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

Abstract

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.

Infectious keratoconjunctivitis and occurrence of Mycoplasma conjunctivae and Chlamydiaceae in small domestic ruminants from Central Karakoram, Pakistan

“…For the detection of Chlamydiaceae species in the eye swabs lysates, an SYBR green-based qPCR assay was performed using the primers Chuni-1F and Chuni-2R. 31 Each reaction consisted of 2.5 µl of DNA sample, 12.5 µl of SYBRGreen PCR Master Mix 2x (Applied Biosystems, Warrington, UK), 400nM of each forward and reverse primer and nuclease-free water to a total volume of 25 µl. PCR was performed following reported cycling conditions.…”

“…PCR was performed following reported cycling conditions. 31 Samples were assayed per duplicate and were assayed with an exogenous Internal Positive Control (IPC; Applied Biosystems, USA) to detect eventual PCR inhibitors.…”

Quantitative PCR Analysis of Gut Disease-Discriminatory Phyla for Determining Shrimp Disease Incidence

“…However, given the conservation of bacterial 16S rRNA genes, it is challenging, if not impossible, to design primers for targeting specific strains, thereby hampering the rapid detection of disease-discriminatory taxa. Instead, a few studies have designed 16S rRNA gene-targeted group-specific primers for rapidly detecting the dominant gut bacterial phyla (15,17,18). Based on these studies, age-discriminatory phyla, Alphaproteobacteria and Verrucomicrobia, were first identified.…”

“…These limitations impede its use in routine monitoring applications. In this regard, cheap, rapid, and convenient features of quantitative PCR (qPCR) might overcome these shortcomings (15). Indeed, qPCR is being used to detect the dominant gut bacterial lineages in human (16) and pig (17).…”

Association of insularity and body condition to cloacal bacteria prevalence in a small shorebird

“…Detection of Campylobacter was based on amplification of a DNA segment within the flaA short variable region (SVR) of Campylobacter jejuni or C. coli, according to Ridley et al [47]. Chlamydia detection centred on amplifying the IGS region and domain I of 23S rRNA gene, following Nordentoft et al [48]. Salmonella detection used primers specific for the invA gene, as described by Rahn et al [49].…”