Evaluation of Two Commercial Loop-Mediated Isothermal Amplification Assays for Detection of Avian Influenza H5 and H7 Hemagglutinin Genes

Abstract

Abstract. Real-time reverse transcription loop-mediated isothermal amplification (real-time RT-LAMP) holds substantial potential as a highly sensitive, specific, and easy-to-perform molecular technique for pathogen detection in clinical samples. In the current study, the analytical and diagnostic performance of 2 commercial realtime RT-LAMP kits, Avian Flu H5 and Avian Flu H7, in detecting Avian influenza virus (AIV) infections were evaluated and compared with validated real-time reverse transcription polymerase chain reaction (RT-PCR) assays using RNA from reference virus isolates of subtypes H5 (n 5 24) and H7 (n 5 25) and of phylogenetically related subtypes (n 5 20). When real-time RT-LAMP was carried out according to the recommendations of the manufacturer, 3 out of 24 H5 isolates and 8 out of 25 H7 reference strains were not detected. Prolonging the amplification phase resulted in detection of all H5 isolates but also in false positive detection of 2 non-H5 isolates. Real-time RT-LAMP specific to H7 failed to detect 2 H7 isolates after prolonged amplification. According to the examination of RNA log dilutions, the sensitivity of the real-time RT-LAMP assays, for a number of historic but also recent strains, was considerably lower compared with subtype-specific real-time RT-PCR assays. Application of the real-time RT-LAMP assays for analysis of diagnostic samples from wild birds confirmed their lower sensitivity. Commercial real-time RT-LAMP as tested in this study with a broad range of AIV H5 and H7 strains of phylogenetically diverse yet recent origin, holds some promise for routine veterinary diagnostic purposes, although real-time RT-LAMP was markedly more vulnerable to a reduction of detection limits because of strainspecific sequence variation than subtype-specific real-time RT-PCR.

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

“…In our study, both the RT-LAMP and qRT-PCR assays showed comparable sensitivity for the detection of DENV in patients’ sera (κ = 0.939), even though the sensitivity of RT-LAMP was slightly lower than that of qRT-PCR. Similar findings have been reported in several other studies for the evaluation of LAMP assay when compared against the qPCR [46,47]. The RT-LAMP assay in this study gave possibly six false negative and one false positive results.…”

Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection

“…Recently, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of the AIV H5 gene (Imai et al, 2006(Imai et al, , 2007Jung et al, 2015;Liu et al, 2013;Postel et al, 2010). The H5 RT-RPA is faster than RT-LAMP (5-7 min in H5 RT-RPA compared to 30-60 min in real time RT-LAMP).…”

Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans

“…Furthermore, as less manipulation of samples is required, it has been suggested that the risks of accidental contamination can be significantly reduced [23]. However, one of the most important factors in optimizing these methods relies on the design of appropriate primers, a more complex procedure than that for conventional PCR [24,25], and 4) and conventional PCR, which is generally considered as the method of choice for the detection of viral DNA present at very low amounts in biological samples [4,26-31], but, just as the above mentioned methods, a number of important disadvantages have been reported by the use PCR for WSSV detection. Thus, the Office of International Epizootic (OIE) recommends, among few other molecular techniques, the use of a two-step nested PCR protocol described by [26,32] for all situations where WSSV diagnosis is required [33].…”