0.25 g of fecal material by using the PowerSoil DNA extraction kit (Mo Bio Inc., Carlsbad, CA) (File S1 in the supplemental material provides details on the sample collection).

TABLE 1.

Specificities of the CGOF1-Bac, CGOF2-Bac, and CG-Prev f5 PCR assays for different species present in Saskatchewan, Canada