Real-Time PCR and Sequencing Assays for Rapid Detection and Identification of Avian Schistosomes in Environmental Samples

Cercarial dermatitis, also known as swimmer''s itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification.     

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.     

Comparison of Two Quantitative Real Time PCR Assays for Rickettsia Detection in Patients from Tunisia

Background and objectives

Quantitative real time PCR (qPCR) offers rapid diagnosis of rickettsial infections. Thus, successful treatment could be initiated to avoid unfavorable outcome. Our aim was to compare two qPCR assays for Rickettsia detection and to evaluate their contribution in early diagnosis of rickettsial infection in Tunisian patients.

Patients and methods

Included patients were hospitalized in different hospitals in Tunisia from 2007 to 2012. Serology was performed by microimmunofluorescence assay using R. conorii and R. typhi antigens. Two duplex qPCRs, previously reported, were performed on collected skin biopsies and whole blood samples. The first duplex amplified all Rickettsia species (PanRick) and Rickettsia typhi DNA (Rtt). The second duplex detected spotted fever group Rickettsiae (RC00338) and typhus group Rickettsiae DNA (Rp278).

Results

Diagnosis of rickettsiosis was confirmed in 82 cases (57.7%). Among 44 skin biopsies obtained from patients with confirmed diagnosis, the first duplex was positive in 24 samples (54.5%), with three patients positive by Rtt qPCR. Using the second duplex, positivity was noted in 21 samples (47.7%), with two patients positive by Rp278 qPCR. Among79 whole blood samples obtained from patients with confirmed diagnosis, panRick qPCR was positive in 5 cases (6.3%) among which two were positive by Rtt qPCR. Using the second set of qPCRs, positivity was noted in four cases (5%) with one sample positive by Rp278 qPCR. Positivity rates of the two duplex qPCRs were significantly higher among patients presenting with negative first serum than those with already detectable antibodies.

Conclusions

Using qPCR offers a rapid diagnosis. The PanRick qPCR showed a higher sensitivity. Our study showed that this qPCR could offer a prompt diagnosis at the early stage of the disease. However, its implementation in routine needs cost/effectiveness evaluation.     

Genotyping of Giardia duodenalis Cysts by New Real-Time PCR Assays for Detection of Mixed Infections in Human Samples

Of the seven genetic groups, or assemblages, currently recognized in the Giardia duodenalis species complex, only assemblages A and B are associated with human infection, but they also infect other mammals. Recent investigations have suggested the occurrence of genetic exchanges among isolates of G. duodenalis, and the application of assemblage-specific PCR has shown both assemblages A and B in a significant number of human infections. In this work, three real-time quantitative (qPCR) assays were developed to target the G. duodenalis triose phosphate isomerase, glutamate dehydrogenase, and open reading frame C4 sequences. Primers were designed to allow the specific amplification of the DNA of assemblage A or B and to generate products distinguishable by their melting curves or, after qPCR, by their sequences, sizes, or restriction patterns. The assays showed full specificity and detected DNA from a single trophozoite (4 to 8 target copies). We applied these assays, as well as a TaqMan assay that targets the β-giardin gene, to genomic DNA extracted from 30 human stools and to Giardia cysts purified by immunomagnetic capture from the same samples. Simultaneous detection of both assemblages was observed in a large number of DNAs extracted from stools, and experiments on the cysts purified from the same samples showed that this was essentially attributable to mixed infections, as only one assemblage was detected when dilutions of cysts were tested. In a few cases, detection of both assemblages was observed even when single cysts were tested. This result, which suggests the presence of recombinants, needs to be confirmed using more accurate methods for cyst separation and enumeration. The assays described in this study can be used to detect Giardia cysts infectious to humans in samples from animals and in water and food.Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) is the only species within the genus Giardia that infects humans, although it is also found in other mammals, including pets and livestock (1). The infection has a global distribution and, with an estimated 2.8 × 10⁸ cases per year, represents the most common gastrointestinal parasitic infection of humans in developed countries (20). In Asia, Africa, and Latin America, about 200 million people have symptomatic giardiasis, with some 500,000 new cases reported each year (35). Several characteristics of G. duodenalis influence the epidemiology of infection: (i) in humans, the infective dose is about 10 to 100 cysts; (ii) cysts are immediately infectious when excreted in feces and can be transmitted by person-to-person or animal-to-animal contact; (iii) cysts are remarkably stable and can survive for weeks to months in the environment; and (iv) environmental contamination can lead to the contamination of drinking water and food (6, 32).A considerable amount of data has shown that G. duodenalis should be considered a species complex whose members show little variation in their morphology yet can be assigned to at least seven distinct assemblages (A to G) based on genetic analyses (7, 34). The analysis of more than a thousand human isolates from different geographical locations, examined by PCR amplification of DNA extracted directly from feces, has demonstrated that in almost all cases, only G. duodenalis assemblages A and B are associated with human infections (6). The prevalence of each assemblage varies considerably from country to country; assemblage B seems more common overall, but no strong conclusions can be drawn from current data. The remaining assemblages (C to G) are likely to be host specific, as assemblages C and D have been identified in dogs, cats, coyotes, and wolves; assemblage E in cattle, sheep, goats, pigs, water buffaloes, and muflons; assemblage F in cats; and assemblage G in rats.The epidemiology of human giardiasis is further complicated by the occurrence of mixed infections and the possibility of genetic exchanges between isolates of assemblage A (10) or even between isolates of assemblages A and B (21, 33). Ideally, genotyping should be performed on single cysts, as this allows a distinction between mixed infections and recombinants. To reach this technically demanding high level of sensitivity and specificity, real-time quantitative PCR (qPCR) appears to be a promising technique.This work describes the development of new qPCR assays that, through the use of assemblage-specific primers, allow the specific and simultaneous detection of DNAs of assemblages A and B. The application of these assays to DNA extracted from human stools and to cysts purified from the same samples is described.     

Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of Mycobacterium ulcerans in Clinical and Environmental Samples

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.     

Real-Time PCR Assays for Quantification of qnr Genes in Environmental Water Samples and Chicken Feces

Real-time PCR assays were developed for the enumeration of plasmid-mediated quinolone resistance (PMQR) determinants, such as the qnrA, qnrB, and qnrS genes, in different water samples and chicken feces. The results indicate that the developed assays are specific and sensitive for the quantification of qnr genes in complex samples.     

Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

Background

Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis.

Methods

From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results.

Results

Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens.

Conclusion

PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml.     

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.     

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.     

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples

BackgroundThe lack of an effective diagnostic tool for Carrion’s disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches.Conclusions/SignificanceFrom the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion’s disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.     

Development of cpn60-Based Real-Time Quantitative PCR Assays for the Detection of 14 Campylobacter Species and Application to Screening of Canine Fecal Samples

Campylobacter species are important organisms in both human and animal health. The identification of Campylobacter currently requires the growth of organisms from complex samples and biochemical identification. In many cases, the condition of the sample being tested and/or the fastidious nature of many Campylobacter species has limited the detection of campylobacters in a laboratory setting. To address this, we have designed a set of real-time quantitative PCR (qPCR) assays to detect and quantify 14 Campylobacter species, C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hyointestinalis, C. jejuni, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, and C. upsaliensis, directly from DNA extracted from feces. By use of a region of the cpn60 (also known as hsp60 or groEL) gene, which encodes the universally conserved 60-kDa chaperonin, species-specific assays were designed and validated. These assays were then employed to determine the prevalence of Campylobacter species in fecal samples from dogs. Fecal samples were found to contain detectable and quantifiable levels of C. fetus, C. gracilis, C. helveticus, C. jejuni, C. showae, and C. upsaliensis, with the majority of samples containing multiple Campylobacter species. This study represents the first report of C. fetus, C. gracilis, C. mucosalis, and C. showae detection in dogs and implicates dogs as a reservoir for these species. The qPCR assays described offer investigators a new tool to study many Campylobacter species in a culture-independent manner.Campylobacter species are important in both the medical and veterinary arenas. Currently, of the 19 Campylobacter species that have been classified (8; http://www.bacterio.cict.fr/), 10 or 11 species/subspecies have well-established associations with animal or human diseases, respectively (4, 20). Of significance is the role campylobacters play as the most common cause of enteric disease worldwide (34). Although most enteric disease is attributed to Campylobacter jejuni and C. coli, advances in cultivation techniques have revealed that other “unusual” campylobacters, such as C. upsaliensis, C. concisus, C. lari, and C. hyointestinalis, may be more commonly associated with gastrointestinal (GI) disease than was previously recognized (6, 17, 21, 27). Beyond being associated with enteric disease, C. rectus and C. gracilis have been directly associated with periodontal disease (24, 31, 32), and of note regarding livestock, C. fetus is an important venereal disease (2). Furthermore, a number of Campylobacter species appear to be part of the normal microbiota of healthy animals; for example, C. jejuni, C. coli, C. helveticus, C. upsaliensis, and C. lari are part of the normal microbiota of domestic dogs and cats (11, 15, 30).Quick and reliable identification and discrimination of Campylobacter species remain challenging. Culture from clinical specimens is often very sensitive but limited by several factors. While a number of different selective media have been specifically designed for Campylobacter species isolation, the fastidious nature and varied requirements of members of this genus mean that there is no single growth condition that is optimal for all species. Transport time from sampling to processing is also an important consideration, especially when sampling is done at some distance from the laboratory or a large number of samples are collected simultaneously. Prolonged time between sample collection and processing can reduce the success of Campylobacter isolation. Processing times as short as 4 h from the time of sample collection may be required for isolation of multiple Campylobacter species (15, 16).Routine identification of cultured Campylobacter species is based on biochemical profiling (29). This, however, has become increasingly problematic, as phenotypic profiles used to distinguish species, such as the hippurate hydrolysis assay to discriminate C. jejuni and C. coli, have