Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood

Abstract

A molecular diagnostic method for robust detection of Ebola virus (EBOV) at the point of care (POC) directly from blood samples is described. This assay is based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) of the glycoprotein gene of EBOV. Complete reaction formulations were lyophilized in 0.2-mL polymerase chain reaction tubes. RT-LAMP reactions were performed on a battery-operated isothermal instrument. Limit of detection of this RT-LAMP assay was 2.8 × 10² plaque-forming units (PFU)/test and 1 × 10³ PFU/test within 40 minutes for EBOV-Kikwit and EBOV-Makona, respectively. This assay was found to be specific for the detection of EBOV, as no nonspecific amplification was detected in blood samples spiked with closely related viruses and other pathogens. These results showed that this diagnostic test can be used at the point of care for rapid and specific detection of EBOV directly from blood with high sensitivity within 40 minutes.

Keywords: Ebola virus; RT-LAMP; diagnostic test; isothermal amplification; point of care.

Figure 1.

Sensitivity of the Ebola virus (EBOV) reverse transcription–loop-mediated isothermal amplification method. Sensitivity was determined against 2 strains of EBOV, EBOV-Kikwit (stock concentration, 1.12 × 10⁷ plaque-forming units [PFU]/mL) and EBOV-Makona (4 × 10⁷ PFU/mL). Ten-fold serial dilutions of each virus were made in human whole blood. Each dilution was added to the lysis buffer at 5%, and 50 µL of this suspension was used to reconstitute lyophilized reagents. Abbreviations: NEG, negative control; POS, positive control (MS2 phage).

Figure 2.

Specificity of the Ebola virus (EBOV) reverse transcription–loop-mediated isothermal amplification (RT-LAMP) method. After completion of the reaction (40 minutes) LAMP reaction products were separated on 2% agarose gel. A, Specificity of EBOV RT-LAMP, using RNA extracts. A total of 5 µL of RNA was used as template in a 25-µL reaction mix. The reaction was performed at 72°C for 40 minutes. CCHFV, Crimean-Congo hemorrhagic fever virus; CHIKV: Chikungunya virus; DENV-1, dengue virus serotype 1; DENV-2, dengue virus serotype 2; DENV-3, dengue virus serotype 3; DENV-4, dengue virus serotype 4; EBOV, Ebola virus-Makona; LASV-J, Lassa virus–Josiah; LASV-S, Lassa virus–Sauer; MARV, Marburg virus–Musoke; WNV: West Nile virus. B, Specificity of EBOV RT-LAMP, using different pathogens. Each pathogen was diluted 1:10 in human whole blood. Each dilution was added to the lysis buffer at 5%, and 50 µL of this suspension was used to reconstitute lyophilized reagents. The reaction was performed at 72°C for 40 minutes. Abbreviations: MS2, MS2 phage (positive control); Neg, negative control (no target); rEBOV, recombinant EBOV-Makona complementary DNA plasmid. A list of other pathogens is provided in Table 2.

Figure 3.

Performance of the Ebola virus (EBOV) reverse transcription–loop-mediated isothermal amplification method using different levels of sample input. A, Human whole blood was spiked with target (E. colni cells and recombinant EBOV) and added to the lysis buffer at various levels (3.5%–6%). A total of 50 µL of this suspension was used to reconstitute lyophilized reagents, followed by incubation at 72°C for 40 minutes. Means with the same letter are not significantly different from each other (P > .05, by the Tukey-Kramer test,). B, Human whole blood was spiked with target (recombinant EBOV) and added to the lysis buffer at 5%. Various volumes (40–60 µL) of this suspension were used to reconstitute lyophilized reagents, followed by incubation at 72°C for 40 minutes. Means with the same letter are not significantly different from each other (P > .05, by the Tukey-Kramer test).

Figure 4.

Recommended workflow for using the Ebola virus (EBOV) reverse transcription–loop-mediated isothermal amplification diagnostic test at the point of care. In step 1, a sample is collected using a 50-µL Minivette POCT (Sarstedt, Germany). In step 2, the sample is added to the lysis buffer predispensed (950 µL) in a sample collection module (SCM; Lucigen, Wisconsin). The nozzle of the SCM is fitted with a 10-μm filter. In step 3, sample is filtered into a clean 1.5-mL tube by squeezing the SCM. In step 4, using a 50-µL fixed-volume pipette, filtered sample is dispensed into reaction tubes. Step 5: Reaction tubes are incubated on AmpliFire (Douglas Scientific, Minnesota). In step 6, after 40 minutes, results are displayed on screen. In step 7, amplification data can be exported onto a USB for further analysis, if needed.