Lyophilised Reverse Transcriptase and Polymerase for Point-of-Care Diagnostics of SARS-CoV-2

Abstract

Early diagnosis of pathogens is key to reducing their spreading and to preventing severe health risks. The COVID-19 pandemic showed us the need for rapid point-of-care tests. Here, we describe the preparation of an amplification master mix for point-of-care diagnostics. Therefore, two off-patent amplification enzymes were designed, expressed and purified. The preparation of the key components enables independence from delivery issues, manufacturer portfolio and product information's. For long-term storage and cold-free transport, our fabricated amplification mix was lyophilised. Finally, we applied our lyophilised master mix on an integrated point-of-care diagnostic system and could detect 10 copies/μL COVID-19 RNA. The combination of stable, cold-free reagents with the mobile and low-cost device will allow molecular diagnostics of pathogens in a field or home setting.

Keywords: RT‐qPCR; SARS‐CoV‐2; lyophilisation; point‐of‐care; pulse controlled amplification; recombinant enzymes.

FIGURE 1

Design of amplification enzymes. (A) Structure of Klentaq. (B) Structure of Taq polymerase (C) Structure of MMLV reverse transcriptase. All structures are predicted with AlphaFold using CATANA (Kuťák et al. 2022). The His₆‐tag is highlighted in red and the linker including the TEV cleavage site is in grey. Introduced mutations are highlighted in purple.

FIGURE 2

RT‐qPCR results. (A) Activity of Klentaq and Taq in PCR amplifying different concentrations of synthetic DNA. (B) Activity of 20 ng μL⁻¹ MMLV RT in combination with AptaTaq or Klentaq in PCR amplifying different concentrations of synthetic RNA visualised with intercalating DNA stain. (C) Activity of 20 ng μL⁻¹ MMLV RT in combination with AptaTaq or Taq in PCR amplifying different concentrations of synthetic RNA visualised with probe. As control AptaTaq polymerase and LunaScript reverse transcriptase were used. All data are fitted with a linear regression line and the corresponding data are presented in Table S3. Each bar represents the mean of three replicated Cq values of each sample. All measurements were performed on a LightCycler480 system (Roche Diagnostics, Switzerland).

FIGURE 3

PCA results with different enzymes. (A) Fluorescence signal representing amplification dynamics over time for different SARS‐CoV‐2 RNA concentrations using the produced Taq polymerase and MMLV RT (B) Fluorescence signal representing amplification dynamics over time for different SARS‐CoV‐2 RNA concentrations using AptaTaq and LUNA. PCA experiments were performed on the Nano device (Staniszewski et al. 2024) and analysed with the PharosMicro software (HP Health Solutions, Planegg, Germany).

FIGURE 4

RT‐qPCR and PCA performance of lyophilised amplification mix composed of 80 ng μL⁻¹ Taq polymerase and 20 ng μL⁻¹ MMLV RT in the presence of 10% trehalose and 2% Ficoll 400. (A) RT‐qPCR amplification curves of different SARS‐CoV‐2 RNA concentrations. (B) PCA amplification curves of different SARS‐CoV‐2 RNA concentrations.