Abstract
Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.
Highlights
Dual-labeled PNA probe used RT-Loop Mediated Isothermal Amplification (LAMP) molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study
Loop Mediated Isothermal Amplification (LAMP) is a molecular technique capable of detecting nucleic acids with high sensitivity within a reduced time compared to classical real-time PCR and has been used widely for the detection of viral infections in a time-effective manner[5]
Since beginning of the COVID-19 outbreak, various molecular detection assays based on real-time PCR or LAMP aimed for detection of SARS-CoV-2 nucleic acid have been developed and received Emergency Use Authorizations (EUA) from the US FDA or the authorizations of the regulatory agencies in the country of production
Summary
Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. SARSCoV-2 LAMP tests have shown inadequate detection performances when testing low positive samples which exhibited Ct values over 30 on real-time PCR assays[14,15,16]; LAMP has been considered that having similar diagnostic accuracy as real-time PCR in detection of SARS-CoV-2 in the acute symptomatic phase of COVID-19 while the sample contains high viral load but not in early or late/clearance stages of the infection[17, 18] To solve those challenges, we developed a Peptide Nucleic Acid based Real Time-LAMP (PNA RT-LAMP) assay “AQ-TOP COVID-19 rapid Detection Kit Plus” which used dual labeled PNA probe that has been reported having superior specificity[19] and sensitivity[20] comparing to the accumulative dye such as SYBR green or sequence specific DNA fluorescent probes which are routinely used in LAMP and other molecular assays. The neutrally charged backbone makes PNA probe to bind its complementary nucleic acid with much higher affinity than the DNA detection probes, Scientific Reports | (2021) 11:20471
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