Universal PCR primer designed for NTNH Sequences used to design a set of universal

PCR primers were obtained from Genbank. All sequences were aligned with MegAlign (DNASTAR, Lasergene, Inc.). Both Primer Express (Applied Biosystems, Foster City, CA) and Ipatasertib Primer3 (http://​frodo.​wi.​mit.​edu/​primer3/​), were used to design a pair of degenerate primers that included base differences to detect all known NTNH gene variants. Primer sequences are designated in Figure 1. Universal PCR for detection of NTNH of all C. botulinum types Purified DNA from C. botulinum, E. coli bacterial DNA (pUC19 plasmid DNA) or crude lysate from human leukocytes were used in the universal

PCR. PCR conditions were as follows: 95°C for 5 minutes, then 35 cycles of 95°C for 15 seconds and 57°C for 1 minute. PCR reaction mixture contained PCR Buffer, BB-94 in vitro 3.5 uM MgCl2, 200 nM dNTP, 1 uM forward or reverse primer, 0.25 U Taq Polymerase (Invitrogen Corp, Carlsbad, CA). 5 μL of DNA (0.25 ng/uL) was used in each 25 μL PCR reaction. PCR products Necrostatin-1 cell line were run on a 2.5% agarose gel to separate the product from any non-specific amplification and visualized for 101 bp bands by UV illumination. Toxin type-specific qPCR primer and probe design Neurotoxin gene sequences, obtained both from Genbank and from sequences provided by Biosciences Division, Los Alamos National Laboratories, were aligned and degenerate primer/probe sets were designed using software packages as above for each toxin type. Each degenerate primer/probe set include all known base differences within each toxin type. Generation of qPCR standards for each C. botulinum toxin type-specific assay Seven samples of purified C. botulinum DNA, one for each toxin type, were

used in the generation of plasmid DNA standards for qPCR. Briefly, primers designed specifically for each toxin type were used to amplify a region of the toxin gene containing Thiamet G the degenerate primer/probe set target sequences. The PCR conditions were as follows: 95°C for 5 minutes, then 35 cycles of 95°C for 15 seconds and 60°C for 1 minute. PCR reaction mixture contained PCR Buffer, 3.5 uM MgCl2, 200 nM dNTPs, 500 nM forward or reverse primer, 0.25 U Taq Polymerase (Invitrogen). 5 μL of DNA (0.25 ng/uL) was used in each 25 μL PCR reaction. PCR products were visualized by UV on a 1.5% agarose gel. Corresponding specific products were gel purified and ligated into pGEM T-easy vector (Promega Corp., Madison, WI). Ligations were transformed into DH5α E.coli bacteria using α-complementation to determine positive colonies. Positive colonies were grown in overnight cultures, plasmid DNA was purified and sequenced for determination of correct subtype insert sequence.