3130 genetic analyzer capillary electrophoresis dna sequencer

The total DNA was extracted from the biopsy samples. DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. Two sets of primers were used in order to amplify different broad‐wide bacterial regions in the gene encoding for the 16S ribosomal RNA in the extracted DNA (Harmsen et al., 2003 (link); Rothman et al., 2002 (link)). PCR products were separated by electrophoresis in ethidium bromide stained 2% agarose gels, and were then sequenced on a 3130 Genetic Analyzer capillary electrophoresis DNA sequencer (Applied Biosystems) and analysed using the basic local alignment search tool (BLAST). Additionally, biopsies were homogenized and plated for aerobic and anaerobic cultures according to clinical microbiology procedures handbook guidelines (Leber, 2016 ).

Identification of cultured Fusarium species was performed at the various institutional microbiology laboratories using standard phenotypic methods. When possible, fresh specimens were sent to a central reference laboratory for DNA sequencing and analysis. DNA was extracted from clinical samples by means of the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA), and amplified with a semi-nested polymerase chain reaction (PCR) assay targeting the 28S rDNA. The PCR products were separated by electrophoresis in ethidium bromide-stained 2% agarose gels, sequenced on a 3130 Genetic Analyzer capillary electrophoresis DNA sequencer (Applied Biosystems, Carlsbad, CA, USA), and analyzed with the Basic Local Alignment Search Tool (BLAST). Testing of antifungal susceptibility was performed locally. The minimal inhibitory concentrations (MIC) of the antifungal agents for each isolate were determined with either broth microdilution or gradient concentration strips (Novamed) on RPMI plates.

Identification of the cultured Mucorales was performed at various institutional microbiology laboratories using standard phenotypic methods. When possible, fresh specimens were sent to a central reference laboratory (Clinical Microbiology Laboratory, Rambam Health Care Campus, Haifa, Israel) for DNA sequencing and analysis. DNA was extracted from clinical samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA), and was amplified using a semi nested polymerase chain reaction (PCR) assay targeting specific zygomycete regions at the 18S rDNA, as well as a fungal-broad range PCR reaction targeting the 28S rDNA. The PCR products were separated by electrophoresis in ethidium bromide stained 2% agarose gels, then sequenced on a 3130 Genetic Analyzer capillary electrophoresis DNA sequencer (Applied Biosystems, Carlsbad, CA, USA) and analyzed using the Basic Local Alignment Search Tool (BLAST).
Antifungal susceptibility testing was performed at the National Mycology Reference Laboratory (Sourasky Medical Center, Tel Aviv, Israel). The minimal inhibitory concentrations (MICs) of the antifungal agents for each isolate were determined using broth microdilution.