Development of a multiplex PCR assay and quantification of microbial markers by ddPCR for the identification of saliva and vaginal fluid.

Dass, M.*, Ghai, M.

Department of Genetics, School of Life Sciences, University of KwaZulu Natal – Westville Campus, Private Bag X 54001, Durban, KwaZulu Natal, South Africa

The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Body fluids display distinct microbial profiles and discrimination of body fluids is achievable based on the microbiome content. 16S rRNA sequencing is the gold standard for microbial analysis, however, next generation sequencing is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. In this study, a multiplex conventional PCR assay was developed to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers and droplet digital PCR (ddPCR) was used to quantify bacterial species alone and in a mixture of body fluids. Two markers each for saliva and vaginal fluid were selected. 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 successfully 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 x 10-9 ng/µl) and S. salivarius (2.5 x 10-6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 x 10-4 ng/µl) and 0.48 copies/µl (2.5 x 10-7 ng/µl). All four 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; hence all four bacteria are suitable and sensitive for the detection of both body fluids. The present research reports for the first time the combination of F. nucleatum, S. salivarius, L. crispatus and G. vaginalis for the detection of saliva and vaginal fluid and use of ddPCR for quantification of these bacterial species.

Keywords: droplet digital PCR, 16S rRNA microbial markers, body fluid identification, Streptococcus salivarius, Fusobacterium nucleatum, Lactobacillus crispatus, Gardnerella vaginalis