RESUMO
Numerous methods for human body fluid identification using microbiological markers specific to different human body parts are well-established in forensic science. However, method for vaginal fluid screening have not been standardized yet. Therefore, in this study, a real-time polymerase chain reaction based assay for vaginal fluid identification was devised using bacteria residing in human vagina. This method employed three markers, namely Lactobacillus iners, Lactobacillus crispatus, and Bacteroides fragilis. L. iners and L. crispatus were chosen due to their high abundance in the vagina, whereas B. fragilis resides in the rectum. To examine the suitability of the new method for forensic microbial applications, a study of the distribution of vaginal flora in 143 Korean women was performed, along with characterization of the specificity, and performance of the new assay. Additionally, a casework study based on 130, 21, 20 and 17 DNA samples collected from the vagina, anus, saliva, blood, respectively, was carried out. L. iners (80.4%) and L. crispatus (55.2%) were detected with high abundance in the vagina of Korean women. The specificity of these markers was verified using microbial DNA from 23 species. This method could detect at least 1,000 copies/µL of microorganisms for all markers, thereby highlighting its robust sensitivity for vaginal fluid identification. The casework study confirmed these findings, with 89.2% (116/130) detection of vaginal fluid-derived DNA samples, and no false positives identified from the other sources studied. In conclusion, the developed method is expected to be efficient for preliminary microbiological analysis of vaginal samples in forensics.
RESUMO
BACKGROUND: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence. METHODS: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing. RESULTS: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing. CONCLUSIONS: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.