Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19

Abstract

Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

Keywords: COVID-19; Colorimetric kit; Lyophilization; RT-LAMP.

Figure 1

Representative image of the “cake” from colorimetric SARS-CoV-2 RT-LAMP lyophilized conditions as follows: (a). 10 mM arginine + 10% PEG 2000; (b). 75 mM trehalose + 10% PEG 2000; (c). 10 mM arginine + 5% PEG 8000; (d). 75 mM trehalose + 5% PEG 8000.

Figure 2

A and B—Result of spectrophotometry for four conditions and two lyophilization times, indicating the difference between absorbance by spectrophotometry at wavelengths of 434 nm and 560 nm (ΔDO), respectively, for positive SARS-CoV-2 samples, negative RNA and NTC. *p < 0.01, with A referring to conditions containing arginine and B referring to conditions containing trehalose.

Figure 3

Representative image of 10% PEG 2,000 + 10 mM arginine and 5% PEG 8,000 + 75 mM trehalose colorimetric SARS-CoV-2 RT-LAMP lyophilized conditions with 10⁴, 10³, and 10² SARS-CoV-2 RNA copies, demonstrating that with the first formulation, the color change occurs in the samples containing 10² copies, while in the second formulation, amplification occurs only up to 10³ copies. NTC Non-template control.

Figure 4

Representative image of 10% PEG 2000 + 10 mM arginine and 5% PEG 8000 + 75 mM trehalose colorimetric SARS-CoV-2 RT-LAMP lyophilized conditions with SARS-CoV-2 clinical samples, characterized by RT-qPCR, and subdivided into high (Ct < 29) and low (Ct > 29) viremia, and negative samples.

Figure 5

Schematic representation of the correlation between SARS-CoV-2 clinical sample detection by RT-qPCR and colorimetric SARS-CoV-2 RT-LAMP lyophilized: a. 10% PEG 2,000 + 10 mM arginine; b. 5% PEG 8,000 + 75 mM trehalose. Positive: samples exhibiting yellow coloration; intermediary: samples exhibiting orange coloration; negative: samples exhibiting red coloration.

Figure 6

Week 1 of colorimetric SARS-CoV-2 RT-LAMP lyophilized conditions 10% PEG 2000 + 10 mM arginine and 5% PEG 8,000 + 75 mM trehalose. RT Room temperature, NTC Non-template control.

Figure 7

Week 2 of colorimetric SARS-CoV-2 RT-LAMP lyophilized conditions: 10% PEG 2,000 + 10 mM arginine and 5% PEG 8,000 + 75 mM trehalose. RT Room temperature, NTC Non-template control.

Figure 8

Week 4 and 6 of colorimetric SARS-CoV-2 RT-LAMP lyophilized condition with 5% PEG 8,000 + 75 mM trehalose, indicating stability at room temperature until week 4. RT Room temperature, NTC Non-template control.

Figure 9

Flowchart indicating the protective reagents and combinations tested.