Computational protocol: Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor

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Protocol publication

[…] mRT-LAMP primers were designed based on the conserved regions of HA gene, M1 gene and NS1 gene for influenza A/H1N1pdm09, A/H3 and influenza B, respectively. RT-LAMP primers were designed using a PrimerExplorer version 4 program on the Eiken website (http://primerexplorer.jp/e/). A set of six primers, including two outer primers (F3/B3), two inner primers (FIP/BIP), and two loop primers (LF/LB), were selected for each of the three gene targets. The specificity and feasibility of the selected primers were subsequently checked by Basic Local Alignment Search Tool search against National Center for Biotechnology Information database. The sequence information of the selected final mRT-LAMP primers containing degenerate bases is shown in , and the details of the primers are shown in .Using the Universal Invader Design Software version 1.2.4 (Third Wave Technologies, Inc., Madison, WI, USA), three sets of cascade invasive reaction upstream and downstream probes were designed based on the sequences of mRT-LAMP amplicons. The sequence information of the selected upstream, downstream and hairpin probes used in secondary invasive reaction, as well as two types of oligonucleotide probes (nanoparticle probes) used for modification of AuNPs is shown in . All the primers and probes were synthesized and modified by TaKaRa Biotechnology Co. Ltd. […]

Pipeline specifications

Software tools PrimerExplorer, BLASTN
Application qPCR