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

Abstract

Influenza virus infections represent a worldwide public health and economic problem due to the significant morbidity and mortality caused by seasonal epidemics and pandemics. Sensitive and convenient methodologies for detection of influenza viruses are essential for further disease control. Loop-mediated isothermal amplification (LAMP) is the most commonly used method of nucleic acid isothermal amplification. However, with regard to multiplex LAMP, differentiating the ladder-like LAMP products derived from multiple targets is still challenging today. The requirement of specialized instruments has further hindered the on-site application of multiplex LAMP. We have developed an integrated assay coupling multiplex reverse transcription LAMP with cascade invasive reaction using nanoparticles (mRT-LAMP-CIRN) as a sensor for the detection of three subtypes of influenza viruses: A/H1N1pdm09, A/H3 and influenza B. The analytic sensitivities of the mRT-LAMP-CIRN assay were 10 1 copies of RNA for both A/H1N1pdm09 and A/H3, and 10 2 copies of RNA for influenza B. This assay demonstrated highly specific detection of target viruses and could differentiate them from other genetically or clinically related viruses. Clinical specimen analysis showed the mRT-LAMP-CIRN assay had an overall sensitivity and specificity of 98.3% and 100%, respectively. In summary, the mRT-LAMP-CIRN assay is highly sensitive and specific, and can be used as a cost-saving and instrument-free method for the detection of influenza viruses, especially for on-site use.

Cyanobacteria as Nanogold Factories: Chemical and Anti-Myocardial Infarction Properties of Gold Nanoparticles Synthesized by Lyngbya majuscula

“…Another popular use of metal nanoparticles is as optical or electrochemical biosensors [ 10 , 11 ]. The use of metal nanoparticles in such techniques enabled their involvement in the medical and clinical diagnosis of many diseases such as cancer [ 12 ], Alzheimer’s disease [ 13 ], HIV [ 14 ], hepatitis B [ 15 ], tuberculosis [ 16 ], diabetes [ 17 ], and influenza [ 18 ]. Metal nanoparticles have also been used as a treatment for many disorders and medical conditions such as cancer [ 19 , 20 , 21 ] and rheumatoid arthritis [ 22 , 23 ], and for several topical and systemic infections as anti-microbial agents [ 24 , 25 , 26 ].…”

A Rapid and Specific Assay for the Detection of MERS-CoV

“…According to a study by Notomi et al (2000) , inserting TTTT into the FIP and BIP primers can enhance LAMP efficiency. However, the inner primers utilized in many studies of the detection of nucleic acids with LAMP assays do not contain TTTT sequences ( Notomi et al, 2000 ; Mori et al, 2001 ; Mekata et al, 2006 ; Wang et al, 2013 ; Ge et al, 2017 ). To investigate the efficiency of the RT-LAMP-VF assay after inserting TTTT into the FIP and BIP primers, two sets of inner primers were designed (Supplementary Table 3 ); one set contained the TTTT sequence and one set did not.…”

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...…”