Design and optimization of a 16S microbial qPCR multiplex for the presumptive identification of feces, saliva, vaginal and menstrual secretions

Abstract

Molecular methods for body fluid identification have been extensively researched in the forensic community over the last decade, mostly focusing on RNA-based methods. Microbial DNA analysis has long been used for forensic applications, such as postmortem interval estimations, but only recently has it been applied to body fluid identification. High-throughput sequencing of the 16S ribosomal RNA gene by previous research groups revealed that microbial signatures and abundances vary across human body fluids at the genus and/or species taxonomic level. Since quantitative PCR is still the current technique used in forensic DNA analysis, the purpose of this study was to design a qPCR multiplex targeting the 16S gene of Bacteroides uniformis, Streptococcus salivarius, and Lactobacillus crispatus that can distinguish between feces, saliva, and vaginal/menstrual secretions, respectively. Primers and probes were designed at the species level because these bacteria are highly abundant within their respective fluid. The validated 16S triplex was evaluated in DNA extracts from thirty donors of each body fluid. A classification regression tree model resulted in 96.5% classification accuracy of the population data, which demonstrates the ability of this 16S triplex to presumptively identify these fluids with high confidence at the quantification step of the forensic workflow using minimal input volume of DNA extracted from evidentiary samples.

A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup

“…Parker et al observed ~2‐fold to ~240‐fold drop in sensitivity when changing from multiple duplex assays in detecting respiratory viral pathogens to a multiplex platform 30 . Many publications are available to guide the optimisation of multiplex PCR or qPCR 35–37 . However, their primary focus on microbial pathogen detection represents a different context (e.g., microbial pathogen can be cultured to higher titre before extraction), and hence, not all of their considerations are specific to the context of doping control.…”

“…Parker et al observed $2-fold to $240-fold drop in sensitivity when changing from multiple duplex assays in detecting respiratory viral pathogens to a multiplex platform 30. Many publications are available to guide the optimisation of multiplex PCR or qPCR [35][36][37]. F I G U R E 1 The qPCR efficiency of each transgene in the multiplex qPCR assay.…”

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids

“…JMP® v14.2.0 (SAS Institute Inc., Cary, NC, USA) was used to analyze data distributions and subsequently perform a non‐parametric Steel‐Dwass test on the ∆Cq data ( α = 0.05). The ∆Cq values were added to the previously reported 16S triplex dataset [17], and all data were randomly split into training and validation sets (70% and 30%, respectively) using the sample() function. A new classification regression tree (CART) model was generated using the rpart package in R version 4.1.1 (R foundation for Statistical Computing, Vienna, Austria).…”

“…Previously, a 16S qPCR triplex that amplifies that 16S rRNA gene of Lactobacillus crispatus (L. crispatus), Bacteroides uniformis (B. uniformis), and Streptococcus salivarius (S. salivarius) to classify vaginal/menstrual secretions, feces, and saliva, respectively, was developed and evaluated [17]. This assay was based on the findings from a large-scale high-throughput sequencing analysis study that identified the above microbial DNA as consistently observed across populations, genders (where appropriate) and ages [18].…”

“…There has never been a combined molecular approach utilizing microRNA and microbial DNA sequences in a qPCR assay to identify forensically relevant body fluids. Therefore, it is proposed in this study to expand the previously reported microbial 16S triplex to incorporate a miRNA reverse transcription‐qPCR (RT‐qPCR) duplex that can now differentiate between vaginal/menstrual secretions, feces, saliva, blood, and semen at the quantification step of the forensic DNA workflow [17]. Our previous work on body fluid classification using miRNAs had previously identified and validated miR‐891a and let‐7g as being useful as markers for distinguishing blood and semen, and as an internal control, respectively [46].…”