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

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.