The TaqMan real-time PCR assay was developed from the Blastomyces dermatitidis BAD1 gene promoter. The assay identified all haplotypes of B. dermatitidis and five of six positive paraffin-embedded tissues. The assay sensitivity threshold was 1 pg genomic DNA of the mold form and 2 CFU of the yeast form of B. dermatitidis. No cross-reactivity was observed against other fungal DNA. The assay allowed rapid (5-h) identification of B. dermatitidis from culture and from clinical specimens. Blastomyces dermatitidis is a dimorphic fungal pathogen that causes blastomycosis. The infection generally starts by inhalation of spores of the mold form of the fungus, found in the environment. Upon entry into the hosts, the spores convert to the yeast form. The infection can be self-limiting to the lungs, or it can disseminate to other body parts, mainly to bones and skin. Apart from humans, dogs are highly susceptible to B. dermatitidis infection. Blastomycosis is endemic in the midwestern and southeastern United States and around the Great Lakes (4). Interestingly, blastomycosis has also been reported in humans and dogs in New York State (6,8), but the ecological niche of this fungus has yet to be established in this region. Similarly, blastomycosis has been reported from Colorado and Nebraska, which are all outside known zones where the infection is endemic (7, 13).The laboratory methods most frequently used to diagnose blastomycosis include serology, direct smear, and histopathology. However, the gold standard for diagnosis remains a positive culture. Traditional confirmation of a suspect culture of B. dermatitidis by conversion to the yeast form in the laboratory can take weeks, but identification can be confirmed on the same day by Gen-Probe (Gen-Probe, Inc., San Diego, CA). The Gen-Probe test can be used only with pure cultures of B. dermatitidis (yeast or mold); hence, it has limited application. To overcome these problems, several conventional PCR assays have been developed for the identification of B. dermatitidis from clinical specimens and soil samples (2, 5). These assays used identical primer pair sequences targeting the putative promoter region of the BAD1 (earlier known as WI-1) gene, which codes for an important adhesin molecule and virulence factor (10). Recently, it has been shown that the BAD1 promoter region has a number of nucleotide polymorphisms, which resulted in the identification of four haplotypes of B. dermatitidis (11,12). In addition to nucleotide polymorphism, a major size disparity due to two large insertions in the BAD1 promoter was also found in many B. dermatitidis strains (12). This can complicate the conventional PCR assay due to either insufficient amplification efficiency or misinterpretation as a nonspecific product.In this study, we describe the development of a TaqMan real-time PCR assay using a specific region of the BAD1 promoter to encompass all known haplotypes of B. dermatitidis. Our results indicate that the BAD1 real-time PCR assay is highly specific and rapid, with a turnaround t...
The M-CAMP™ (Microbiome Computational Analysis for Multiomic Profiling) Cloud Platform was designed to provide users with an easy-to-use web interface to access best in class microbiome analysis tools. This interface allows bench scientists to conduct bioinformatic analysis on their samples and then download publication-ready graphics and reports. The core pipeline of the platform is the 16S-seq taxonomic classification algorithm which provides species-level classification of Illumina 16s sequencing. This algorithm uses a novel approach combining alignment and kmer based taxonomic classification methodologies to produce a highly accurate and comprehensive profile. Additionally, a comprehensive proprietary database combining reference sequences from multiple sources was curated and contains 18056 unique V3-V4 sequences covering 11527 species. The M-CAMPTM 16S taxonomic classification algorithm was validated on 52 sequencing samples from both public and in-house standard sample mixtures with known fractions. Compared to current popular public classification algorithms, our classification algorithm provides the most accurate species-level classification of 16S rRNA sequencing data.
Background: The M-CAMPTM (Microbiome Computational Analysis for Multi-omic Profiling) Cloud Platform was designed to provide users with an easy-to-use web interface to access best in class microbiome analysis tools. This interface allows bench scientists to conduct bioinformatic analysis on their samples and then download publication-ready graphics and reports. Objective: In this study we aim to describe the M-CAMPTM platform and demonstrate that the taxonomic classification is more accurate than previously described methods on a wide range of microbiome samples. Methods: The core pipeline of the platform is the 16S-seq taxonomic classification algorithm which provides species-level classification of Illumina 16s sequencing. This algorithm uses a novel approach combining alignment and kmer based taxonomic classification methodologies to produce a highly accurate and comprehensive profile. Additionally, a comprehensive proprietary database combining reference sequences from multiple sources was curated and contains 18056 unique V3-V4 sequences covering 11527 species. Results and Discussion: The M-CAMPTM 16S taxonomic classification algorithm was evaluated on 52 sequencing samples from both public and in-house standard sample mixtures with known fractions. The same evaluation process was also performed on 5 well-known 16S taxonomic classification algorithms including Qiime2, Kraken2, Mapseq, Idtaxa and Spingo using the same dataset. Results have been discussed in term of evaluation metrics and classified taxonomic levels. Conclusion: Compared to current popular public classification algorithms, M-CAMPTM 16S taxonomic classification algorithm provides the most accurate species-level classification of 16S rRNA sequencing data.
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