Detection of Streptococcus equi subsp. equi in guttural pouch lavage samples using a loop-mediated isothermal nucleic acid amplification microfluidic device

Abstract

Background: Rapid point-of-care (POC) detection of Streptococcus equi subsp. equi (S. equi) would theoretically reduce the spread of strangles by identifying index and carrier horses.

Hypothesis: That the eqbE isothermal amplification (LAMP) assay, and the same eqbE LAMP assay tested in a microfluidic device format, are comparable to a triplex real-time quantitative polymerase chain reaction (qPCR) assay that is commonly used in diagnostic labs.

Samples: Sixty-eight guttural pouch lavage (GPL) specimens from horses recovering from strangles.

Methods: Guttural pouch lavage specimens were tested for S. equi retrospectively using the benchtop eqbE LAMP, the eqbE LAMP microfluidic device, and compared to the triplex qPCR, that detects 2 S. equi-specific genes, eqbE and SEQ2190, as the reference standard using the receiver operating characteristic area under the curve (ROC).

Results: The 27/68 specimens were positive by benchtop eqbE LAMP, 31/64 by eqbE LAMP microfluidic device, and 12/67 by triplex qPCR. Using the triplex PCR as the reference, the benchtop eqbE LAMP showed excellent discrimination (ROC Area = 0.813, 95% confidence interval [CI] = 0.711-0.915) as did the LAMP microfluidic device (ROC Area = 0.811, 95% CI = 0.529-0.782). There was no significant difference between the benchtop LAMP and LAMP microfluidic device (ROC Area 0.813 ± 0.055 vs 0.811 ± 0.034, P = .97).

Conclusions: The eqbE LAMP microfluidic device detected S. equi in GPL specimens from convalescent horses. This assay shows potential for development as a POC device for rapid, sensitive, accurate, and cost-efficient detection of S. equi.

Keywords: DNA amplification; Streptococcus equi; diagnostics; equine; point-of-care; strangles.

FIGURE 1

The smart cup is a customized Thermos bottle that provides the inserted single‐use chip with constant temperature heating. The smart cup has a built‐in mount for positioning a smartphone. The smartphone flash excites fluorescence emission, the smartphone CCD camera monitors the reaction progress. Heating is due to an exothermic reaction of Mg‐Fe powder initiated by addition of water. A phase‐change material (PCM) maintains the chip at a constant ~65°C temperature for about 60 minutes. A, Cross‐section of Smart Cup showing Mg‐Fe alloy pouch that is activated by adding water to initiate heating, PCM, heat sink, and slot holding microfluidic chip. Smartphone is held for optimal focus of smartphone CCD camera on chip amplification chamber to measure fluorescence; B, Field unit and companion chip (inset). The smart cup can be optionally made of Styrofoam, making it entirely disposable. ⁹ Permission obtained from Elsevier for reprint of figure and legend License number 4850770852787, June 16, 2020

FIGURE 2

Limit of detection of known positive S. equi CFU measured in threshold time (minutes) via the LAMP assay on microfluidic device. CFU, colony forming unit; LAMP, loop‐mediated isothermal nucleic acid amplification

FIGURE 3

Normalized fluorescence intensity of S. equi amplicons as a function of time (minutes) LAMP assay on microfluidic device. LAMP, loop‐mediated isothermal nucleic acid amplification

FIGURE 4

Fluorescence emission from the microfluidic device following 40 minutes of S. equi DNA LAMP amplification. There is equal fluorescence at 40 minutes whether LAMP detects 1 CFU or 100 CFU. CFU, colony forming unit; LAMP, loop‐mediated isothermal nucleic acid amplification

FIGURE 5

(A) Fluorescent by‐product of LAMP distinguished a positive vs negative result of S. equi DNA detection on dry storage microfluidic device (B) as seen via a smartphone screen. LAMP, loop‐mediated isothermal nucleic acid amplification