A novel method for the identification of saliva by detecting oral streptococci using PCR

Abstract

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva. Our data indicated that S. salivarius is more reliable than S. mutans as an indicator of saliva presence, because the detection rates for S. salivarius and S. mutans by this method were 100% and 90%, respectively. Furthermore, S. salivarius was detected in all saliva stain samples, whereas S. mutans was only identified in 60% of the stains. Finally, using this method we were able to successfully detect S. salivarius and S. mutans in mock forensic samples. We therefore suggested that this method is useful for the identification of saliva in forensic science.

Introduction

Recent developments in forensic practices have contributed to the investigation of crimes and the verification of criminal identity. The discrimination of body fluids in forensic examinations is important to determine the events that took place at a crime scene. The detection of saliva is particularly important for understanding the details of a crime. For example, the detection of a criminal's saliva left on a victim's skin provides evidence that the criminal had contact with a victim. Furthermore, the criminal's DNA may be isolated from the saliva, thereby verifying identity. The conventional method for testing for the presence of saliva is still a test for the presence of α-amylase [1], [2]. However, α-amylase may be present in other body fluids such as urine, and semen [3], [4]. Therefore, it is necessary to develop a new assay that can reliably discriminate saliva from other body fluids. An RNA-based assay targeting saliva-specific gene expression products was recently reported [5]. We also aimed to establish a further assay system for saliva that does not depend on α-amylase.

Numerous bacteria exist in the oral cavity. For example, the average total microscopic count is approximately 750 million oral bacterial cells per milliliter of saliva [6]. Of these, streptococci are the most abundant [7]. Specifically, Streptococcus salivarius is one of the most common streptococci in oral bacteria [6], and S. mutans is the prime causative bacterium for dental caries [8]. The detection and identification of oral bacteria has generally been performed by culturing samples on nutritive media. However, methods that incorporate the polymerase chain reaction (PCR) have recently been used to detect and identify oral bacteria such as S. salivarius or S. mutans, and have been applied in the diagnosis of caries risk [9], [10], [11], [12], [13], [14], [15].

With respect to forensics, the detection of oral streptococci has only been used to verify bite marks [16], [17], [18]. However, these reports did not discuss the identification of saliva presence. If it were possible to detect the presence of oral-specific bacteria by PCR from a forensic specimen, this could be used to verify the presence of saliva. Therefore, we evaluated whether the detection of S. salivarius and S. mutans by PCR is sufficient to confirm saliva presence.