RESUMEN
Identifying body fluids from forensic samples can provide valuable evidence for criminal investigations. Messenger RNA (mRNA)-based body fluid identification was recently developed, and highly sensitive parallel identification using reverse transcription polymerase chain reaction (RT-PCR) has been described. In this study, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple, rapid assay for identifying three common forensic body fluids, namely blood, semen, and saliva, and evaluated its specificity and sensitivity. Hemoglobin beta (HBB), transglutaminase 4 (TGM4), and statherin (STATH) were selected as marker genes for blood, semen, and saliva, respectively. RT-LAMP could be performed in a single step including both reverse transcription and DNA amplification under an isothermal condition within 60 min, and detection could be conveniently performed via visual fluorescence. Marker-specific amplification was performed in each assay, and no cross-reaction was observed among five representative forensically relevant body fluids. The detection limits of the assays were 0.3 nL, 30 nL, and 0.3 µL for blood, semen, and saliva, respectively, and their sensitivities were comparable with those of RT-PCR. Furthermore, RT-LAMP assays were applicable to forensic casework samples. It is considered that RT-LAMP is useful for body fluid identification.
Asunto(s)
Líquidos Corporales/química , Genética Forense/métodos , Marcadores Genéticos , Técnicas de Amplificación de Ácido Nucleico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sangre , ADN/genética , Subunidades de Hemoglobina/genética , Humanos , Cinética , Límite de Detección , Masculino , Saliva/química , Proteínas y Péptidos Salivales/genética , Espectrometría de Fluorescencia , Espermatozoides/química , Transglutaminasas/genéticaRESUMEN
In forensics, body fluid identification plays an important role because it aids in reconstructing a crime scene. Therefore, it is essential to develop simple and reliable techniques for body fluid identification. Nucleic acid aptamers are useful tools in analytical chemistry that can be used to improve conventional forensic analytical techniques. They have numerous advantages over antibodies including their low cost, long shelf life, and applicability for chemical modification and PCR amplification. A DNA aptamer against a human prostate-specific antigen (PSA), which is a well-known protein marker for semen identification in forensics, has been reported previously. In this study, as a proof-of-concept for nucleic acid aptamer-based identification of body fluids, we developed a technique of aptamer-based PSA assays for semen identification that employed enzyme-linked oligonucleotide assay (ELONA) and real-time PCR. We evaluated their sensitivity and specificity for semen compared with those for blood, saliva, urine, sweat, and vaginal secretion. The assays have equivalent procedures compared to enzyme-linked immunosorbent assay; their results were consistent with those produced by the conventional immunochromatographic assay. The minimum volume of semen required for detection was 62.5 nL in ELONA and 5 nL in real-time PCR, making this assay applicable for semen detection in actual criminal investigation. Aptamers can be a cost-effective and versatile tool for forensic body fluid identification.