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 10¹ copies of RNA for both A/H1N1pdm09 and A/H3, and 10² 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.

Keywords: LAMP; gold nanoparticles; influenza virus; multiplex; on-site detection.

Figure 1

Schematic showing location of multiplex RT-LAMP primer binding sites. Notes: (A) HA gene of influenza A/H1N1pdm09. Assay spans region from nucleotide 757–937 with reference to the HA gene sequence of the H1N1pdm09 virus strain A/Jiangsu/2/2009(H1N1). (B) M1 gene of influenza A/H3N2. Assay spans region from nucleotide 425–606 with reference to the M1 gene sequence of the H3N2 virus strain A/Nanjing/1/2009(H3N2). (C) NS1 gene of influenza B. Assay spans region from nucleotide 442–667 with reference to the NS1 gene sequence of the influenza B virus strain B/Jiangsu/01/2015. Abbreviations: HA, hemagglutinin; M1, matrix protein 1; NS1, nonstructural protein 1; RT-LAMP, reverse-transcription loop-mediated isothermal amplification.

Figure 2

The principles of cascade invasive reaction and signal detection with AuNPs for detecting multiplex RT-LAMP products. Notes: The system contains three steps: primary invasive reaction, secondary invasive reaction and nanoparticle hybridization. Primary invasive reaction: the Up, Dp and RT-LAMP amplicon form a one-base overlapping structure at the 3′end of the Up. AfuFEN enzyme specifically recognizes the structure and cleaves the 5′ flap of the Dp. Secondary invasive reaction: the cleaved 5′ flap from the target-specific primary invasive reaction hybridizes with the Hp and drives a secondary invasive reaction. Then the Hp is cleaved by AfuFEN. Nanoparticle hybridization: if the target of RT-LAMP product is present, the cleaved Hp cannot trigger the aggregation of AuNPs, resulting in red-colored dispersions of AuNPs. Conversely, if the target of RT-LAMP product is absent, the Hp remains intact without DNA cleavage, yielding nanoparticle aggregation-induced precipitation, and then the reaction mixture becomes colorless. Abbreviations: Up, upstream probe; Dp, downstream probe; Hp, hairpin probe; AfuFEN, Archaeoglobus fulgidus flap endonuclease; AuNPs, gold nanoparticles; RT-LAMP, reverse-transcription loop-mediated isothermal amplification.

Figure 3

Sensitivity test results of the mRT-LAMP-CIRN assay for detecting influenza A/H1N1pdm09, A/H3 and influenza B. Notes: Ten-fold serial dilutions of A/H1N1pdm09 (A), A/H3 (B) and influenza B (C) RNA transcripts (ranging from 10⁴ to 10⁰ RNA copies) were detected by the mRT-LAMP-CIRN assay. The multiplex RT-LAMP amplification reactions for A/H1N1pdm09 (D), A/H3 (E) and influenza B (F) RNA transcripts were also real-time monitored by a turbidimeter and the corresponding curves of concentrations of templates were marked in the figure. Abbreviations: mRT-LAMP-CIRN, multiplex reverse-transcription loop-mediated isothermal amplification coupled with cascade invasive reaction using nanoparticles as a sensor; DW, distilled water.

Figure 4

Specificity test results of the mRT-LAMP-CIRN assay for detection of influenza viruses. Notes: The analytical specificity of the mRT-LAMP-CIRN assay was assessed by testing the RNA extracts from influenza A/H1N1pdm09, A/H3N2 and influenza B, and from various control viruses separately. RNA extracts from the three influenza virus strains were also tested in different combinations in a single reaction. (A) The cascade invasive reactions were performed with 09H1-Up, 09H1-Dp and Hp. (B) The cascade invasive reactions were performed with H3-Up, H3-Dp and Hp. (C) The cascade invasive reactions were performed with FluB-Up, FluB-Dp and Hp. 1: no-template control; 2: A/H1N1pdm09; 3: A/H3N2; 4: influenza B; 5: A/H1N1pdm09 and A/H3N2; 6: A/H1N1pdm09 and influenza B; 7: A/H3N2 and influenza B; 8: A/H1N1pdm09, A/H3N2 and influenza B; 9: A/H1N1; 10: A/H5N1; 11: A/H7N9; 12: A/H9N2; 13: respiratory syncytial viruses type A; 14: respiratory syncytial viruses type B; 15: parainfluenza viruses types 1; 16: parainfluenza viruses types 2. Abbreviations: mRT-LAMP-CIRN, multiplex reverse-transcription loop-mediated isothermal amplification coupled with cascade invasive reaction using nanoparticles as a sensor; Up, upstream probe; Dp, downstream probe; Hp, hairpin probe.