Comparison of Reverse Transcription Loop-Mediated Isothermal Amplification Method with SYBR Green Real-Time RT-PCR and Direct Fluorescent Antibody Test for Diagnosis of Rabies

Naji, Elahe; Fadajan, Zohreh; Afshar, Davoud; Fazeli, Maryam

doi:10.7883/yoken.jjid.2019.009

Cited by 10 publications

(8 citation statements)

References 24 publications

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“…Prior to use, and for the stability (shelf life) test of the paper-based LAMP device, the LAMP reaction mixtures (for 15 μL reaction) including the rest of LAMP primers (Table 1), MgSO 4 , Bst DNA polymerase large fragment, dNTPs, betaine, 1× Thermopol buffer, (optional) 0.08 × SYBR Green I, and 3% poly(vinyl alcohol) (PVA) Fluorescence Monitoring and Analysis. Either SYBR Green I was added together with the LAMP reagents or to the paper-based LAMP product; 33,34 the product visualization was allowed under an ultraviolet (UV) transilluminator (Labnet International Inc., New Jersey) at 302 nm wavelength and 15 min in a dark room. Real-time analysis of kinetic fluorescence intensity through time (min) of LAMP products was performed using QuantStudio 3 Real-Time PCR.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…Fluorescence Monitoring and Analysis. Either SYBR Green I was added together with the LAMP reagents or to the paper-based LAMP product; 33,34 the product visualization was allowed under an ultraviolet (UV) transilluminator (Labnet International Inc., New Jersey) at 302 nm wavelength and 15 min in a dark room. Real-time analysis of kinetic fluorescence intensity through time (min) of LAMP products was performed using QuantStudio 3 Real-Time PCR.…”

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confidence: 99%

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Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA)

et al. 2021

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Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36−43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25°C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.

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Section: ■ Experimental Sectionmentioning

confidence: 99%

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confidence: 99%

Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA)

et al. 2021

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“…RT-LAMP products may be read upon fluorometric or colorimetric signals. Fluorometric signals are usually generated by SYBR green (Naji et al 2019 ) or CYTO9 (Tangkanchanapas et al 2018 ) which require equipped instruments for detection. On the other hand, colorimetric RT-LAMP products are detectable by the naked eye.…”

Section: Resultsmentioning

confidence: 99%

Malachite Green-Based Detection of SARS-CoV-2 by One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

et al. 2023

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The pandemic of severe acute respiratory syndrome 2 (SARS-CoV-2) revealed the necessity of diagnosis of the infected people to prevent the prevalence infection cycle. Many commercial pathogen diagnosis methods are based on the detection of genomic materials. Isothermal amplification methods such as loop-mediated-isothermal amplification (LAMP) are the method of choice in these cases. Reverse transcription steps are efficiently coupled to LAMP for the detection of pathogens with genomic RNAs such as SARS-CoV-2. Many detection systems for LAMP include fluorescent readout systems. Although such systems result in desirable limits of detection, the need for special instrumentation is the main dispute of such systems to become real point of care assays. In contrast, colorimetric detection methods would reduce costs and improve the applicability of the system. In this study one-step reverse transcription-LAMP reaction was established that enables visual detection of the SARS-CoV-2 genome. Nasopharyngeal RNA samples were first validated by reverse transcription quantitative polymerase chain reaction and then subjected to RT-LAMP. To lower the cost associated with the readout system equipment, malachite green (MG) was used. The color change of MG to blue allowed visual detection of the virus. Firstly, experiments were set up as two-step RT-LAMP reaction to identify the best primer sets. In addition, MG concentration was optimized with the significant colorimetric signal for the positive samples. Next, a one-step colorimetric method was developed for the detection of SARS-CoV-2 based on MG color shift in 2 h.

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“…Fluorescent dyes can be used in the LAMP reaction for fluorescence monitoring which improves detection and solves the problem of non-primer-derived signals [26,27]. Thus, RT-LAMP has several advantages over RT-qPCR, and when fluorescence is used as an endpoint for RT-LAMP there is also improved detection arguing fluorescent RT-LAMP superiority [28]. Further, fluorescent RT-LAMP can be deployed as a point-of-care (POC) test filling a role that is vital for disease management and control.…”

Section: Introductionmentioning

confidence: 99%

Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

et al. 2021

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The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

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Comparison of Reverse Transcription Loop-Mediated Isothermal Amplification Method with SYBR Green Real-Time RT-PCR and Direct Fluorescent Antibody Test for Diagnosis of Rabies

Cited by 10 publications

References 24 publications

Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA)

Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA)

Malachite Green-Based Detection of SARS-CoV-2 by One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

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