SYBR Green-based PCR Assay Research Articles

Rapid Molecular Identification and Quantification of Allergenic Pollen By Real-Time PCR

Identifying and quantifying airborne pollen is important for causal assessment of allergic symptoms, for determining which pollens should be tested and for interpreting the results of allergy tests. Present pollen counting methods are time-consuming, require specific expertise and training, and are prone to subjective variability. Our goal is to devise a sensitive, high throughput method of quantifying pollen load in environmental samples. 9 different pure pollen samples, including grass (Paspalum notatum), trees (Morella cerifera, Morus alba, Juniperus ashei, Pinus strobus, Quercus virginiana, Taxodium distichum, Ulmus americana), and ragweed (Ambrosia artemisifolia) were used for analysis (courtesy of Tom Greer PhD, Greer Laboratories). Genomic DNA (gDNA) was extracted from pure pollens using a modification of the DNAzol ES procedure. Primers specific for the 5.8S rRNA genes of the different pollens were designed for quantitative real-time PCR (qPCR) using a SYBRGreen-based PCR assay. Individual gDNA from the pollens was detectable to sub nanogram levels. Individual pollen gDNA was detected in samples consisting of a mixture of all 9 pollen gDNAs. Specific pollens were detected in gDNA extracted from randomly admixed samples of the various pollen samples. The selected primers provided accurate identification in mixed samples with variable efficiency. qPCR detection of the pollen gDNA is sensitive and specific. The current assay can be expanded to include additional species and/or modified to detect pollens native to distinct geographical regions. Actual environmental sample quantification is necessary to prove applicability.

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

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.