Development and practical evaluation of an RT-PCR procedure using a real-time PCR instrument for saliva identification

Abstract

 STAGE DOI: 10.3408 / jafst.790 ) Saliva is often left at crime scenes and its identiˆcation can prove useful in in-vestigating criminal cases such as sexual assault. In this study, we developed a rever-se-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 speciˆcity, sensitivity, and applicability to forensic casework. The assay per-formance of QS5 was nearly identical to that of the SmartCycler II system ( SCII ) , which has been discontinued. Our proposed cutoŠ cycle quantiˆcation ( Cq ) values for the positive detection of saliva were Cq ＜ 40, 38, and 40 for ACTB , STATH , and HTN3 , respectively. The cutoŠ D Cq value for STATH was also set at 12. When the proposed criteria were applied, the developed procedure showed higher speciˆcity for saliva compared with conventional presumptive or conˆrmatory tests. Detection sensitivity was comparable to that of SCII but lower than that of a -amylase activity-based presumptive tests. An evaluation was then made using saliva samples under various storage conditions though Cq and D Cq values were drastically changed. In conclusion, the developed RT-PCR procedure has higher speciˆcity and lower sensitivity for saliva, suggesting its potential eŠectiveness for more precisely identifying saliva when performed in conjunction with current presumptive and conˆrmative saliva tests.