Direct detection of Campylobacter jejuni in human stool samples by real-time PCR

Our purpose was to establish a quick and accurate real-time PCR (rtPCR) method to detect Campylobacter jejuni directly from human diarrheal stool as an alternative to traditional culture methods. To determine the consistency of rtPCR and culture method, 256 clinical diarrheal stool samples and 50 normal stool samples from healthy individuals were examined, and the whole process was double-blinded. Our data showed that the sensitivity of rtPCR in pure cultures and stool was 102 CFU.mL-1 and 103 CFU.g-1, respectively. Of the 256 diarrheal samples, 10 specimens were successfully detected by both methods, whereas two specimens were PCR positive but culture negative. No positive results were found by these two methods in 50 normal specimens. Our data suggested that rtPCR was convenient in operation and time-saving (turnaround time 3.5-4 h), so it could be used for clinical diagnostic and epidemiological purposes.     

A multi-well filtration assay for quantitation of inositol phosphates in biological samples

The quantitation of inositol phosphates (IPs), mediators of certain signal transduction processes, typically involves laborious and time consuming conventional ion-exchange chromatography procedures. We have developed a high throughput microtiter plate-based IP assay that utilizes vacuum rather than gravitational flow and has significant advantages over existing methods. The response of recombinant HEK-293 cells expressing human LHRH receptor cDNA to LHRH agonists was used as a model system to develop the assay conditions. Cell lysates containing labeled IPs were applied in 96-well plates fitted with filtration discs containing regenerated Dowex AGI-X8 resin. Specifically bound inositol phosphates were eluted with 1 M ammonium formate in 0.1 M formic acid directly into a fresh 96-well plate and an aliquot of the eluate from each well is transferred into a 96-well plate and counted. The results were comparable to those obtained with the conventional column method and the variation among replicates was significantly improved. This assay facilitates rapid quantitation of inositol phosphates from a large number of samples with relative ease and reduced generation of radioactive waste.     

Rapid detection and quantification of bisulfite reductase genes in oil field samples using real-time PCR

Sulfate-reducing bacteria (SRB) pose a serious problem to offshore oil industries by producing sulfide, which is highly reactive, corrosive and toxic. The dissimilatory sulfite reductase ( dsr ) gene encodes for enzyme dissimilatory sulfite reductase and catalyzes the conversion of sulfite to sulfide. Because this gene is required by all sulfate reducers, it is a potential candidate as a functional marker. Denaturing gradient gel electrophoresis fingerprints revealed the presence of considerable genetic diversity in the DNA extracts achieved from production water collected from various oil fields. A quantitative PCR (qPCR) assay was developed for rapid and accurate detection of dsrB in oil field samples. A standard curve was prepared based on a plasmid containing the appropriate dsrB fragment from Desulfomicrobium norvegicum . The quantification range of this assay was six orders of magnitude, from 4.5 × 10⁷ to 4.5 × 10² copies per reaction. The assay was not influenced by the presence of foreign DNA. This assay was tested against several DNA samples isolated from formation water samples collected from geographically diverse locations of India. The results indicate that this qPCR approach can provide valuable information related to the abundance of the bisulfite reductase gene in harsh environmental samples.     

Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes

Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.     

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

Aims: A new real‐time polymerase chain reaction‐based method was developed for the detection of Salmonella enterica in food. Methods and Results: The method consisted of a novel two‐step enrichment involving overnight incubation in buffered peptone water and a 5‐h subculture in Rappaport–Vassiliadis medium, lysis of bacterial cells and a Salmonella‐specific 5′‐nuclease real‐time PCR with an exogenous internal amplification control. Because a two‐step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10⁷ CFU (25 g)^?1, eliminating potential false‐positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real‐time PCR‐based method and by the standard microbiological method, according to EN ISO 6579. When the real‐time PCR‐based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10⁰ CFU (25 g)^?1, identical results were obtained from both methods. Conclusions: The real‐time PCR‐based method involving a two‐step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. Significance and Impact of the Study: The developed method is suitable for rapid detection of S. enterica in food.     

LightCycler real-time PCR for rapid detection and quantitation of Mycobacterium leprae in skin specimens

Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.     

