TY - JOUR
T1 - A novel application of real-time RT-LAMP for body fluid identification
T2 - using HBB detection as the model
AU - Su, Chih Wen
AU - Li, Chiao Yun
AU - Lee, James Chun I
AU - Ji, Dar Der
AU - Li, Shu Ying
AU - Daniel, Barbara
AU - Syndercombe-Court, Denise
AU - Linacre, Adrian
AU - Hsieh, Hsing Mei
PY - 2015/6/1
Y1 - 2015/6/1
N2 - 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.
AB - 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.
KW - 18S rRNA
KW - Body fluid identification
KW - Hemoglobin beta (HBB)
KW - Real-time RT-LAMP
UR - http://www.scopus.com/inward/record.url?scp=84937761869&partnerID=8YFLogxK
U2 - 10.1007/s12024-015-9668-6
DO - 10.1007/s12024-015-9668-6
M3 - Article
C2 - 25877518
AN - SCOPUS:84937761869
SN - 1547-769X
VL - 11
SP - 208
EP - 215
JO - Forensic Science, Medicine, and Pathology
JF - Forensic Science, Medicine, and Pathology
IS - 2
ER -