Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP)

Lai, Meng Yee; Bukhari, Fatma Diyana Mohd; Zulkefli, Nur Zulaikha; Ismail, Ida Suriya; Mustapa, Nur Izati; Soh, Tuan Suhaila Tuan; Hassan, Afifah Haji; Peariasamy, Kalaiarasu M; Lee, Yee Leng; Suppiah, Jeyanthi; Thayan, Ravindran; Isa, Mohd Khairi Mat; Wahid, Nur Zafirah Abdul; Lau, Yee Ling

doi:10.1186/s12879-022-07684-w

Cited by 3 publications

(2 citation statements)

References 22 publications

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“…Out of all these isothermal techniques, RT-LAMP has been most extensively compared to RT-PCR using parameters such as sample pre-treatment (RT-LAMP is more flexible to biological and chemical inhibitors than RT-PCR), analytical time and robustness (RT-LAMP amplification takes less than 30 min, unlike RT-PCR, which takes >3 h), and assay efficacy (RT-LAMP nears the sensitivity of RT-PCR, which is 68-80% sensitive and 90-95% specific, according to WHO guidelines). These characteristics make RT-LAMP equivalent to RT-PCR and pave the way for developing RT-LAMP-based POC technological biosensor platforms [8,17,18].…”

Section: Introductionmentioning

confidence: 99%

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

Agarwal,

Hamidizadeh,

Bier

2023

Biosensors

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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.

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Section: Introductionmentioning

confidence: 99%

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

Agarwal,

Hamidizadeh,

Bier

2023

Biosensors

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show abstract

“…These characteristics place RT-LAMP as equivalent to qRT-PCR and lead the way towards developing a RT-LAMP based PoC setup. [16,17] Modifications to the amplification product (amplicon) during reaction with isothermal amplification techniques (LAMP and RPA) have been demonstrated. Introduction of biotin and FITC/FAM labels [18], enabling the readout on a paper-based, sensitive immunochromatographic Lateral Flow Assay (LFA) platform were achieved with high efficacy.…”

mentioning

confidence: 99%

Biotin labelled DNA-probe based detection of nucleic acid in reverse transcription-LAMP amplified oropharyngeal viral swab samples via a lateral flow assay

Agarwal

Hamidizadeh

Bier

2023

Preprint

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This study focuses on three key aspects: a) crude throat swab sample as 30 template for RT-LAMP reaction, b) biotinylated DNA probes enhanced specificity for LFA readout and c) digital semi-quantification of LFA readout. The throat swab samples from SARS-CoV-2 positive and negative patients have been used in their crude (no cleaning or pre-treatment) form for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. RT-LAMP (20 min processing time) result was readout out via a LFA approach, using two labels: FITC and Biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with LF-LAMP-primer and biotin was later introduced using biotinylated DNA probes specific for the amplicon region. DNA probes based LFA readout of RT-LAMP amplicon was 97.73% sensitive and 93.10% specific. The LFA result was further analysed via a smartphone based IVD-device wherein the test line intensity was recorded. The LFA test line intensity was then correlated with the qRT-PCR CT-value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA is reported with the correlation coefficient R2= 0.7. The overall RT-LAMP-LFA assay time was recorded to be 35 min with the LoD of 4,000 RNA copies/ml.

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Effectiveness of mRNA COVID-19 vaccines against symptomatic SARS-CoV-2 infections during the SARS-CoV-2 Omicron BA.1 and BA.2 epidemic in Japan: vaccine effectiveness real-time surveillance for SARS-CoV-2 (VERSUS)

Maeda

Saito

Igarashi

et al. 2023

Expert Review of Vaccines

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Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP)

Cited by 3 publications

References 22 publications

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

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

Biotin labelled DNA-probe based detection of nucleic acid in reverse transcription-LAMP amplified oropharyngeal viral swab samples via a lateral flow assay

Effectiveness of mRNA COVID-19 vaccines against symptomatic SARS-CoV-2 infections during the SARS-CoV-2 Omicron BA.1 and BA.2 epidemic in Japan: vaccine effectiveness real-time surveillance for SARS-CoV-2 (VERSUS)

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