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

ABSTRACT 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.

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Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

Applied and Environmental Microbiology ◽

10.1128/aem.68.5.2420-2427.2002 ◽

2002 ◽

Vol 68 (5) ◽

pp. 2420-2427 ◽

Cited By ~ 126

Author(s):

Teresa Requena ◽

Jeremy Burton ◽

Takahiro Matsuki ◽

Karen Munro ◽

Mary Alice Simon ◽

...

Keyword(s):

Real Time ◽

16S Rdna ◽

Quantitative Pcr ◽

Gene Sequence ◽

Denaturing Gradient Gel Electrophoresis ◽

Bifidobacterium Longum ◽

Bifidobacterium Adolescentis ◽

Fecal Samples ◽

Real Time Quantitative Pcr ◽

Pcr Targeting

ABSTRACT Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

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Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Human Fecal Samples

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01684-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 606-609 ◽

Cited By ~ 63

Author(s):

Pablo Quirós ◽

Marta Colomer-Lluch ◽

Alexandre Martínez-Castillo ◽

Elisenda Miró ◽

Marc Argente ◽

...

Keyword(s):

Antibiotic Resistance ◽

Real Time ◽

Quantitative Pcr ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Fecal Samples ◽

Real Time Quantitative Pcr ◽

Healthy Humans ◽

Phage Dna

ABSTRACTA group of antibiotic resistance genes (ARGs) (blaTEM,blaCTX-M-1,mecA,armA,qnrA, andqnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs.blaTEM,qnrA, and,blaCTX-M-1were the most abundant, andarmA,qnrS, andmecAwere less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra- and extraintestinal environments.

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Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR

Applied and Environmental Microbiology ◽

10.1128/aem.00107-10 ◽

2010 ◽

Vol 76 (17) ◽

pp. 5693-5701 ◽

Cited By ~ 41

Author(s):

Lejla Imamovic ◽

Elisenda Ballesté ◽

Juan Jofre ◽

Maite Muniesa

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Shiga Toxin ◽

Enteric Bacteria ◽

Fecal Samples ◽

Urban Sewage ◽

Real Time Quantitative Pcr ◽

Gene Copies ◽

Animal Wastewater ◽

Wastewater Samples

ABSTRACT Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 103 gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 1010 GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 105 GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 104 GC per g of sample). The stx 2 genes and stx 2 variants were detected in the viral fraction of some of the samples after sequencing of stx 2 fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.

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