Reliance one step multiplex supermix

To estimate the minimum read depth required to confidently identify pathogens in a given sample, we performed an incremental analysis of reads using 4,000 read increments. Bioinformatic analysis was performed as above using incremental datasets of 4,000, 8,000, 12,000, and 16,000 reads and the taxonomic and pathogen distribution was monitored for each data set to identify read thresholds.
In addition to read-depth analysis, conventional real-time PCR analysis was performed to determine viral presence as described previously with minor modification (11 (link), 14 (link), 17 (link), 22 (link), 23 (link)). Briefly, the real-time PCR assays were slightly modified and optimized to use a commercial master mix Reliance One-Step Multiplex super mix (Bio-Rad Laboratories, Inc) or TaqMan™ Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) as well as additional internal control system on the Biorad CFX96 Real-time PCR (Bio-Rad Laboratories, Inc.) or ABI 7500 FAST Real-time PCR system (Thermo Fisher Scientific). All assays were performed as part of approved protocols under the quality system of the Nebraska Veterinary Diagnostic Center that have been validated for use.

For detection of pathogen RNA by qRT-PCR, a Reliance One-step Multiplex Supermix (Bio-Rad, Hercules, CA) was used, with 5μL of kit master mix combined with 4.5μL of either primer combination “A” or “B”, using the sequences listed in Table 1. Ten and a half microliters of extracted tick RNA was used for each test, loaded mechanically from the QIAcube output plates with a Qiagen QIAgility robotic plate loader. Control DNA fragments were synthesized by (Azenta, formerly Genewiz, South Plainfield, NJ, USA) to include the area of the target sequence enclosed by the primers listed, along with an additional flanking region of 20bp on either end. Equal quantities of each of the appropriate primer-specific sequence tested were combined into controls for primer mixtures mixes “A” and “B”. Each plate was run with one positive control and one no template control (NTC). PCR was run using CFX96 Touch Real-time qPCR systems (Bio-Rad, Hercules, CA). The cycle began with 50°C for 10min, followed by 95°C for 10min. Then 45 cycles of 95°C (10sec) and 60°C (30sec) were conducted, with fluorescence measured in the 60°C step using the all the excitation/emission frequencies allowable by the machine’s internal color filters, optimized for FAM, HEX, TEX-615 (Texas Red), CY-5, and CY-5.5.