Abstract
Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5′ backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 103 copies and 102 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.
Highlights
Influenza, which is caused by the influenza virus, is a significant cause of morbidity and mortality that has major social and economic impacts throughout the world [1, 2]
Optimization of the multiplex influenza A/B/IC loop-mediated isothermal amplification (LAMP) primer set Before optimization of multiplex LAMP assay, each Influenza A, B and internal control (IC, actin beta) LAMP primer set were tested with clinical samples (Influenza A H1, H1N1, H3N2, Influenza B, human serum RNA and distilled water)
For optimization of the multiplex influenza A/B/IC LAMP primer set, different concentration ratios of the influenza A, influenza B, and internal control primer sets (1:1:1, 1:1:0.5, and 1:0.5:0.5, respectively) were tested using human serum RNA samples spiked with influenza A and B plasmids (107 copy, ratio of 1:1) (Fig 1A)
Summary
Influenza, which is caused by the influenza virus, is a significant cause of morbidity and mortality that has major social and economic impacts throughout the world [1, 2]. Influenza A and B viruses comprise the major respiratory pathogens in humans that cause seasonal flu epidemics [9, 10]. For multiple detection using the LAMP assay, an assimilating probe consisting of the fluorescently tagged probe and its complementary sequence probe tagged with quencher was developed. We present a rapid multiplex RT-LAMP diagnosis method for influenza A, influenza B, and an internal control using a newly designed target-specific assimilating probe and fluorescently-tagged strand displaceable probes. This multiplex influenza A/B/IC RT-LAMP assay had high sensitivities for influenza A and influenza B without interference from one another. The assay showed no cross-reactivity against other respiratory and hemorrhagic fever viruses
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