Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques.Methods
We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.
We designed FungiQuant, a TaqMan(R) qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant's is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged fromConclusions
FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.
Cindy M Liu, Sergey Kachur, Michael G Dwan, Alison G Abraham, Maliha Aziz, Po-Ren Hsueh, Yu-Tsung Huang, Joseph D Busch, Louis J Lamit, Catherine A Gehring, Paul Keim and Lance B Price
A nested polymerase chain reaction (PCR) assay for detecting Valsa malivar. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. maliisolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsaspp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.
Rui Zang, Zhiyuan Yin, Xiwang Ke, Xiaojie Wang, Zhengli Li, Zhensheng Kang, and Lili Huang Plant Disease November 2012, Volume 96, Number 11, Pages 1645-1652
We developed automated software, entitled TALENSeek, to identify unique TALEN binding sites of a user-specified gene or genomic locus. The program is outlined in Figure 1, where (a) automated annotation of genetic regions are used to identify start and end sites of a gene using the farthest protein coding regions across all isoforms (b) potential TALEN binding sites are identified; (c) TALEN binding sites are evaluated for uniqueness across the genome; (d) if a binding site is non-unique, the binding sites are shifted into the coding region of the gene and the algorithm iterates from step (b). Chromosomal locations and genomic sequences of the TALEN binding site are output. In addition, chromosomal locations of TALEN binding sites can be visualized in genome browsers. Homology arms to generate gene knock-outs, knock-ins or correction are also output by the program and can be used to generate donor constructs to direct homologous dependent repair. The TALENSeek program has been applied to generate genome-wide TALEN binding sites for each isoform of each gene in the human genome (hg19; 509,262 unique binding sites generated). Eight TALEN pairs were assembled and demonstrated activity in K562 cells; one of these eight has been used in human embryonic stem cells to generate hetero- and homozygously targeted cell lines. TALENSeek can be easily modified to any species with an available genome sequence. An application of the program to mouse (mTALENSeek) is also currently available. TALENSeek is a user-friendly bioinformatics tool that allows for the design of unique TALEN binding sites in the genome. TALENs are efficient genome editing tools, which create the possibility of direct modeling of disease-associated mutations in developing human stem cells.
Diginomica SAP Announces Strategic Alliances to Drive Forward Genomic Research MarketWatch "SAP HANA has the potential to drive breakthrough innovation in genomics research and the healthcare industry overall," said Barbara Stortz, senior vice...
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