Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus

Abstract

A B S T R A C TInfluenza virus and respiratory syncytial virus cause acute upper and lower respiratory tract infections, especially in children and the elderly. Early treatment for these infections is thought to be important, so simple and sensitive detection methods are needed for use at clinical sites. Therefore, in this study, real-time reverse transcription loop-mediated isothermal amplification assays with quenching primer for influenza virus and respiratory syncytial virus were developed. Evaluation of a total of 113 clinical specimens compared to real-time RT-PCR assays showed that the novel assays could distinguish between the types and subtypes of influenza virus and respiratory syncytial virus and had 100% diagnostic specificity. The diagnostic sensitivity of each assay exceeded 85.0% and the assays showed sufficient clinical accuracy. Furthermore, positive results could be obtained in around 15 min using the novel assays in cases with high concentrations of virus. The developed assays should be useful for identifying influenza virus and respiratory syncytial virus cases not only in experimental laboratories but also in hospital and quarantine laboratories.

Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection

“…The insertion of biotin into amplicons by biotin-labelled primers is unproblematic regarding the efficiency of amplication due to Aedes aegypti (Wolbachia infection) 85,86 Ammonia-oxidizing enzyme in environmental bacteria 64 Avian reovirus 70 Bacteriophage MS2 73 Bemisia tabaci 60 BRAF allele (V600E) 83 Brucella 72 Caenorhabditis elegans 68 Chikungunya virus 73 Chlamydia trachomatis 61 Dengue virus 87,112,156 Diarrheal disease 82 Escherichia coli 68 Fomitiporia torreyae 59 Fulviformes umbrinellus 59 Fusarium oxysporum f. sp. lycopersici (point mutations in xylem 3 (SIX3) gene) 66 Genetically modied maize [77][78][79][80][81] Haemophilus ducreyi 101 Haemophilus inuenzae 105 hBRCA1 68 HeLa 68 Hepatitis B and C virus 62,87,107 Herpes simplex virus 1 (HSV1) US4 83 Human immunodeciency virus 87,101 HLA-B*15:02 allele 163 Human papillomavirus 153,171 Human T-lymphotropic virus 1 101 Inuenza virus 63,114 Lambda DNA 68 Leptospira 148 Leishmania 155 Listeria ivanovii 103 Listeria monocytogenes 103 Methicillin-resistant Staphylococcus aureus (MRSA) 71,166 Middle east respiratory sy...…”

“…So-called QPrimers 63 were used for the detection of inuenza virus and respiratory syncytial virus in singleplex assays. 63 The analytical sensitivity was between 25-250 copies per reaction, and the diagnostic sensitivity and specicity were determined to be 85.0% and 100% respectively. One big drawback is that signal generation depends heavily on the target sequence.…”

Detecting amplicons of loop‐mediated isothermal amplification

“…In addition, use of QPrimers reduces the LAMP detection time by several minutes relative to turbidity detection, and positive signals are typically detected within 20 min. 45 Although QPrimer can resolve concerns regarding detection of primer-derived signals directly, nonspecific DNA extension at the annealed 3′ end and/or primer dimers can result in false positives, so strict screening for primer integrity is still necessary. To overcome this, LAMP using a quenching probe (QProbe) 44 was developed.…”

Development of real‐time fluorescent reverse transcription loop‐mediated isothermal amplification assays for rhinovirus detection

“…Previously, real‐time fluorescent RT‐LAMP was established to detect viral RNA using a quenching primer (QPrimer) or a quenching probe (QProbe). By using a QPrimer or QProbe, nonspecific reactions can be decreased and the target can be detected in a real‐time manner . In this study, two assays were developed to detect RVs by a real‐time fluorescent RT‐LAMP method, and the competence of both assays was compared with real‐time RT‐PCR assays for the detection of RV infection using clinical specimens.…”