Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription

Lee, Hyojin; Lee, Hyobeen; Hwang, Sang‐Hyun; Jeong, Woong; Kim, Dong-Eun

doi:10.1016/j.aca.2022.339909

Cited by 9 publications

(5 citation statements)

References 35 publications

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“…El límite de detección es muy bajo situándose entre 1-10 copias/µl (44). Además de su uso para COVID-19, SDA está aprobada para la detección de enfermedades de transmisión sexual (C. trachomatis, N. gonorrhoeae y HHV-1/2) (12,45).…”

Section: Sda-strand Displacement Amplification Y Near-nicking and Ext...unclassified

Diagnóstico por amplificación isotérmica de ácidos nucleicos. Oportunidad para la farmacia comunitaria

Montero-Gómez

2024

Farm Comunitarios

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Esta revisión se centra en describir nuevos sistemas de diagnóstico molecular de tipo POC disponibles en el mercado que pueden implementarse fácilmente en farmacias comunitarias y tienen el potencial de ampliar la cartera de servicios farmacéuticos y hacer una contribución significativa a la mejora de la salud pública.El conocimiento de nuevas técnicas de diagnóstico molecular distintas de la PCR es relativamente desconocido. Sin embargo, las opciones disponibles son diversas y han alcanzado suficiente madurez tecnológica para su uso a gran escala. La pandemia de SARS-CoV-2 ha sacado al mercado pruebas de diagnóstico que, en algunos casos, se han utilizado exclusivamente en investigación durante décadas.La tecnología isotérmica de amplificación de ácidos nucleicos sigue evolucionando y es probable que en los próximos años seamos testigos de un aumento exponencial de su uso, así como del desarrollo de nuevas mejoras que simplifiquen y reduzcan aún más el coste de cada ensayo.Igualmente, no podemos obviar el hecho de que durante la pandemia de COVID-19, el público se ha habituado a autodiagnosticarse a través de canales de distribución masiva como las farmacias comunitarias, lo que puede abrir el sector a otras enfermedades -como las de transmisión sexual o salud animal-, el control de alimentos, la contaminación del agua y del aire (hongos) o la presencia de alérgenos.El conocimiento de estas nuevas tecnologías es esencial estrategia de vigilancia tecnológica e inteligencia competitiva del sector farmacéutico.

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Section: Sda-strand Displacement Amplification Y Near-nicking and Ext...unclassified

Diagnóstico por amplificación isotérmica de ácidos nucleicos. Oportunidad para la farmacia comunitaria

Montero-Gómez

2024

Farm Comunitarios

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“…Utilizing the conformational changes of G4s, several other studies converted the concentration of SARS‐CoV‐2 RNA to the signal visualized by G4‐specific dye molecules or chromogenic reaction catalyzed by G4s combining with hemin. [ 84 , 85 , 86 ] Chen et al . integrated multi‐branch rolling circle amplification (mbRCA) signal amplification with the signal readout of the G4/iridium(III) system, achieving the rapid and specific detection of SARS‐CoV‐2.…”

Section: G4 For Sars‐cov ‐2 Detectionmentioning

confidence: 99%

“…develop a SARS‐CoV‐2 RNA detection system by integrating ternary rolling circle amplification (t‐RCA) and subsequent strand displacement amplification (SDA) coupled with G4‐generating RCA, which has high sensitivity and accuracy as an alternative to detection strategy based on qRT‐PCR. [ 85 ]…”

Section: G4 For Sars‐cov‐2 Detectionmentioning

confidence: 99%

Unlocking G‐Quadruplexes as Targets and Tools against COVID‐19

et al. 2022

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Comprehensive Summary The applicability of G‐quadruplexes (G4s) as antiviral targets, therapeutic agents and diagnostic tools for coronavirus disease 2019 (COVID‐19) is currently being evaluated, which has drawn the extensive attention of the scientific community. During the COVID‐19 pandemic, research in this field is rapidly accumulating. In this review, we summarize the latest achievements and breakthroughs in the use of G4s as antiviral targets, therapeutic agents and diagnostic tools for COVID‐19, particularly using G4 ligands. Finally, strength and weakness regarding G4s in anti‐SARS‐CoV‐2 field are highlighted for prospective future projects.

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“…During the SDA procedure, NE generates a nick by cleaving one strand of the target double-stranded DNA at the recognition site, and KF then extends the DNA from the 3 end of the nick and displaces the downstream strand. Through perpetual cycling of these enzymatic reactions, target nucleic acids are amplified as multitudes of single-stranded DNAs (ssDNA) that report the presence of target nucleic acids, which can be subsequently utilized as primers or templates in downstream workflows [14][15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

“…Ligation of a linear padlock DNA with splint target nucleic acid sequence is a critical target decision step in most of the RCA-based target gene detection methods. However, this ligation process is likely to be a bottleneck because the alignment of target sequences with padlock DNA is often hindered by factors such as the limited concentration of target molecules, intramolecularly structured nucleic acids, or nonspecific interactions [17]. Thus, this ligation step is often hampered in application to direct detection of long RNAs (>3000 nucleotides; nt), such as viral RNA [22,23].…”

Section: Introductionmentioning

confidence: 99%

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification

Kyung,

Ku,

Cho

et al. 2023

Bioengineering

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Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5′-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

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Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription

Cited by 9 publications

References 35 publications

Diagnóstico por amplificación isotérmica de ácidos nucleicos. Oportunidad para la farmacia comunitaria

Diagnóstico por amplificación isotérmica de ácidos nucleicos. Oportunidad para la farmacia comunitaria

Unlocking G‐Quadruplexes as Targets and Tools against COVID‐19

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification

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