A new RT-LAMP-on-a-Chip Instrument for SARS-CoV-2 diagnostics

Abstract

This work describes the development of a Point-of-Care (POC) Lab-on-a-Chip (LOC) instrument for diagnosis of SARS-CoV-2 by Reverse-Transcription Loop-mediated isothermal amplification (RT-LAMP). The hardware is based on a Raspberry Pi computer ($35), a video camera, an Arduino Nano microcontroller, a printed circuit board as a heater and a 3D printed housing. The chips were manufactured in polymethyl methacrylate (PMMA) using a CO2 laser cutting machine and sealed with a PCR optic plastic film. The chip temperature is precisely controlled by a proportional-integral-derivative (PID) algorithm. During the RT-LAMP amplifications the chip was maintained at ∼ (65.0 ± 0.1) °C for 25 minutes and 5 minutes cooling down, totaling a 30 minutes of reaction .The software interpretation occurs in less than a second. The chip design has four 25 µL chambers, two for clinical samples and two for positive and negative control-samples. The RT-LAMP master mix solution added in the chip chambers contains the pH indicator Phenol Red, that is pink (for pH ∼ 8.0) before amplification and becomes yellow (pH ∼ 6.0) if the genetic material is amplified. The RT-LAMP SARS-CoV-2 diagnostic was made by color image recognition using the OpenCV machine vision software library. The software was programmed to automatically distinguish the HSV color parameter distribution in each one of the four chip chambers. The instrument was successfully tested for SARS-CoV-2 diagnosis, in 22 clinic samples, 11 positives and 11 negatives, achieving an assertiveness of 86% when compared to the results obtained by RT-LAMP standard reactions performed in conventional PCR equipment.

Keywords: COVID-19; LAMP-on-a-chip; OpenCV; Raspberry Pi; SARS-CoV-2; diagnostic.

Graphical abstract

Fig. 1

PMMA Chip device fabrication through a laser ablation process. (a) Engraving the grooves. (b) Engraving the chambers and channels. (c) Cutting the holes. (d) Cutting the chip limits. (e) Chamber in detail.

Fig. 2

Lab-on-a Chip RT-LAMP Instrument parts. (a) LAMP-on-a-Chip instrument. (b) Identification block. (c) Heating board. (d) Rendered image of the equipment showing its main components.

Fig. 3

Lamp-on-a-Chip Image recognition process: (a) Photo taken after the RT-LAMP reaction of 2 positive samples of SARS-CoV-2, plus a positive and a negative control. (b) Diagnostic result showing the negative and positive controls on the top left and right of the chip respectively and the two positive results below to sample 1 and 2. (c and d) Masks created during the identification process to identify negative and positive samples respectively.

Fig. 4

Chip sealing and loading process. (a) Sealing the front of the chip with PCR optic plastic sealing film. (b) Fixing the sealing film in the grooves. (c) inserting samples. (d) Sealing the back side. (e) chip ready for reaction.

Fig. 5

RT-LAMP Diagnostic verification. The chip is considered “valid” when the negative control (NTC) remains pink after reaction and the positive control (Ctrl +) changes its color to yellow. The sample is considered “negative” when the color is pink and “positive” when color changes to yellow