出版社：Japanese Association of Forensic Science and Technology
摘要：Saliva is often left at crime scenes and its identification can prove useful in investigating criminal cases such as sexual assault. In this study, we developed a reverse-transcription polymerase chain reaction (RT-PCR) procedure for detection of STATH and HTN3 as markers characteristic of saliva, using the QuantStudio 5 real-time PCR system (QS5). Discrimination criteria were then proposed and evaluated on the specificity, sensitivity, and applicability to forensic casework. The assay performance of QS5 was nearly identical to that of the SmartCycler II system (SCII), which has been discontinued. Our proposed cutoff cycle quantification (Cq) values for the positive detection of saliva were Cq<40, 38, and 40 for ACTB , STATH , and HTN3 , respectively. The cutoff ΔCq value for STATH was also set at 12. When the proposed criteria were applied, the developed procedure showed higher specificity for saliva compared with conventional presumptive or confirmatory tests. Detection sensitivity was comparable to that of SCII but lower than that of α-amylase activity-based presumptive tests. An evaluation was then made using saliva samples under various storage conditions and mock casework samples. Unfortunately, positive results were difficult to obtain from saliva samples stored for long periods. Cq values of the analyzed genes were increased in samples stored under high humidity and temperature conditions, although ΔCq values for STATH and HTN3 were unchanged. In contrast, ΔCq values were significantly increased in saliva samples collected from a licked body surface, depending on the collection time. Positive results were obtained from saliva stains mixed with 10 times the volume of other body fluids, even though Cq and ΔCq values were drastically changed. In conclusion, the developed RT-PCR procedure has higher specificity and lower sensitivity for saliva, suggesting its potential effectiveness for more precisely identifying saliva when performed in conjunction with current presumptive and confirmative saliva tests.