Detection of bioterror agents in air samples using real-time PCR

Aims:  To use real-time PCR for the detection of bacterial bioterror agents in a liquid air sample containing potential airborne interferences, including bacteria, without the need for DNA extraction.
Methods and Results:  Bacteria in air were isolated after passive sedimentation onto R2A agar plates and characterized by 16S rRNA sequencing. Real-time PCR was used to identify different bacterial bioterror agents in an artificial air sample consisting of a liquid air sample and a mixture of miscellaneous airborne bacteria showing different colony morphology on R2A agar plates. No time-consuming DNA extraction was performed. Specifically designed fluorescent hybridization probes were used for identification.
Conclusions:  Fourteen different bacterial genera were classified by 16S rRNA gene sequencing of selected bacterial colonies showing growth on R2A agar plates. Real-time PCR amplification of all the bacterial bioterror agents was successfully obtained in the artificial air sample containing commonly found airborne bacteria and other potential airborne PCR interferences.
Significance and Impact of the Study:  Bacterial bioterror agents can be detected within 1 h in a liquid air sample containing a variety of commonly found airborne bacteria using real-time PCR. Airborne viable bacteria at Kjeller (Norway) were classified to the genera level using 16S rRNA gene sequencing.     

Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR

M. pneumoniae is a common causative agent of community-acquired pneumonia in children. The diagnosis of such infections is usually based on serology using complement fixation or, more recently, enzyme-immuno assays. PCR has been shown to be a promising alternative. We have evaluated a real-time PCR assay targeting the P1 adhesion protein gene and compared it to a conventional semi-nested PCR assay with the 16S rDNA as target. Comparison of 147 specimens from 48 patients showed an overall agreement of 97.4%. Real-time PCR proved to be of equal value on clinical specimens as conventional PCR regarding sensitivity and specificity, but is clearly advantageous regarding speed, handling and number of samples that can be analyzed per run.     

Precise quantitation of human parvovirus B19 DNA in biological samples by PCR.

An application of a quantitative PCR-based method was developed for the detection of human parvovirus B19 DNA. The procedure was characterised according to guidelines for the validation of analytical procedures. Furthermore, the reliability was demonstrated by the correct quantitation of samples of an international collaborative study. This application might be useful for studies focussed on removal and/or inactivation procedures of human parvovirus B19 as well as for general screening purposes of biological materials.     

A real-time PCR assay for the detection and quantitation of Campylobacter jejuni using SYBR Green I and the LightCycler

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional biochemical identification for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable (VNC) state. Nucleic acid-based tests have emerged as a useful alternative to traditional testing. In this article, we present fluorescent quantitative PCR assay for quantitative detection of C. jejuni, the assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double-stranded DNA binding dye SYBR Green I. When this assay was applied, the assay positive for all of the isolates of C. jejuni tested (11 isolates, including type strain ATCC33560) and negative for all other Campylobacter spp. (three isolates) and several other bacteria (five species tested). The total assay could be completed in 60 min with a detection limit of approximately 1 CFU, and a correlation coefficient was 1.000. Result indicated that fluorescent quantitative detection methods provided a special, sensitive, rapid, reproducible and accurate method for quantitative detection of C. jejuni.     

Trapped in keratin; a comparison of dermatophyte detection in nail, skin and hair samples directly from clinical samples using culture and real-time PCR

Traditionally, laboratory detection and identification of dermatophytes consists of culture and microscopy which yields results within approximately 2-6 weeks. In 2007 our medical microbiological diagnostic laboratory implemented a molecular method for the detection of dermatophytes. A real-time PCR assay was developed which simultaneously detects and identifies the most prevalent dermatophytes directly in nail, skin and hair samples and has a turnaround time of less than two days. For 1437 clinical samples, received by our diagnostic laboratory, we compared the results obtained from both culture and real-time PCR. This study showed that real-time PCR significantly increased the detection rate of dermatophytes compared to culture. Furthermore, excellent concordance between culture and real-time PCR identification was achieved.     

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.     

A sensitive analytical method for the detection and quantitation of adenosine in biological samples

A new method for the measurement of adenosine in biological materials has been developed. The method is based on the combined principles of isotope dilution and enzymatic catalysis using a highly specific adenosine kinase isolated from rat heart. By differential centrifugation and gel filtration, this adenosine kinase was obtained free of adenosine deaminase and other enzymes which would have been a source of error in the use of this enzyme in the adenosine assay. The cardiac adenosine kinase was shown to be highly specific and to exhibit an apparent K_m for adenosine of 0.35 μM. Using this enzyme, unknown quantities of adenosine could be detected by measuring the effect of their addition on the conversion of radioisotopic adenosine to 5′-AMP in the kinase reaction. In this procedure, as little as 20 pmoles of adenosine could be detected. To test the applicability of the assay, measurements of the tissue content of this nucleoside were made in samples of dog and rat hearts frozen in situ under control, hypoxic, or ischemic conditions. The assay has several advantageous features when compared to other existing methods used to measure adenosine: a minimum of sample preparation is required before the actual assay procedure; many samples can be processed per day by a single operator; single determinations can be done on as little as 5 μl of sample, and the specificity of the assay can be readily checked by treatment of samples with adenosine deaminase.     

Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.     

Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay

Batrachochytrium dendrobatidis is a major pathogen of frogs worldwide, associated with declines in amphibian populations. Diagnosis of chytridiomycosis to date has largely relied upon histological and immunohistochemical examination of toe clips. This technique is invasive and insensitive particularly at early stages of infection when treatment may be possible. We have developed a real-time PCR Taqman assay that can accurately detect and quantify one zoospore in a diagnostic sample. This assay will assist the early detection of B. dendrobatidis in both captive and wild populations, with a high degree of sensitivity and specificity, thus facilitating treatment and protection of endangered populations, monitoring of pristine environments and preventing further global spread via amphibian trade.     

Direct immunoassay for detection of salmonellae in foods and feeds

A direct enzyme immunoassay (EIA) with polyclonal antibodies was developed for detecting salmonellae in foods and feeds. Salmonella cells were attached firmly to the wells of polystyrene microtitration plates with a capture-antibody technique. Spicer-Edwards anti-H immunoglobulin G was bound to protein A-beta-D-galactosidase to serve as the signal; 4-methylumbelliferyl-beta-D-galactoside was used as the substrate. The sensitivity threshold was 10(7) cells per ml. Direct EIA, indirect EIA, and pure-culture techniques were compared by using 48 samples of naturally contaminated foods and feeds. The direct EIA was more sensitive than the indirect EIA or pure-culture technique. Food samples were analyzed within 3 working days, and 32 samples were tested simultaneously in a single 96-well microtitration plate. False-positive or false-negative results did not pose a problem. This direct EIA is sensitive, rapid, and amenable to automation.     

High-performance cation-exchange chromatography and pulsed amperometric detection for the separation, detection, and quantitation of N-alkylated imino sugars in biological samples

The use of imino sugars for the potential treatment of lysosomal glycolipid storage diseases and hepatitis virus infections requires accurate, quantitative measurement of these compounds in biological samples. We demonstrate here the versatility of cation-exchange chromatography and pulsed amperometric detection of a range of compounds that differ in both isometric structure and N-alkyl chain length. Although column retention appears dependent upon residual charge on the imine function, successful isocratic separation can be achieved by secondary hydrophobic interactions. A series of N-alkylated deoxynojirimycin compounds containing C(1-10) alkyl chains are readily separated and detected by pulsed amperometry after cation suppression. Using experimentally derived response factors for imino sugars and measurement of peak areas we have developed a reliable method for quantitatively determining concentrations in solution. A rapid protocol for the removal of protein and contaminants in biological samples is described. This has allowed the successful measurement of imino sugars in animal tissues and will be useful for understanding the factors involved in compound bioavailability and in the design of novel therapeutics.     

Identification and quantification of arsC genes in environmental samples by using real-time PCR

The arsC gene is responsible for the first step in arsenate biotransformation encoding the enzyme arsenate reductase. The quantitative real-time PCR method was developed to quantify the abundance of the arsC genes in environmental samples contaminated with arsenic. Two sets of primers that showed high specificity for the target arsC gene were designed based on consensus sequences from 13 bacterial species. The arsC gene was used as an external standard instead of total DNA in the calibration curve for real-time PCR, which was linear over six orders of magnitude and the detection limit was estimated to be about three copies of the gene. Soil samples from arsenic contaminated sites were screened for arsC genes by using PCR and showed the presence of this gene. The copy numbers of the gene ranging from 0.88 x 10(4) to 1.56 x 10(5) per ng total DNA were found in eight arsenic contaminated samples. Soil samples from a bioreactor containing pulp mill biomass and high concentration of arsenate showed a tenfold higher count of arsC gene copies than soil samples collected underground from an arsenic-rich gold mine.     

Direct detection of Shigella flexneri and Salmonella typhimurium in human feces by real-time PCR

We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was 1 x 10(4) CFU/g feces for S. flexneri and 2 x 10(4) CFU/g feces for S. typhimurium this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.