Lyophilized visually readable loop-mediated isothermal reverse transcriptase nucleic acid amplification test for detection Ebola Zaire RNA

Abstract

Recent viral outbreaks highlight the need for reliable, yet broadly deployable diagnostics for detection of epidemic and emerging pathogens. In this study we designed and optimized methods to visually detect viral nucleic acid by isothermal amplification and SYBR dye intercalation. We designed and tested loop-mediated isothermal amplification (LAMP) primers and lyophilized reactions to optimize the detection of Zaire Ebola Virus (ZEBOV) and further evolved the LAMP platform to allow room-temperature storage for deployment in resource limited settings. Our results demonstrated excellent sensitivity and specificity for viral nucleic acid sequences with lower limits of detection of less than 100 copies. Moreover, lyophilized reaction mixtures retained activity for prolonged periods under dry conditions at room temperature. This approach offers a way for detection of emerging viruses in resource limited settings.

DNA-Detection Based Diagnostics for Taenia solium Cysticercosis in Porcine

“…Hayashida et al [37] developed a direct dry LAMP format for human African trypanosomiasis ideal for bedside or sampling site operation. e reagents maintain the same reactivity as freshly prepared reagents and remain stable for up to 3 months when stored at 4°C [52]. A study by Chander et al [53] reported that dry LAMP formulation for bacteria and viruses remain stable at room temperature for at least 6 months with no apparent loss in activity and the sensitivity remains at about 12 copies which was similar to the freshly prepared reagents for at least 3 months [53].…”

“…e requirement for pointof-care diagnostic test in resource limited settings requires the ability for long shelf life without any means of a cold chain [21] and as a result lyophilized LAMP formats have been developed [52,53]. is method eliminates the need to open the reaction tubes as it requires only sample addition, reducing the possibility of contamination [45,54].…”

Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection of Strongyloides Larvae in Different Specimen Matrices

“…While this is yet to be compared with standard Bst (large fragment) using stool extracts, there may be benefit to its use. Lyophilization may not necessarily increase assay sensitivity, but it can contribute to the stability of reagents and facilitate diagnostics in settings where resources are limited (48,49). Syto 82 intercalating dye is an effective means of detecting amplified DNA without the need to open the reaction tube, minimizing the risk of contamination (18,50,51).…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…One of the latest development of the lyophilization method of RT-LAMP reaction was reported by Carter et al (2017) with its application in the detection of Zaire Ebola Virus (ZEBOV). LAMP primers targeting L gene of ZEBOV were designed based on the alignment of isolates from 1976, 1995, 2007 and 2014 outbreaks as well as corresponding fragments were synthesized and cloned into plasmid which served as the template for in vitro transcription of the sense strand.…”

“…The master mix was then subjected to cool in liquid nitrogen followed by freezedrying. In this study, they highlighted key modifications of the reaction components in order to maintain the lyophilized reagents stability, which was the addition of a nonreducing sugar trehalose and dialysis of the used enzyme preparations (Carter et al 2017).…”