Restriction fragment length polymorphisms of mutans streptococci in forensic odontological analysis
University of Chester, Chester, UK
28 Sept 2009
21 Apr 2010
13 May 2010
Streptococcus mutans, restriction fragment length polymorphism, polymerase chain reaction, mitis salivarius bacitracin agar, restriction endonuclease HaeIII, 16S ribosomal RNA gene
Humans bite each other with alarming frequency. Such injuries can result in forensic investigation and, ultimately, criminal prosecution. Bite mark analysis techniques include odontometric measurement, ABO serotyping and DNA profiling, none of which is infallible. Research into additional techniques would be advantageous. Biting involves, also, bacterial transfer from teeth to skin and vice versa. The principal species found on anterior teeth is Streptococcus mutans, which is universal among humans. The aim of this research was to establish, in the context of forensic odontology, whether chromosomal DNA profiles of this ubiquitous oral bacterium would vary significantly among a sample of Caucasian individuals (P = 0.05). Hence, if sufficient discriminatory power were present, the technique could be deemed useful to forensic investigation. Oral fluid was recovered from the lower incisors of 10 adults. Samples were cultured selectively using mitis salivarius bacitracin agar and microbiological tests carried out for the purpose of differential identification. These included visual assessment of colony morphology, Gram staining and microscopic analysis, followed by chemical testing for the enzyme catalase. Chromosomal DNA was extracted from subcultured cells, resolved by agarose gel electrophoresis and viewed using ultraviolet transillumination. The presence of DNA was confirmed. Subsequently, the 16S ribosomal RNA gene was amplified by polymerase chain reaction using specific 27 forward and 1492 reverse primers. Amplicons were resolved and viewed as previously. Amplified products were digested by incubation with restriction endonuclease HaeIII, resolved by polyacrylamide gel electrophoresis and viewed. Linear regression analysis of gel profiles was used to calculate restriction fragment lengths. A Kruskal–Wallis (analysis of variance) test was performed on ranked data (H = 8.161, df = 9, P = 0.518). Consequently, the null hypothesis (no inter-subject variation) was accepted. However, all but two profiles were proved to be unique. Proof of principle was provided regarding the application of oral bacterial genotyping to forensic bite mark cases.