Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

Abstract

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 μl) TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum-caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.

Cleavage-based signal amplification of RNA

“…Thermal cycling of the PCR technique imposes instrumental constraints, limiting the technique to a laboratory setting, and dual-labelled fluorescent probes, such as Taqman probes 12 , are usually needed to determine the specificity of amplification. Therefore, isothermal amplification of RNA, such as nucleic acid sequence-based amplification [13][14][15] , rolling-cycle amplification 16,17 and loopmediated isothermal amplification 18,19 have emerged as alternative amplification techniques. As the above mentioned reactions can be preceded at a constant temperature, there is no need of specialized instruments for RNA detection, and in addition, they have potential for 'on-site' testing.…”

“…Two kinds of catalytic DNA sequences, RNA-cleaving DNAzyme (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) 26,27 and G-quadruplex horseradish peroxidase-mimicking DNAzyme (PW17) [28][29][30] were applied in our research. The 10-23 RNAcleaving DNAzyme has been reported to cleave any purinepyrimidine (RY) junction under simulated physiological conditions 26 ; hence, it was applied in our strategy for the cleaving of target RNA molecule to initiate the following exponential amplification.…”

Visual Detection of High-Risk Human Papillomavirus Genotypes 16, 18, 45, 52, and 58 by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye

“…Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method developed by Notomi et al (18) and has emerged as a powerful gene amplification tool due to its simplicity, speed, specificity, and cost effectiveness. The use of this technique with hydroxynaphthol blue (HNB) dye was first developed in our laboratory and is being used increasingly for rapid and visual detection and typing of emerging viruses (1,16,27).In this study, a simple and visual type-specific LAMP assay for the detection of high-risk HPV genotypes 16, 18, 45, 52, and 58 is described, in which the reaction was carried out in a single tube by mixing primers and DNA polymerase together with the tested samples at 63°C for 65 min. The LAMP assay was further evaluated with HPV DNAs from 294 clinical cervical scrape samples, and the results demonstrate that this assay is sensitive and specific.…”

“…Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method developed by Notomi et al (18) and has emerged as a powerful gene amplification tool due to its simplicity, speed, specificity, and cost effectiveness. The use of this technique with hydroxynaphthol blue (HNB) dye was first developed in our laboratory and is being used increasingly for rapid and visual detection and typing of emerging viruses (1,16,27).…”

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

“…RT-qPCR remains the gold standard for RNA quantification; however, this technique is limited by time and equipment constraints and can be prone to contamination. To minimize these limitations, isothermal RNA amplification techniques have been developed [7][8][9][10][11][12][13][14], but these still remain dependent on nucleic acid replication and are therefore hindered by polymerase speed and fidelity. Recent RNA detection methods that have employed duplex specific nuclease (DSN) isolated from the Paralithodes camtschaticus crab [15,16], remain limited to microRNA [17] and are thus unsuitable for longer RNA templates like virus genomic or mRNA targets.…”