Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification

The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R² = 0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs.     

Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345^T and Lactobacillus fermentum JCM 1173^T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 10³ cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10⁴ cells/g feces on day 4 and 10⁵ cells/g feces on days 11 to 18. However, cell populations of 10⁶ to 10⁷ cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 10¹¹ cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.     

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.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1 pg of purified genomic DNA, 5 EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when ≥10³ CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥10⁴ CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥10² CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥10⁴ CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.     

Detection of Brucella canis from inguinal lymph nodes of naturally infected dogs by PCR

The aim of this study was to standardize and evaluate a PCR assay for the detection of Brucella canis (B. canis) in lymph node samples of naturally infected dogs. The performance of the PCR was compared with the results of bacteriological culture as reference method. Forty-eight inguinal lymph node samples were collected from 48 dogs (18 males and 30 females) that died in the city's pound in the years 2007-2008 and were examined by microbiological culture and the PCR assay. B. canis was isolated from 4 (8.3%) of 48 lymph node samples. Forty-four (91.7%) of the samples were bacteriological culture negative. B. canis DNA was directly detected from all culture positive lymph node samples (n = 4) by PCR. All of the culture negative samples were confirmed as negative by PCR. When the culture method was used as a gold standard, sensitivity and specificity of the PCR assay were found to be 100%. The limit of PCR detection of B. canis DNA was 1.4 × 10¹ CFU/g at least. In conclusion, the PCR assay has been shown to have a diagnostic performance equal to bacteriological culture for detection of B. canis. By a non-hazardous protocol for laboratory workers, the assay can be performed in one day.     

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10⁶ to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10⁶ cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1 g feces (3.25 CFU/reaction). The assay was completed within 2 h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.     

Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.     

Evaluation of a Real-Time PCR Kit for Detecting Escherichia coli O157 in Bovine Fecal Samples

A commercially available real-time, rapid PCR test was evaluated for its ability to detect Escherichia coli O157. Both the sensitivity and specificity of the assay were 99% for isolates in pure culture. The assay detected 1 CFU of E. coli O157:H7 g⁻¹ in artificially inoculated bovine feces following enrichment.     

Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture,human sera seeded with MRSA and from patient's serum

The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient''s serum. One hundred and thirty-five clinical isolates of MRSA strains and different species were utilised in this study. In addition, a pilot study with 9 patients'' serum samples was performed. The sensitivity and specificity values for this assay were 99% and 100% respectively. The detection limit for this method was 1.23×10² CFU/ml from the serum seeded with MRSA cells and the limiting concentration of DNA for detection was 18 fg, which equates to 5.14 genomic DNA copies. In addition, this assay detected MRSA from patient''s serum (7 out of 9) with sensitivity of 77.8%. Overall, the assay was rapid, efficient, sensitive and easy to perform.     

Prion protein detection via direct immuno-quantitative real-time PCR

We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32 × 10² prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06 × 10⁴ to 3.24 × 10² epitopes (R² = 0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.     

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.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

Development of A Loop-Mediated Isothermal Amplification (LAMP) for the Detection of F5 Fimbriae Gene in Enterotoxigenic Escherichia coli (ETEC)

The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 10² cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC.     

Simultaneous Detection of Salmonella Strains and Escherichia coli O157:H7 with Fluorogenic PCR and Single-Enrichment-Broth Culture

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in ≤4 h after enrichment.     

Development of a Highly Sensitive Method for Detection of Clarithromycin-Resistant Helicobacter pylori from Human Feces

Gastric infection of clarithromycin (CAM)-resistant Helicobacter pylori is one of the major causes of failure to eradicate this organism. A noninvasive and useful method for the detection of CAM-resistant H. pylori from human feces by restriction fragment length polymorphism (RFLP)-nested polymerase chain reaction (PCR) targeting the mutation of the 23S rRNA gene that confers CAM-resistance in H. pylori was developed in this study. Our nested PCR method detected DNA of H. pylori in feces with high sensitivity and specificity compared with both an enzyme-linked immunoadsorbent assay (ELISA) of H. pylori in feces and the isolation of H. pylori from gastric biopsy. Furthermore, the results of mutation analysis of the H. pylori 23S rRNA gene amplified from feces completely correlated with both that of the H. pylori 23S rRNA gene amplified from the isolates of gastric biopsy and the susceptibility of H. pylori isolates to CAM. Therefore, our results show that this RFLP/nested PCR method is useful for the accurate diagnosis of CAM-resistant H. pylori infection from feces.     

Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 2 to 10⁷ spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60 min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 10³ spores and 10⁴ spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.