Rapid oral bacteria detection based on real-time PCR for the forensic identification of saliva

Ju Yeon Jung,Si-Keun Lim,Kyungmyung Lee,Jee Won Lee,Hyun Kyu Yoon,Yeon-Ji Kim,Sanghyun An,Jung Ho Hwang,Eu-Ree Ahn,Hyun-Chul Park

doi:10.1038/s41598-018-29264-2

Ju Yeon Jung, Si-Keun Lim + Show 8 more

Open Access

https://doi.org/10.1038/s41598-018-29264-2

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Abstract

This study developed a new method for forensic saliva identification using three oral bacteria, Streptococcus salivarius, Streptococcus sanguinis, and Neisseria subflava, combined with a real-time polymerase chain reaction (RT-PCR) system we called OB mRT-PCR. Analytical sensitivity results showed that the target bacteria were amplified at 102–107 copies/reaction, and analytical specificity was assessed using 24 other viruses, bacteria, and protozoa. To evaluate the OB mRT-PCR kit for forensic applications, saliva from 140 Korean individuals was tested, and at least two target bacteria were detected in all the samples. Additional studies on non-saliva samples demonstrated the specificity of the kit. Comparison of the kit with two conventional saliva test methods, the SALIgAE and RSID-Saliva assays, indicated that it was more sensitive and applicable to saliva samples in long-term storage (up to 14 weeks). Additionally, through amplification of mock forensic items and old DNA samples (isolated without lysis of the bacterial cells, regardless of their Gram-positivity), we found that the kit was applicable to not only saliva swabs, but also DNA samples. We suggest that this simple RT-PCR-based experimental method is feasible for rapid on-site analysis, and we expect this kit to be useful for saliva detection in old forensic DNA samples.

Highlights

The identification of saliva from stains on evidence is an important procedure in forensic investigations, in those related to sex crimes
For the evaluation of the analytical performance of the OB mRT-polymerase chain reaction (PCR) kit, synthetic genes prepared at various concentrations were used
The types and amounts of oral bacteria vary among individuals; the OB multiplex real-time PCR (mRT-PCR) kit will improve the accuracy of saliva identification by simultaneously analysing three species of bacteria as targets

Summary

Introduction

The identification of saliva from stains on evidence is an important procedure in forensic investigations, in those related to sex crimes. Detection of oral bacterial DNA based on polymerase chain reaction (PCR) has been studied as a strategy and reported to be simple and sensitive[1,11,12,13]. We developed an oral bacteria multiplex real-time PCR kit, named OB mRT-PCR, as a new forensic saliva identification tool. The environment (eating and drinking habits, tobacco use, age, and chronic nail-biting habits) can change the complex community of oral bacteria[19,20,21,22,23]. Our data showed that the OB mRT-PCR kit was useful as a saliva identification tool and could be applicable to swabs, and to isolated DNA, even that isolated without any particular Gram-positive bacterial cell lysing process

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