title: A novel application of real-time RT-LAMP for body fluid identification: using HBB detection as the model.
abstract: We report on a novel application of real-time reverse transcription-loop-mediated isothermal amplification (real-time RT-LAMP) to identify the presence of a specific body fluid using blood as a proof-of-concept model. By comparison with recently developed methods of body fluid identification, the RT-LAMP assay is rapid and requires only one simple heating-block maintained at a single temperature, circumventing the need for dedicated equipment. RNA was extracted from different body fluids (blood, semen, saliva, menstrual blood, sweat, and urine) for use in real-time RT-LAMP reaction. The 18S rRNA locus was used as the internal control and hemoglobin beta (HBB) as the blood-specific marker. Reverse transcription and LAMP reaction were performed in the same tube using a turbidimeter for real-time monitoring the reaction products within a threshold of 60 min. HBB LAMP products were only detected in blood and not in any of the other body fluid, but products from the 18S rRNA gene were detected in all the tested body fluids as expected. The limit of detection was a minimum of 10(-5) ng total RNA for detection of both 18S rRNA and HBB. Augmenting the detection of RT-LAMP products was performed by separation of the products using gel electrophoresis and collecting the fluorescence of calcein. The data collected indicated complete concordance with the body fluid tested regardless of the method of detection used. This is the first application of real-time RT-LAMP to detect body fluid specific RNA and indicates the use of this method in forensic biology.
authors: Department of Forensic Science, Central Police University, 56 Shu-Jen Road, Kwei-San, Taoyuan, 33304, Taiwan, ROC.
authors: Chih-Wen
authors: CW
authors: Su
authors: Chiao-Yun
authors: CY
authors: Li
authors: James Chun-I
authors: JC
authors: Lee
authors: Dar-Der
authors: DD
authors: Ji
authors: Shu-Ying
authors: SY
authors: Li
authors: Barbara
authors: B
authors: Daniel
authors: Denise
authors: D
authors: Syndercombe-Court
authors: Adrian
authors: A
authors: Linacre
authors: Hsing-Mei
authors: HM
authors: Hsieh
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood; D001774:Blood Chemical Analysis; D004587:Electrophoresis, Agar Gel; D005260:Female; D005453:Fluorescence; D005554:Forensic Medicine; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012313:RNA / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry*; D012661:Semen / Q000737:chemistry*; D013542:Sweat / Q000737:chemistry*; D014556:Urine / Q000737:chemistry*; D055815:Young Adult; D055544:beta-Globins / Q000235:genetics
chemical_list: D015415:Biomarkers; D012337:RNA, Ribosomal, 18S; D055544:beta-Globins; D012313:RNA