Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay

by Selleckchem | Feb 06 2024

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.

Comments:

This study sounds impressive! It seems to demonstrate a streamlined and efficient approach for detecting SARS-CoV-2 using throat swab samples without the need for extensive sample preparation or purification steps.

The use of crude throat swab samples directly in the RT-LAMP reaction without prior cleaning or treatment simplifies the testing process significantly. Additionally, incorporating a biotinylated DNA probe for LFA readouts, along with the use of FITC and biotin labels, enhances both the specificity and sensitivity of the assay.

The combination of these techniques not only provides high sensitivity (98.11%) and specificity (96.15%) but also allows for semi-quantitative analysis through a smartphone-based IVD device, correlating LFA T-line intensity with qRT-PCR Ct values. Achieving a digital semi-quantification of the RT-LAMP-LFA with a correlation coefficient of R2 = 0.702 is a notable advancement, enabling a better understanding of the viral load in samples.

Moreover, the assay's quick processing time of 35 minutes and a Limit of Detection (LoD) of three RNA copies/µL (Ct-33) are impressive, showcasing the potential for this technique to be used as a rapid point-of-care testing method. The elimination of the need for sample storage, transportation, or extensive pre-processing further emphasizes its suitability as a versatile biosensor platform for pathogen detection.

Overall, this study seems to offer a promising approach that could significantly impact the efficiency and accessibility of nucleic acid testing at point-of-care facilities, particularly in the context of detecting infectious diseases like SARS-CoV-2.