Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

Background

Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of ±25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4–8 determinations from each amplification reaction.

Findings

Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp) is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy.

Conclusions

Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the potential to fundamentally transform how real-time qPCR is conducted.     

TaqMan-Based Real-Time PCR Method for Detection of Yersinia pseudotuberculosis in Food

A sensitive and specific assay for detection of food-borne pathogenic Yersinia pseudotuberculosis was developed. The primer-probe set was designed to target a 157-bp sequence of the chromosomally located gene ail. The complete method, including an internal amplification control, was evaluated for several different food items.     

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r² = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

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

Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

Comparison of Cryptosporidium parvum Viability and Infectivity Assays following Ozone Treatment of Oocysts

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4°C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4′,6′-diamidino-2-phenylindole–propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a “gold standard” until suitable alternative viability surrogates are identified for disinfection studies.     

Identification and Validation of Suitable Housekeeping Genes for Normalizing Quantitative Real-Time PCR Assays in Injured Peripheral Nerves

Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.     

实时定量PCR法检测生物技术药物中宿主基因组DNA残留

利用基于SYBR GreenⅠ荧光染料的实时定量PCR方法检测酵母表达生物技术药物产品中宿主DNA残留量。该方法检测灵敏度可达到1.0 fg/μL, DNA浓度在1.0 fg/μL～1.0 ng/μL范围内线性良好,其标准曲线的相关系数为099以上。应用该方法对3批不同实验样本进行测定,宿主DNA残留量分别为8.635×105 fg/μL、6.265×102 fg/μL和1436 fg/μL 。实验表明该方法操作简便、灵敏度高,可用于生物技术药物产品中酵母DNA残留的定量测定。     

Fate of Cryptosporidium Oocysts and Giardia Cysts in the Norwegian Aquatic Environment over Winter

We investigated the survival of Cryptosporidium oocysts and Giardia cysts during winter in an aquatic environment (approximate temperature measurements between 1 and 7°C) in Norway, using morphology and uptake of dyes as indicators of viability. Previous research has shown that in the terrestrial environment, shear forces caused by freeze and thaw cycles probably cause the parasites to be inactivated. Such forces occurred infrequently in the aquatic environment, as freezing of the water around the parasites was not observed during the study period (although freezing of the water surface did occur). The rate of decline in viability (log₁₀ N _t/N ₀) was similar in control and experimental environments for both parasites; no Cryptosporidium oocysts with viable morphology were detected after approximately 20 weeks and no Giardia cysts with apparently viable morphology could be detected after 1 month. These results suggest that infection with these parasites in Norway is not usually from transmission stages that have overwintered in the Norwegian environment.     

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R² > 0.98) over a 7-log-unit dynamic range down to 10¹ B. atrophaeus cells or spores. Quantification of S. marcescens (R² > 0.98) was linear over a 6-log-unit dynamic range down to 10² S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.     

Development of TaqMan-Based Quantitative PCR for Sensitive and Selective Detection of Toxigenic Clostridium difficile in Human Stools

Background

Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.

Methods

TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).

Results

The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA⁺TcdB⁺, TcdA⁻TcdB⁺, and TcdA⁻TcdB⁻ types), TcdB-producing strains (TcdA⁺TcdB⁺ and TcdA⁻TcdB⁺ types), and TcdA-producing strains (TcdA⁺TcdB⁺ type), respectively, with a lower detection limit of 10³ cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA⁺TcdB⁺ strain in six specimens and TcdA⁻TcdB⁻ strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 10^4.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.

Conclusion

Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.     

Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.Toxoplasma gondii is a ubiquitous parasite found in all classes of warm-blooded vertebrates. Nearly one-third of humans have been exposed to this parasite (15). In immunocompetent adults, acute infection normally results in transient influenza-like symptoms, but in immunocompromised persons retinochoroiditis and encephalitis are more common. Infected individuals can retain the parasite as quiescent tissue cysts for long periods, but invasive infection can occur if the immune status of the infected person deteriorates (42). If women become infected during pregnancy, the parasite can cause abortion or seriously damage the fetus. The potential morbidity from the ingestion of oocysts of T. gondii and the organism''s low infectious dose are a great concern for public health. There are at least four reported waterborne outbreaks of toxoplasmosis (2, 3, 14, 44), and endemic toxoplasmosis in Brazil is associated with the consumption of water or ice contaminated with T. gondii oocysts (1, 23), demonstrating the potential for the waterborne transmission of this disease (15).There is no rapid detection method for T. gondii oocysts recovered from water or other environmental samples. Traditionally, the detection of protozoa in water required their concentration from large volumes of water by filtration or centrifugation, isolation from concentrated particulates by immunomagnetic separation (IMS) or other methods, and detection by immunofluorescence microscopy, the infection of cultured cells, biochemistry, animal infection tests, molecular techniques, or combinations of these (17, 58). For T. gondii oocysts there are no commercially available IMS techniques, no widely available immunofluorescent staining reagents, and no standardized cultivation protocols. The identification of oocysts from environmental samples has included differential floatation and mouse inoculation (27). Recently, IMS techniques have been developed for the isolation of T. gondii oocysts and sporocysts in water (16, 18). Both the oocyst and sporocyst IMS assays, however, had poor specificity, because antibodies cross-reacted with water debris and the sporocyst wall of Hammondia hammondi, Hammondia heydorni, and Neospora caninum (16).PCR is becoming a favored technique for the detection of T. gondii oocysts in water (32, 35, 36, 46, 49, 55) over the conventional mouse bioassay (27, 55), as it reduces the detection time from weeks to 1 to 2 days. Although they have been developed for the detection of T. gondii in clinical specimens (50), no real-time PCR assays have been adapted for the detection of oocysts in water samples, possibly because of expected high concentrations of PCR inhibitors and low numbers of T. gondii oocysts in environmental samples (55).There are several unresolved issues regarding the effectiveness of the PCR detection of T. gondii oocysts in water. The most readily available method for the isolation of T. gondii oocysts from water samples is flocculation or sucrose floatation prior to DNA extraction (35, 36, 49, 55). Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor. For DNA extraction, the phenol-chloroform method or QIAamp mini kit frequently is used (16, 35, 36, 46, 55). When oocysts are recovered from water either by the Environmental Protection Agency (EPA) information collection rule method (53) or EPA Method 1623 (54) without purification by IMS, neither the conventional phenol-chloroform DNA extraction nor the QIAamp mini kit is effective at removing PCR inhibitors (30, 55, 57).Recently, a method was used effectively in the analysis of Cryptosporidium oocysts in surface water, storm water, and wastewater samples (30). This method extracted DNA directly from water concentrates without pathogen IMS, differential flotation, or enrichment cultures, and it utilized a commercial DNA extraction kit, the FastDNA spin kit for soil, and a high concentration of nonacetylated bovine serum albumin in PCR. The FastDNA soil kit has a higher capacity for PCR inhibitor removal than several other commercial extraction kits designed for environmental samples. The use of nonacetylated bovine serum in the PCR neutralizes residual PCR inhibitors that are coextracted with the DNA (30).In the present study, the performance of two published LightCycler real-time PCR assays based on the multicopy B1 gene and 529-bp repetitive element (13, 45) and a newly developed LightCycler real-time PCR assay using a common primer set were analyzed for the detection of T. gondii, using pure DNA and DNA extracted by the aforementioned extraction method (30) from water sample concentrates seeded with known number of oocysts.     

Real-Time PCR Assays for Monitoring Anaerobic Fungal Biomass and Population Size in the Rumen

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.     

Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.     

Quantification of Bacterial Uropathogens in Preclinical Samples Using Real-Time PCR Assays

Uropathogenic Escherichia coli (UPEC) and Staphylococcus saprophyticus (S. saprophyticus) are responsible for the majority of community-acquired urinary tract infections (UTI). Agar plating, a gold standard for detection of bacterial uropathogens, is labor intensive, limited for distinguishing between environmental contaminants and pathogens, and fails to effectively detect mixed infections. A reliable method for specific and sensitive quantitative assessment of infections would allow cost-effective evaluation of large numbers of experimental samples. A methodology such as quantitative PCR (qPCR) addresses the limitations of agar plating. We developed and validated highly specific and sensitive qPCR assays to assist researchers in the evaluation of potential vaccines and interventions in preclinical models of UPEC and S. saprophyticus UTI. The developed UPEC PCR targeted a highly conserved region of the UPEC hemolysin D (hlyD) gene that reproducibly detected type strains CFT073 and J96 over a 9 log range with high precision. To quantify S. saprophyticus genomes, a separate qPCR assay targeting the Trk transport gene was developed with an 8 log range. Neither assay detected bacterial species predicted to be sample contaminants. Using our optimized workflow that includes automated steps, up to 200 urine or tissue samples can be processed in as few as 3 h. Additionally, sequence comparisons of our primers and probe to other UTI bacterial strains indicated the broad applicability of these assays. These optimized qPCR assays provide a cost-effective and time-saving method for quantification of bacterial burdens in tissues and body fluids to assess the effectiveness of candidate vaccines or interventions.     

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

Development of Real-Time PCR Assays for Rapid Detection of Pfiesteria piscicida and Related Dinoflagellates

Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.