A validation study was performed on the new STR (Short tandem repeat) multiplex PCR Kit for human identification: AmpFℓSTR® Identifiler® Plus PCR Amplification Kit (IDPlus) released from Applied Biosystems. IDPlus contains the same fifteen loci as AmpFℓSTR® Identifiler® PCR Amplification Kit (ID) which is currently widely utilized in forensic DNA analysis. Consequently, IDPlus has the same discrimination ability as ID. According to the manufacturer, this kit has higher sensitivity, more resistant to PCR inhibitors and less background noise in electrophoresis. Thus, IDPlus was expected to be applied to forensic samples difficult to DNA profiling using ID. An applicability of IDPlus to forensic STR analysis was evaluated. Our study confirmed that IDPlus was more sensitive than ID. IDPlus has about 1.4 times to 1.5 times higher peak than ID when using the same PCR cycle number (28) for both kits. While ID has only one PCR protocol, IDPlus has two PCR protocols differing in cycle number: 28 and 29. To clarify the basic ability we compared heterozygous peak height ratio, stutter, intra-color balance, inter-locus balance among IDPlus to both protocols and ID from 94 individual DNA samples. Lower peak height ratio and the larger standard deviation in heterozygous samples were observed when using IDPlus 29-cycle compared to IDPlus 28-cycle. There was no significant difference between ID and IDPlus 28-cycle and between ID and IDPlus 29-cycle about the peak height ratio. Stutter ratios significantly were different in some loci between ID and IDPlus. Although Applied Biosystems supplies one stutter ratio to filter peaks for each locus to IDPlus in spite of having two PCR protocols, it was confirmed that the same stutter ratio could proper filter out stutter peaks for both cycle numbers. With regard to intra-color balance and inter-locus balance, IDPlus 28-cycle tended to be the highest in balance and ID tended to be the lowest in balance across all the samples and colors. Our validation study indicates IDPlus kit is useful in forensic applications, especially for analysis of trace DNA samples when using 29 cycles. However, forensic biologists must be cautious in interpreting DNA profiles obtained using IDPlus 29 cycles, because peak balance in heterozygous samples tended to be imbalanced when using 29 cycles compared to 28 and ID.
