Rapid and specific detection of Asian- and African-lineage Zika viruses

Abstract

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers.

Fig. 1. Design and implementation of LAMP primers

(A) LAMP target sequences in the Puerto Rican strain (PRVABC59) of Zika virus aligned with sequences from the Malaysia (P6-740), Uganda MR-766NIID (MR-766), and Senegal (41525) strains. Primer sequences are indicated by black bars. BIP and FIP primers are fusions of B1 and B2c and F1c and F2, respectively (B1, B2c, F1c, and F2 labels are shown). Alu I restriction sites are indicated. Sequence variations are in gray. (B) Asian-lineage–specific primers detect Zika virus RNAs exclusively from Vero cells infected with Asian strains, PRVABC59 and P6-740. (C) African-lineage–specific primers detect Zika virus RNAs exclusively from Vero cells infected with African strains, MR-766 and 41525. (B and C) PRVABC59 (circles), P6-740 (boxes), MR-766 (diamonds), and 41525 (crosses). The threshold of detection is defined by a horizontal gray line. X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, relative fluorescence units (RFUs).

Fig. 2. Direct detection of Zika virus RNA in cells and supernatants

(A) Lysates of 1000 infected Vero or C6/36 cells in water. Vero cell lysate (black triangles), C6/36 cell lysate (black squares), mock-infected Vero cells (no symbols; line below threshold), mock-infected C6/36 cells (no symbols; line below threshold), infected Vero cell RNA (positive control; red circles), buffer only (no symbols; line below threshold). (B) Duplicate aliquots of infected Vero cell supernatant amplified directly (circles) or diluted 10-fold in water (squares). X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.

Fig. 3. Direct detection of Zika virus in mosquitoes

A LAMP assay was performed on 2.5 μl (1/100th of total) from PRVABC59 strain–infected mosquitoes split sagittally; one-half of the mosquito carcass was homogenized in mosquito diluent (red circles) and the other half in water (blue squares). Blue and red crosses, single mosquito with low virus load (table S2, mosquito #3); x axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.

Fig. 4. Limit of detection of Zika virus RNA

(A) A LAMP assay was performed on 2 μl of 10-fold dilutions of Vero cell supernatants containing 6.4 × 10⁵ PFU/ml. Virus input: 1280 PFU (black circles), 128 PFU (black triangles), 12.8 PFU (black squares), 1.28 PFU (black diamonds), 0.128 PFU (black crosses), and 0.0128 PFU (no symbols; line below threshold). (B) A LAMP assay was performed on 10-fold dilutions of Zika virus genome copies: 10⁵ copies (black circles), 10⁴ copies (black triangles), 10³ copies (black squares), 10² copies (black diamonds), 10 copies (black crosses), and 1 copy (line, no symbols). (A and B) Control infected Vero cell RNA (positive control; red circles) and no RNA (no symbols; line below threshold). Results are representative of a minimum of six replicates of each dilution series. X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.

Fig. 5. Detection of Zika virus spiked into human blood, plasma, saliva, urine, and semen samples

Healthy human biofluids were spiked with the Zika virus PRVABC59 strain from infected Vero cell supernatants at a final concentration of 10⁶ PFU/ml. (A) Amplification of 2 μl of serial 10-fold dilutions of plasma containing 10⁶ PFU/ml of the PRVABC59 strain followed by dilution to 1:100 or 1:1000 in water. (B) Matching samples of the 1:1000 plasma dilutions were incubated in tubes in a heat block for 70 min and examined for turbidity. (C) Tenfold dilutions of blood, saliva, urine, and semen each spiked with 10⁶ PFU/ml of PRVABC59 Zika virus strain followed by dilution to 1:1000 in water. Virus input: (A and C) 2000 PFU (black circles), 200 PFU (black triangles), 20 PFU (black squares), 2 PFU (black diamonds), 0.2 PFU (black crosses; below threshold), 0.02 PFU (no symbols; gray line below threshold), infected Vero cell RNA (positive control; red circles), and diluted biofluids without virus (no symbols; black line below threshold). Results are representative of a minimum of six replicates of each dilution series. X axis, minutes to LAMP amplicon incorporation of fluorescent dye; y axis, RFUs.