Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A/H1, A/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity

作者：Mahony James^*; Chong Sylvia; Bulir David; Ruyter Alexandra; Mwawasi Ken; Waltho Daniel

来源：Journal of Clinical Virology, 2013, 58(1): 127-131.

DOI：10.1016/j.jcv.2013.06.006

• Influenza
• Rapid detection
• LAMP

Background: Rapid isothermal amplification methods have recently been introduced and they offer significant advantages over PCR. %26lt;br%26gt;Objective: To develop a rapid and sensitive M-LAMP assay for the detection of influenza A (H1 and H3) and B that does not require RNA extraction. %26lt;br%26gt;Study design: We designed six primers targeting the matrix genes of influenza H1 and H3 and the NS1 gene of influenza B and developed a M-LAMP assay using a commercially available Master Mix and a real time fluorometer (Genie II, Optigene, UK) that displays real time amplification, time to positivity and amplicon annealing temperature (Tm). M-LAMP was evaluated against PCR by testing 202 nasopharyngeal (NP) specimens. %26lt;br%26gt;Results: Optimized M-LAMP was rapid with a mean amplification time of 12 min (compared with 90-120 min for PCR), had an analytical sensitivity of 1 genome equivalent (ge), and could distinguish influenza A including subtypes A/H1 and A/H3 from influenza B by Tm. M-LAMP detected 26/28 influenza A/H1, 27/27 influenza A/H3 and 39/39 influenza B specimens and had a combined sensitivity and specificity for detecting influenza (A and B) of 97.9% (92/94) and 100% (108/108), respectively. The rapid amplification time of LAMP coupled with a novel 10-min specimen preparation procedure consisting of vortexing and heating in M-Swab diluent (Copan Italia) provided a rapid result. %26lt;br%26gt;Conclusions: M-LAMP had excellent sensitivity and specificity for detecting influenza A and B in NP specimens and when used together with a rapid specimen processing method provided a specimen-to-result diagnosis in 30 min.

• 出版日期2013-9