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.

Forensic body fluid identification: state of the art

“…59,74 Recently, new methods have emerged, such as real-time RT loop-mediated isothermal amplification, offering equal sensitivity and specificity but significantly simpler and quicker analyses. 75 Constitutively expressed housekeeping genes provide a reference point and assess the performance of a reaction when using capillary electrophoresis-based methods and are essential when using quantitative methods. 67,76 An ideal housekeeping gene is one that is expressed in all tissues, does not vary significantly amongst and within individuals, and is not affected significantly by physiological/pathological conditions.…”

Detection of Novel duck reovirus (NDRV) using visual reverse transcription loop-mediated isothermal amplification (RT-LAMP)

“…A reverse transcription loop mediated isothermal amplification (RT-LAMP) provides a rapid and precise detection assay for viral pathogens with high sensitivity and specificity 4 . This technique is profitable and convenient, only requires a constant temperature water bath and averts some deficiencies, including high necessity for equipment, tremendous cost, lengthened examination period and low sensitivity 5 . In comparison with the equipment required for traditional polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR) assays, RT-LAMP is easy to conduct in resource-limited laboratory settings in underdeveloped countries 6 .…”

A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification

“…Currently, available methods for detection of PDCoVs include conventional reverse transcription—polymerase chain reaction (RT-PCR), nested RT-PCR, real-time RT-PCR, and ELISA [ 6 , 10 – 13 ]. However, these techniques have some shortcomings, such as higher requirements for equipment, high cost, extended detection period, and/or low sensitivity [ 14 ]. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a potential effective tool for rapid and accurate identification of viral pathogens, which amplifies nucleic acids under isothermal conditions with high sensitivity and specificity.…”

“…This novel gene detection technique is cost-effective and time-saving, and only requires a constant temperature water bath. RT-LAMP has been widely used in clinical diagnosis for detection of the presence of several important viral pathogens, including PEDV, TGEV, classic swine fever virus (CSFV), and H10N8 subtype of influenza A virus [ 14 – 17 ]. The improved LAMP assays, such as visual detection of amplified products by adding SYBR Green I or hydroxynaphthol blue (HNB), have made it easier to apply in primary clinical settings or for field use.…”