Sensitive and rapid detection of Zika virus by loop-mediated isothermal amplification

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus, which is a pathogen affecting humans in Africa, Asia, and America. It is necessary to detect ZIKV with a rapid and sensitive molecular method to guide timely treatment. In this study, a loop-mediated isothermal amplification (LAMP) assay was described, which is an attractive option as a fast, sensitive, and specific method for ZIKV detection using the NS5 protein coding region and the envelope protein (EP) coding region as target sequences. Two different techniques, a calcein/Mn²⁺ complex chromogenic method and real-time turbidity monitoring, were employed. The specificity and sensitivity of the LAMP assay were determined. The assay's detection limit was 0.5 × 10^-9 pmol/µl DNA for NS5 protein coding region and 1.12 × 10^-11 pmol/µl DNA for E coding region, respectively, which is a 100-fold increase in sensitivity compared with real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional PCR. All 12 non-ZIKA respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for ZIKV. In conclusion, a visual detection LAMP assay was developed, which could be a useful tool for primary quarantine purposes and clinical screening, especially in situations where resources are poor and in point-of-care tests.

Keywords: LAMP; Rapid detection; Sensitivity; Specificity; Zika virus.

Fig. 1

The most appropriate primers for the loop-mediated isothermal amplification assay. a A total of five sets of primers including Zika(NS5)-2, Zika(NS5)-3, Zika(NS5)-5, Zika1(NS5-Natal1), and Zika2(NS5-Natal2) were designed to detect the recombinant plasmid containing NS5 gene. b A total of three sets of primers including Zika-1, Zika-10, and Zika-15 were designed to detect the recombinant plasmid containing E gene. Turbidity was monitored and recorded every 6 s for five sets of primers used to amplify the target gene with a Loopamp Real-time Turbidimeter at 650 nm

Fig. 2

The most appropriate reaction temperatures for the loop-mediated isothermal amplification assay for NS5 (a) and E (b). Reaction temperatures ranged from 55 to 69 °C with 2 °C intervals. Turbidity was monitored every 6 s with a Loopamp Real-time Turbidimeter at 650 nm

Fig. 3

Specificity of ZIKV NS5 (a, b) and E (c, d) detection by LAMP. a, c Turbidity was monitored every 6 s with a Loopamp Real-time Turbidimeter at 650 nm. b, d Visual detection using a calcein fluorescent detection reagent. Lane 1, H1N1; 2, H3N2; 3, SWL; 4, BV; 5, BY; 6, H7N9; 7, OC43; 8, NL63; 9, 229E; 10, SARS; 11, Dengue; 12, positive control (the recombinant plasmids containing NS5 gene or E gene); 13, Ebola; 14, negative control (double-distilled water)

Fig. 4

Comparison of the sensitivities of the LAMP reaction and conventional PCR in detecting the ZIKV NS5 gene (a–c). a Turbidity monitoring, b visual detection, c PCR detection. The recombinant plasmid containing NS5 gene was serially diluted 10-fold from 0.5 × 10⁻² to 0.5 × 10⁻⁹ pmol/µl. 1, negative control (double-distilled water); 2, 0.5 × 10⁻² pmol/µl; 3, 0.5 × 10⁻³ pmol/µl; 4, 0.5 × 10⁻⁴ pmol/µl; 5, 0.5 × 10⁻⁵ pmol/µl; 6, 0.5 × 10⁻⁶ pmol/µl; 7, 0.5 × 10⁻⁷ pmol/µl; 8, 0.5 × 10⁻⁸ pmol/µl; 9, 0.5 × 10⁻⁹ pmol/µl. Comparison of the sensitivities of the LAMP reaction and conventional PCR in detecting the ZIKV E gene (d–f). d Turbidity monitoring, e visual detection, f PCR detection. The recombinant plasmid containing E gene was serially diluted 10-fold from 1.12 × 10⁻⁴ pmol/µl to 1.12 × 10⁻¹¹ pmol/µl. 1, negative control (double-distilled water); 2, 1.12 × 10⁻⁴ pmol/µl; 3, 1.12 × 10⁻⁵ pmol/µl; 4, 1.12 × 10⁻⁶ pmol/µl; 5, 1.12 × 10⁻⁷ pmol/µl; 6, 1.12 × 10⁻⁸ pmol/µl; 7, 1.12 × 10⁻⁹ pmol/µl; 8, 1.12 × 10⁻¹⁰ pmol/µl; 9, 1.12 × 10⁻¹¹ pmol/µl