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Development and application of a multiplex PCR system for forensic salivary identification

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International Journal of Legal Medicine Aims and scope Submit manuscript

Abstract

In forensics, accurate identification of the origin of body fluids is essential for reconstructing a crime scene or presenting strong evidence in court. Microorganisms have demonstrated great potential in body fluid identification. We developed a multiplex PCR system for forensic salivary identification, which contains five types of bacteria:Streptococcus salivarius, Neisseria subflava, Streptococcus. mutans, Bacteroides thetaiotaomicron, and Bacteroides. uniformis. And the validated studies were carried out following the validation guidelines for DNA analysis methods developed by the Scientific Working Group on DNA Analysis Methods (SWGDAM), which included tests for sensitivity, species specificity, repeatability, stability, and mixed samples, trace samples, case samples, and a population study. Our result depicted that the lowest detection limit of the system was 0.01 ng template DNA. Moreover, the corresponding bacteria can still be detected when the amount of saliva input is low to 0.1 μL for DNA extraction. In addition, the target bacteria were not detected in the DNA of human, seven common animals, and seven bacteria DNA and in nine other body fluid samples (skin, semen, blood, menstrual blood, nasal mucus, sweat, tears, urine, and vaginal secretions). Six common inhibitors such as indigo, EDTA, hemoglobin, calcium ions, alcohol and humic acid were well tolerated by the system. What is more, the salivary identification system recognized the saliva component in all mixed samples and simulated case samples. Among 400 unrelated individuals from the Chinese Han population analyzed by this novel system, the detection rates of N. subflava, S. salivarius, and S. mutans were 97.75%, 70.75%, and 19.75%, respectively, with 100% identification of saliva. In conclusion, the salivary identification system has good sensitivity, specificity, stability, and accuracy, which can be a new effective tool for saliva identification.

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Data Availability

The data that supports the findings of this study are available in the supplementary material of this article.

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Acknowledgements

We are grateful to all volunteers who contributed samples for this study.

Funding

This study was supported by the Natural Science Foundation of Guangdong Province (Grant no.2022A1515012011), Qian Duansheng Distinguished Scholars Program of China University of the Political Science and Law (Grant no: 01140065140), and Innovative training program for College Students (Grant no: 202212121272).

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Author notes

  1. Xiaomin Liang, Xueyuan Liu, and Linying Ye contributed equally to this work.

Authors and Affiliations

  1. Guangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou, 510515, Guangdong, China

    Xiaomin Liang, Xueyuan Liu, Linying Ye, Litao Huang, Guichao Xiao, Manling Huang, Chao Liu & Ling Chen

  2. Guangdong Province Key Laboratory of Forensic Genetics, Guangzhou Forensic Science Institute, Guangzhou, 510030, China

    Xueyuan Liu, Changhui Liu & Chao Liu

  3. Guangdong Homy Genetics Ltd, Foshan, 528000, China

    Weian Du & Yangyang Zheng

  4. Criminal Justice College of China University of Political Science and Law, Beijing, 100088, People’s Republic of China

    Meisen Shi

Authors
  1. Xiaomin Liang
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  2. Xueyuan Liu
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  11. Chao Liu
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Corresponding authors

Correspondence to Meisen Shi, Chao Liu or Ling Chen.

Ethics declarations

The project was approved by the Biomedical Ethics Committee of Southern Medical University, Guangzhou, China (NO. 2019–011), and it was carried out in strict accordance with the ethical research principle of Southern Medical University.

Conflict of interest

The authors declare no competing interests.

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Highlight

1. The salivary identification system comprises three salivary core bacteria and two fecal core bacteria, providing a novel tool for salivary identification.

2. The salivary identification system overcomes errors in saliva recognition caused by shared salivary bacteria in feces, which has high sensitivity, stability, and accuracy for saliva identification.

3. The 10-year-old indoor saliva stains have been successfully identified.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 73 KB)

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Liang, X., Liu, X., Ye, L. et al. Development and application of a multiplex PCR system for forensic salivary identification. Int J Legal Med 137, 961–969 (2023). https://doi.org/10.1007/s00414-023-03004-2

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  • DOI: https://doi.org/10.1007/s00414-023-03004-2

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