become varied within species (29). There are also cases where multiple species share the same basic biochemical characteristics (such as C. mucosalis and C. concisus), leading to disputes over the definitive identities of isolated Campylobacter strains (9, 18, 19). The similar phenotypic and biochemical profiles of other closely related gram-negative curved rods, such as Arcobacter and Helicobacter spp., may further complicate the identification of Campylobacter isolates.To circumvent some of the limitations of biochemical profiling, various DNA-based identification methods have been developed. Methods with high discrimination potential for Campylobacter strains, like macrorestriction analysis of the whole genome by pulsed-field gel electrophoresis or of certain loci by restriction fragment length polymorphism, amplified fragment length polymorphism, and multilocus sequence typing, are available (20). However, these identification techniques are best suited for short- and long-term epidemiological studies in which the identity of an individual strain is of interest for source tracking or population phylogenies. These techniques also require considerable operator skill to generate reproducible patterns for comparison or the sequencing of multiple regions for identification. For targeted identification, a number of PCR strategies, both conventional and quantitative, based on the 16S rRNA gene or unspecified species- or subspecies-specific regions, have been designed (3, 22, 23, 26, 28). Unfortunately, strategies to date have focused on the detection of a single species or subspecies (26, 28), the genus as a whole (3), or a subset of species to the exclusion of others (22, 23). At the present, there is simply no way to quickly and reliably detect and identify many Campylobacter species.We have designed a set of real-time quantitative PCR (qPCR) assays to identify and quantify 14 Campylobacter species, C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hyointestinalis, C. jejuni, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, and C. upsaliensis, directly from DNA extracted from feces. These assays are based on the cpn60 gene, which encodes the universal 60-kDa chaperonin (also known as HSP60 or GroEL). The utility of this target has been demonstrated through its ability to identify and differentiate Campylobacter species from each other and from Helicobacter and Arcobacter species (12). This target is also supported by the reference database cpnDB (13; http://cpndb.cbr.nrc.ca), a curated collection of cpn60 sequences from thousands of type strains, reference strains, and clinical isolates. To validate and test our qPCR assays, we applied them in a survey of fecal samples from a population of dogs from a rural community of northern Saskatchewan, Canada.     

Detection and Quantification of Wallemia sebi in Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 10⁷ m⁻³ by real-time PCR and 10⁶ m⁻³ by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment.     

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.     

Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves

Background

Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples.

Methodology/Principal Findings

In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the “comparative Ct method”. Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy.

Conclusions/Significance

The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.     

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.Phytoplasmas are wall-less bacteria of the class Mollicutes that inhabit plant phloem and insect vectors. They are known to cause disease in hundreds of plant species worldwide and are classified into 16Sr groups and “Candidatus Phytoplasma” species based on their 16S rRNA gene sequences (14, 21). Since it is not possible to isolate and study phytoplasmas in pure cultures, conventional PCR has become the method of choice for detection and diagnosis. Numerous PCR primer combinations have been devised to amplify the 16S rRNA gene for diagnostics; some of these are universal primers that work on DNA from all phytoplasma phylogenetic groups, while others are group specific (8, 10, 17). However, diagnostics based on these primers can be problematic, with occasional false positives through amplification of other bacteria that might be present in samples (11).Real-time PCR assays have also been developed for both generic and specific phytoplasma detection. In general, the aims have been to produce very specific and sensitive assays for detection of a group-specific nature. For example, TaqMan and SYBR green chemistries have been applied in various diagnostic assays for 16SrX group (1, 3, 18), 16SrV and -XII group (2, 9, 15), 16SrI group (16, 20), and 16SrVI group (5) phytoplasmas. However, several of these specific assays cross-react with phytoplasmas from other groups (5, 16). The most successful attempt to develop a fully universal assay has been a TaqMan assay that was demonstrated to amplify all 16Sr groups except 16SrIV, -XIII, and -XIV, which were not tested (4).     

Evaluation and Validation of a Real-Time PCR Assay for Detection and Quantitation of Human Adenovirus 14 from Clinical Samples

In 2007, the Centers for Disease Control and Prevention (CDC) reported that Human adenovirus type 14 (HAdV-14) infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10² to 10⁷ copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.     

Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.