{"title":"Development of a multiplex PCR assay and quantification of microbial markers by ddPCR for identification of saliva and vaginal fluid","authors":"","doi":"10.1016/j.forsciint.2024.112147","DOIUrl":null,"url":null,"abstract":"<div><p>The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. <em>Lactobacillus crispatus</em> and <em>Streptococcus salivarius</em> were selected because of high abundance within vaginal fluid and saliva respectively. While <em>Fusobacterium nucleatum</em> and <em>Gardnerella vaginalis</em>, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected <em>L. crispatus</em> and <em>G. vaginalis</em> in vaginal fluid while <em>F. nucleatum</em> and <em>S. salivarius</em> was detected in saliva. Multiplex PCR detected <em>F. nucleatum</em>, <em>S. salivarius</em> and <em>L. crispatus</em> in mixed body fluid samples while, <em>G. vaginalis</em> was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, <em>L. crispatus</em> and <em>G. vaginalis</em> were detected in vaginal swabs while only <em>S. salivarius</em> was detected in saliva swabs. The limit of detection was 0.06 copies/µl for <em>F. nucleatum</em> (2.5 ×10<sup>−9</sup> ng/µl) and <em>S. salivarius</em> (2.5 ×10<sup>−6</sup> ng/µl). <em>L. crispatus</em> and <em>G. vaginalis</em> had detection limits of 0.16 copies/µl (2.5 ×10<sup>−4</sup> ng/µl) and 0.48 copies/µl (2.5 ×10<sup>−7</sup> ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.</p></div>","PeriodicalId":12341,"journal":{"name":"Forensic science international","volume":null,"pages":null},"PeriodicalIF":2.2000,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0379073824002287/pdfft?md5=4a7245b2454e68b6332f30293e407744&pid=1-s2.0-S0379073824002287-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Forensic science international","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0379073824002287","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MEDICINE, LEGAL","Score":null,"Total":0}
引用次数: 0
Abstract
The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10−9 ng/µl) and S. salivarius (2.5 ×10−6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10−4 ng/µl) and 0.48 copies/µl (2.5 ×10−7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.
期刊介绍:
Forensic Science International is the flagship journal in the prestigious Forensic Science International family, publishing the most innovative, cutting-edge, and influential contributions across the forensic sciences. Fields include: forensic pathology and histochemistry, chemistry, biochemistry and toxicology, biology, serology, odontology, psychiatry, anthropology, digital forensics, the physical sciences, firearms, and document examination, as well as investigations of value to public health in its broadest sense, and the important marginal area where science and medicine interact with the law.
The journal publishes:
Case Reports
Commentaries
Letters to the Editor
Original Research Papers (Regular Papers)
Rapid Communications
Review Articles
Technical Notes.