ABSTRACT
Background: Formalin-fixed paraffin-embedded (FFPE) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. Unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology FFPE tissue archives in Africa have been a largely unexploited genetic resource, with the usability of DNA obtainable from these samples being unknown.Intervention: The study, conducted from January 2015 to August 2016, determined the usefulness of FFPE tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. Formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the University of Pretoria (Pretoria, South Africa). Deoxyribonucleic acid from FFPE tissue samples and control blood samples was amplified by high-resolution melt real-time polymerase chain reaction before sequencing. The procurement parameters and fixation times were compared with the quantity and quality of the extracted DNA and the efficiency of its subsequent molecular applications.Lessons learnt: This study has shown that FFPE samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics.Recommendations: FFPE samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs.
Subject(s)
DNA , Resolutions , Paraffin , Archives , Autopsy , Tissues , Pain Measurement , Genetic Testing , Polymerase Chain Reaction , Pathology, Molecular , Molecular Docking SimulationABSTRACT
The identification of human remains plays a big role in solving legal and social challenges. To date; significant strides have been made to help positively identify human body remains following both natural and man-made disasters as well as reported cases of missing individuals. Thorough anthropological examination and DNA analysis of the remains can be used to conclusively link the profiles of the remains to persons if a potential living match is available even after a long period of time. We present cases of excavated human remains and samples from Rwanda that were part of both legal and social disputes. Following anthropological examination and DNA analysis; the disputes were conclusively settled. This case report also highlights the possibilities as well as challenges of identifying victim remains of larger calamities such as the 1994 Genocide perpetrated against the Tutsis in Rwanda in which an estimated one million Tutsis lost their lives
Subject(s)
DNAABSTRACT
Background: To achieve early diagnosis and effective treatment of pulmonary tuberculosis; simple and sensitive methods that enhance the detection of Mycobacterium tuberculosis (M. tuberculosis) from clinical specimens are needed. This study compared the effectiveness and suitability of an insertion sequence (IS 6110) based polymerase chain reaction (PCR) assay with conventional methods for the detection of M. tuberculosis from clinical specimens in a resource-limited setting. Methods: Sputa from 101 HIV-positive patients and 40 clinical specimens (sputa; gastic wash out; ascitic fluid; pleural fluid and cerebrospinal fluid) collected from children (HIV status unknown); all suspected for pulmonary tuberculosis at the Jos University Teaching Hospital; Jos; (JUTH) Nigeria; were examined by Ziehl Neelsen (ZN) smear microscopy; Lowenstein Jensen's (LJ) egg-based culture; and PCR methods for the detection of M. tuberculosis Results: Mycobacteria was detected in 45/101 (44.6) of the specimens from the HIV-positive patients and comprised of 6ZN+culture+PCR+; 4ZN-culture+ PCR-; 16ZN-culture+ PCR+ and 19ZN-culture-PCR+. Twenty-two of forty (55) children were positive with 0smear microscopy; 4/40 (10) culture+PCR+; and 18/40 (45) culture- PCR+. The sensitivity and specificity of the PCR for the HIV-positive patients were 85and 74respectively against 23and 100for ZN smear microscopy. Conclusion: The IS6110 PCR is a rapid and sensitive method that is specific for the M. tuberculosis complex group. It is simple in our experience and increased the detection of M. tuberculosis from the specimens examined. We suggest its use for the detection of M. tuberculosis in high TB and HIV burden areas
Subject(s)
DNA , HIV Infections , Polymerase Chain Reaction , TuberculosisABSTRACT
The human genome comprises of abundant DNA sequences related to endogenous retroviruses (ERV) and a variety of solitary long terminal repeats (LTRs). Substantial numbers of intact retroviral particles have been detected by electron microscopy in normal human placental villous tissue particularly in syncytiotrophoblast. Understanding the molecular structure; organisation and distribution of these ERV sequences may lead to elucidation of their possible dual function at the foetal-maternal interface; proliferation and differentiation of cytotrophoblast and induction of local pregnancy-associated immune suppression thus allowing survival of the foetal allograft. In this study; antibody probes were used to screen a human placental expression library and cDNA clones isolated were characterized by polymerase chain reaction; Southern blot hybridisation; DNA cloning and partial nucleotide sequencing. A specific 1.7kb-cDNA clone was isolated from a human placental expression library. Further characterisation showed this clone represents a single copy gene; approximately 9-10kb and did not hybridise to the env region of ERV3 human endogenous retrovirus. The 1.7kb-cDNA clone may represent a provirus co-expressed with cellular sequences