On Chip Detection of Zika Virus Based on Loop Mediated Isothermal Amplification

Abstract

There was a world-wide Zika virus (ZIKV) epidemic occurred in 2015, with the major concern of about 20-fold increase in fetuse microcephaly rate in Brazil. To improve the ZIKV point-of-care (POC) molecular diagnostic, we established a rapid and sensitive real-time fluorescence quantitative loop mediated isothermal amplification (LAMP) method and further applied it on a self-fabricated microfluidic chip. After optimization of LAMP reaction conditions, the assay achieved the detection limit of single copy of the standard plasmid in a reaction. Linear regression analysis revealed that the correlation coefficients (R²) were 0.9931. No cross reaction was observed in the controls of yellow fever clinical specimen and several known human influenza viruses, including seasonal A/H1N1, A/H7N9, A/H9N2 and B. To evaluate the performance characteristics of the ZIKV-LAMP assay, we detected 39 clinical specimens by both LAMP assay and real-time RT-PCR, which obtained with completely consistent results. The sensitivity, specificity and performance characteristics of the ZIKV-LAMP assay conformed its utility in ZIKV determination. Moreover, we had also developed a real-time fluorescence detection biomedical system with microfluidic chips. The microfluidic chips were designed with four microcells and the volume of the LAMP reaction was greatly reduced from about 25μL to 10μL. Our newly established real-time fluorescence LAMP detection system with microfluidic chips has the potential for ZIKV POC diagnostics with the advantages of low cost, short analytical time, disposability, low reagent and sample consumption and so on.