PCR cycling parameters around the standard set of conditions were tested on one instrument, except cycle number which was done on a second instrument. Six 1000 M control swabs with 100,000 cells were used to test each of the thermal cycling parameters. The following thermal cycling parameters were examined, with the standard conditions indicated in bold: activation temperature: 94 °C, 96 °C, and 98 °C; denaturation temperature:

94 °C, 96 °C, and 98 °C; annealing temperature: 58 °C, 60 °C, and selleckchem 62 °C; final extension time: 4 min, 8 min and 12 min; cycle number: 27, 28, and 29 cycles. Specificity was tested using 8 ng of DNA from five non-primate sources (bovine, chicken, horse, porcine and rabbit) and pooled microorganisms (ca. 105 copies each from Streptococcus mitis, Streptococcus salivariu, Latobacillus SB203580 molecular weight casei, Fusobacterium nucleatum, Enterococcus faecalis, Streptococcus mutans). Three replicates for each species listed and five replicates of the microbial pool were tested. Sensitivity was tested in three ways by

running the following sets of swabs on three systems: (1) Two-fold serial dilutions of 1000 M cells from 200,000 down to 3125 cells (1.2 μg–18.75 ng based on 6 pg/diploid cell) were prepared and added to swabs (6 replicates/dilution, n = 42 total swabs); (2) Mock swab collection to simulate potential DNA amounts from two donors, across the range from 1 touch to inside of cheek, 1 swipe, 2 swipes, 5 swipes, 10 3-mercaptopyruvate sulfurtransferase swipes to 20 swipes of the inside of the cheek (3 replicates/collection/donor, n = 36 total swabs). (3) Two-fold dilutions of blood (20–2.5 μL and 1 μL) from three donors applied to cotton swabs (n = 3/dilution/donor, n = 15 total swabs). Percentage of alleles detected at each dilution and average peak height was determined.

A mixture study was performed to verify the analysis software will appropriately flag a sample that may be a mixture before expert review. Mixture of two cell lines, 1000 M and 1000 F, were examined at the following ratios (1:0, 1:1, 1:2.5, 1:4, and 1:9) while maintaining the total amount of cells at 50,000 (low range of cells on buccal swabs). These two cell lines were selected to minimize both the number of overlapping alleles and alleles occurring in stutter positions. Each mixture series was tested in six separate runs on the RapidHIT. A subset of donor buccal swabs (150 individuals) was processed on four RapidHIT Systems. Genotype concordance was checked against reference profiles generated from the GlobalFiler Express runs on the ABI 9700/3130xL instruments and analyzed with GeneMapper ID-X v1.4. In addition, DNA (∼1–2 ng/20 μL) from the NIST SRM 2391c DNA Profiling standard (components A–D) were added directly to the STR reagent vials prior to insertion onto the cartridge and run on the RapidHIT System. Concordance was checked against the NIST certified genotypes.