Characterization of<i>Chlamydiaceae</i>species using PCR and high resolution melt curve analysis of the 16S rRNA gene

Robertson, Timothy James; Bibby, Summa; O’Rourke, Denise; Belfiore, T.; Lambie, H.; Noormohammadi, Amir H.

doi:10.1111/j.1365-2672.2009.04388.x

Cited by 51 publications

(36 citation statements)

References 35 publications

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“…One, applying a family-specific PCR targeting the ribosomal genes, was able to distinguish six species by subjecting generated amplicons to high-resolution melt curve analysis (19). However, as this is a highly sensitive method allowing discrimination on the basis of a single base pair polymorphism, it also requires highly standardized conditions for preparing the master mix and clean DNA to be reproducible.…”

Section: Discussionmentioning

confidence: 99%

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Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

Nordentoft

Kabell

Pedersen

2011

Appl Environ Microbiol

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Infections caused by members of the Chlamydiaceae family have long been underestimated due to the requirement of special laboratory facilities for the detection of this group of intracellular pathogens. Furthermore, new studies of this group of intracellular pathogens have revealed that host specificity of different species is not as clear as recently believed. As most members of the genus Chlamydophila have shown to be transmissible from animals to humans, sensitive and fast detection methods are required. In this study, SYBR green-based real-time assays were developed that detect all members of Chlamydiaceae and differentiate the most prevalent veterinary Chlamydophila species: Cp. psittaci, Cp. abortus, Cp. felis, and Cp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs. Cp. psittaci was detected in 47 birds, Cp. felis was found in 10 cats, Cp. caviae was found in one guinea pig, and Cp. abortus was detected in one sheep. The screening assay appeared more sensitive than traditional microscopical examination of stained tissue smears. By combining a fast, robust, and cost-effective method for sample preparation with a highly sensitive family-specific PCR, we were able to screen for Chlamydiaceae in veterinary specimens and confirm the species in positive samples with additional PCR assays.

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Section: Discussionmentioning

confidence: 99%

“…Two real-time PCR detection systems for identification of Chlamydiaceae have been described (17,19). One, applying a family-specific PCR targeting the ribosomal genes, was able to distinguish six species by subjecting generated amplicons to high-resolution melt curve analysis (19).…”

Section: Discussionmentioning

confidence: 99%

Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

Nordentoft

Kabell

Pedersen

2011

Appl Environ Microbiol

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“…HRM analysis has been used in numerous studies since the mid-2000s on a wide range of human pathogens, including those within the genera Campylobacter, Brucella, Leishmania, Bordetella, Clostridium, Mycobacterium, Mycoplasma, Chlamydophila, Cryptosporidium, and Staphylococcus, among others (445)(446)(447)(448)(449)(450)(451)(452). Recent studies have shown HRM analysis to be a powerful technique for characterizing antibiotic resistance and typing respiratory disease agents, including M. pneumoniae, C. pneumoniae, and Chlamydophila psittaci (453)(454)(455)(456)(457)(458)(459). To our knowledge, HRM analysis for intraspecies or interspecies Legionella discrimination has been included in only a few studies (460)(461)(462)(463); however, this approach seems promising for future Legionella diagnostics or typing.…”

Section: Emerging Methods and Technologiesmentioning

confidence: 99%

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

2015

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SUMMARY Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic “blind spot” for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed.

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“…Extracted DNA was tested for the presence of Chlamydia spp. using the Chlamydia 16SG nucleotide set qPCR first described by Robertson et al (2009). The resulting melt curves from this qPCR, coupled with appropriate controls, can be used to distinguish detected Chlamydia species (Fig.…”

Section: Methodsmentioning

confidence: 99%

Identification of unusual Chlamydia pecorum genotypes in Victorian koalas (Phascolarctos cinereus) and clinical variables associated with infection

Legione

Patterson

Whiteley

et al. 2016

Journal of Medical Microbiology

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Characterization ofChlamydiaceaespecies using PCR and high resolution melt curve analysis of the 16S rRNA gene

Abstract: This technique allowed for the rapid detection and identification of the six Chlamydiaceae reference species and may be useful for identification of uncharacterized Chlamydiaceae species or for use in animal species where occurrence of the disease has not been fully investigated.

Cited by 51 publications

References 35 publications

Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

Identification of unusual Chlamydia pecorum genotypes in Victorian koalas (Phascolarctos cinereus) and clinical variables associated with infection

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