Instrument-Free Point-of-Care Molecular Detection of Zika Virus

Abstract

The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors' offices, clinics, and at home.

Figure 1

(A) Schematic of saliva sample preparation. Saliva samples are first collected in a saliva collection tube and then lysed in Qiagen binding/lysis (AVL) buffer. (B) The lysed sample is filtered through the isolation membrane of our microfluidic cassette for nucleic acid extraction. (C) Exploded view of the chemically heated cup. The cup consists of a thermos cup body, a 3D-printed cup lid, a chip holder, PCM material, heat sink and single-use Mg–Fe alloy pack heat source. (D) A photograph of the chemically heated cup for point of care molecular diagnostics of ZIKV.

Figure 2

(A) RT-LAMP amplification curves in the presence of 500, 50, 5, and 0 PFU of ZIKV per reaction. (B) The threshold time t_T (in minutes) as a function of the PFU (n = 3).

Figure 3

Photographs of the isothermal amplification reactors (A) before and (B) after 40 min incubation in the chemically heated cup. Leuco crystal violet dye is used as an amplification indicator. Amplification reactors 1, 2, 3, and 4 contain 0, 5, 50, and 500 PFU of ZIKV.