Amplification of Oral Streptococcal DNA from Human Incisors and Bite Marks

Abstract

Challenges to the evidentiary value of morphometric determinations have led to a requirement for scientifically substantiated approaches to the forensic analysis of bite marks. Human teeth support genotypically distinctive populations of bacteria that could be exploited for forensic purposes. This study explored the feasibility of directly amplifying bacterial DNA from bite marks for comparison with that from teeth. Samples from self-inflicted experimental bite marks (n = 24) and human incisors were amplified by PCR using primers specific for streptococcal 16S ribosomal DNA. Amplicon profiles (resolved by denaturing gradient gel electrophoresis) from bite mark samples aligned significantly more closely with profiles generated from the teeth responsible than with those from other teeth. Streptococcal amplicons were generated from dental samples applied to excised porcine skin for up to 48 h. These findings indicate that streptococcal DNA can be amplified directly from bite marks, and have potential application in bite mark analysis.

Microbial Analysis of Bite Marks by Sequence Comparison of Streptococcal DNA

“…Sixteen unrelated adult participants recruited from the staff and students of the University of Otago generated self-inflicted bites on their upper arms [20], [21], [22]. Participants were healthy adults who had not used mouthwash in the preceding month or antibiotics in the preceding three months.…”

“…However, nucleases, such as deoxyribonuclease I, present in saliva at relatively high concentrations [16], [17], [18] contribute to the rapid degradation of exposed DNA [19]. Because of the difficulties that can be encountered in recovering salivary DNA of sufficient quality and quantity to generate a DNA profile, an alternative objective approach to bite mark analysis has been directed toward a bacterial genotyping method [20], [21], [22].…”

“…This intraspecies diversity provides the premise that oral streptococci isolated from a bite mark inflicted on human skin may be genotypically matched, with a high degree of assurance, to those from the teeth responsible [20], [21]. These observations were reiterated in a third study [22] that circumvented the need for prior culturing by amplifying bacterial DNA directly from teeth and experimental bite marks. In that study, streptococcal DNA, amplified with primers specific for hypervariable region 9 of streptococcal 16S rRNA gene, was resolved by denaturing gradient gel electrophoresis (DGGE), and a comparison of the amplicon profiles from the bite marks and teeth matched most bite marks to the teeth responsible.…”

“…In that study, streptococcal DNA, amplified with primers specific for hypervariable region 9 of streptococcal 16S rRNA gene, was resolved by denaturing gradient gel electrophoresis (DGGE), and a comparison of the amplicon profiles from the bite marks and teeth matched most bite marks to the teeth responsible. However, there was a concomitant risk of false positives with the sole use of this relatively conserved locus [22].…”

“…Phylogenetic analysis and identification of bacterial species have been conventionally based on 16S rRNA gene sequence comparison; however, the variable regions contained within this locus are generally insufficient for distinguishing closely related streptococcal species [22], [36]. Alternative gene targets that discriminate between closely related streptococci include ITS (stretch of non-coding DNA that lies between the 16S and the 23S rRNA genes) [37], [38], [39], [40], rnpB (encoding endoribonuclease P) [41], [42] and rpoB (encoding the beta subunit of the bacterial RNA polymerase) [43], [44].…”