PATHPOD - A loop-mediated isothermal amplification (LAMP)-based point-of-care system for rapid clinical detection of SARS-CoV-2 in hospitals in Denmark

Abstract

Sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a vital goal in the ongoing COVID-19 pandemic. We present in this comprehensive work, for the first time, detailed fabrication and clinical validation of a point of care (PoC) device for rapid, onsite detection of SARS-CoV-2 using a real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) reaction on a polymer cartridge. The PoC system, namely PATHPOD, consisting of a standalone device (weight less than 1.2 kg) and a cartridge, can perform the detection of 10 different samples and two controls in less than 50 min, which is much more rapid than the golden standard real-time reverse-transcription Polymerase Chain Reaction (RT-PCR), typically taking 16-48 h. The novel total internal reflection (TIR) scheme and the reactions inside the cartridge in the PoC device allow monitoring of the diagnostic results in real-time and onsite. The analytical sensitivity and specificity of the PoC test are comparable with the current RT-PCR, with a limit of detection (LOD) down to 30-50 viral genome copies. The robustness of the PATHPOD PoC system has been confirmed by analyzing 398 clinical samples initially examined in two hospitals in Denmark. The clinical sensitivity and specificity of these tests are discussed.

Keywords: COVID-19 pandemic; Clinical sensitivity; Limit of detection (LOD); Point of care (PoC) device; Reverse-transcription, loop-mediated isothermal amplification; SARS-CoV-2.

Fig. 1

(i) The advantages of PoC testing in terms of rapid detection (figure inspired by Bissonnette et al. [36]). (ii) Potential uses of our pathogen detection PoC device (PATHPOD). (iii) The amplification reaction (reaction 1), and the formation of the precipitation of Magnesium Pyrophosphate (reaction 2), which inhibits the transmittance of light passing through the reaction solution.

Fig. 2

(i) The designs (3D) of the polymer cartridge, the diameter of the cartridge is 50 mm. (ii) The components of the cartridge before filling reagents and samples. (iii) Schematic (not to scale) of the working principle of the cartridge inside the PoC device. (iv) Schematic showing the detection cycle. By continuously turning on and off the LEDs and paired phototransistors, it is possible to measure all 12 wells with only 6 LEDs and 5 phototransistors.

Fig. 3

(i) Schematic for the exploded view of the point-of-care device (the PATHPOD). (ii) Digital image of the PATHPOD prototype.

Fig. 4

(a) Studying sensitivity of the developed RT-rLAMP assay to detect COVID 19 RT-LAMP (left). 2% agarose gel electrophoresis of LAMP amplification (right) (L:DNA molecular leader, lanes 2, 3, 4, 5,6 and 7 are plasmid copy numbers and NC: negative control. (b): Sensitivity of the RT-rLAMP to detect SARS-CoV-2 adapting to the PATHPOD system.

Fig. 5

(i) Standard graph of SARS-COV-2 plasmid control in a thermocycler to determine LOB. (ii) The sensitivity (%) of RT-rLAMP reaction corresponding to the copy of plasmid/reaction to determine LOD.

Fig. 6

Specificity of the RT-LAMP on PATHPOD device. 1 A: E. coli, 2 A: S. pyogenes, 3 A: AIV, 1B: K. pneumonia, 2B: P. auruginosa, 1 C: S. pneumonia, 2 C: Sars-Cov control, 1D: S. aureus, 2D: Mers-Cov control, 3D: Paramyxovirus, 4 A: negative control and 4D: positive control.

Fig. 7

. (a) PATHPOD system responses graph of the sensitivity study with clinical sample A-39. Negative control was loaded to well 4 A and 4D. (b) PATHPOD system responses graph of the sensitivity study with clinical sample A-39. All the wells were loaded with dilutions of clinical sample A-39. As expected, sample A-39 was negative below the dilutions 1:8000.

Fig. 8

PATHPOD response graph of the RLOD study. Positive control was loaded to well 4D, and negative control was loaded to well 4 A in both experiments. All other wells were loaded with 1:50,000 dilution of the heat lysed inactivated SARS-CoV-2 virus.

Fig. 9

(a) Assessment of the sensitivity (SE) and negative predictive value (NPV) of the PATHPOD against different intervals of reaction time. (b) Assessment of the sensitivity of the PATHPOD against samples with different viral load.