Oral gram-positive bacterial DNA-based identification of saliva from highly degraded samples

Forensic Sci Int Genet. 2019 Sep;42:103-112. doi: 10.1016/j.fsigen.2019.06.016. Epub 2019 Jul 6.

Abstract

Analyzing degraded evidence is an important challenge in forensic casework. Saliva remaining at a crime scene may deteriorate, due to various factors, making it difficult to identify. This study aims to clarify the efficacy of oral gram-positive and -negative bacterial DNA-based identification of saliva for analyzing highly degraded samples. Saliva samples were subjected to three different degradation treatments (heat denaturation: 40-80 °C in wet conditions; microbial decomposition: 1-5 days in humid soil; and ultraviolet (UV) irradiation: 0.01-1 J/cm2). We compared saliva markers' detectability from the degraded samples-oral gram-positive bacterial DNA (Streptococcus salivarius and Streptococcus oralis), oral gram-negative bacterial DNA (Veillonella atypica and Prevotella maculosa) and salivary α-amylase. Oral bacterial DNA was detected using a melting curve analysis following real-time PCR. The efficacy of short tandem repeats (STR) and mitochondrial DNA (mtDNA) analyses were also compared. All oral bacterial DNA were detected with specific melting peaks from the heat-denatured samples, while neither catalytic nor immunochromatographic tests detected salivary α-amylase from the heat (80 °C) samples. The gram-positive bacterial DNA (S. salivarius and S. oralis) was detected from the microbial degradation (1-5 days) samples. In contrast, the gram-negative bacterial DNA (V. atypica and P. maculosa) and salivary α-amylase were not detected from samples treated for more than two days. UV exposure made bacterial DNA-based saliva identification difficult in a dose-dependent manner; however, UV irradiation did not influence protein-based saliva tests using salivary α-amylase as an indicator. As a result of STR and mtDNA typing, partial or null STR profiles were generated from the severely degraded (microbial (2-5 days) and UV (0.1-1 J/cm2) degradation) samples, but full mtDNA profiles were obtained from all degraded samples. The forensic applicability of bacterial DNA test evaluated, using mock case samples, indicates that the oral gram-positive bacterial DNA was more resistant to degradation than the other markers. We conclude that the oral gram-positive bacterial DNA-based examination could be useful for identifying saliva from severely environmentally-exposed forensic samples as well as mtDNA typing.

Keywords: Degradation; Melting curve analysis; Prevotella maculosa; Saliva identification; Streptococcus oralis; Streptococcus salivarius; Veillonella atypical.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Biomarkers / metabolism
  • DNA Fingerprinting
  • DNA, Bacterial / genetics*
  • DNA, Mitochondrial / genetics
  • Environmental Exposure / adverse effects
  • Female
  • Gram-Negative Bacteria / genetics*
  • Gram-Positive Bacteria / genetics*
  • Hot Temperature / adverse effects
  • Humans
  • Male
  • Microsatellite Repeats
  • Middle Aged
  • RNA, Ribosomal, 16S / genetics
  • Real-Time Polymerase Chain Reaction
  • Saliva / metabolism
  • Saliva / microbiology*
  • Ultraviolet Rays / adverse effects
  • Young Adult
  • alpha-Amylases / metabolism

Substances

  • Biomarkers
  • DNA, Bacterial
  • DNA, Mitochondrial
  • RNA, Ribosomal, 16S
  • alpha-Amylases