Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay
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-C...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-11-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/13/11/988 |
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| author | Saloni Agarwal Mojdeh Hamidizadeh Frank F. Bier |
| author_facet | Saloni Agarwal Mojdeh Hamidizadeh Frank F. Bier |
| author_sort | Saloni Agarwal |
| collection | DOAJ |
| description | 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 R<sup>2</sup> = 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. |
| first_indexed | 2024-03-09T16:58:22Z |
| format | Article |
| id | doaj.art-265e6c78d1ec4df9a9691579595bd365 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-09T16:58:22Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-265e6c78d1ec4df9a9691579595bd3652023-11-24T14:32:28ZengMDPI AGBiosensors2079-63742023-11-01131198810.3390/bios13110988Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow AssaySaloni Agarwal0Mojdeh Hamidizadeh1Frank F. Bier2Institute for Biochemistry and Biology, Chair of Molecular Bioanalysis and Bioelectronics, University of Potsdam, Karl-Liebknecht-Strasse 24/25, 14476 Potsdam, GermanyInstitute for Biochemistry and Biology, Chair of Molecular Bioanalysis and Bioelectronics, University of Potsdam, Karl-Liebknecht-Strasse 24/25, 14476 Potsdam, GermanyInstitute for Biochemistry and Biology, Chair of Molecular Bioanalysis and Bioelectronics, University of Potsdam, Karl-Liebknecht-Strasse 24/25, 14476 Potsdam, GermanyThis 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 R<sup>2</sup> = 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.https://www.mdpi.com/2079-6374/13/11/988RT-LAMPLFANAAT-LFAsemi-quantitativesurveillance-based diagnostics |
| spellingShingle | Saloni Agarwal Mojdeh Hamidizadeh Frank F. Bier Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay Biosensors RT-LAMP LFA NAAT-LFA semi-quantitative surveillance-based diagnostics |
| title | Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay |
| title_full | Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay |
| title_fullStr | Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay |
| title_full_unstemmed | Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay |
| title_short | Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay |
| title_sort | detection of reverse transcriptase lamp amplified nucleic acid from oropharyngeal viral swab samples using biotinylated dna probes through a lateral flow assay |
| topic | RT-LAMP LFA NAAT-LFA semi-quantitative surveillance-based diagnostics |
| url | https://www.mdpi.com/2079-6374/13/11/988 |
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