Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from<i>Geobacillus stearothermophilus</i>by Site-Directed Mutagenesis at the Active Site

Ma, Yi; Zhang, Beilei; Ou, Yanghui; Wang, Jufang; Li, Shan

doi:10.1155/2016/2906484

Cited by 28 publications

(19 citation statements)

References 30 publications

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“…This added sensitivity is important considering that a significant number of COVID-19 patients have presented with negative qPCR ( 7 ) results or the “relapse after negative” phenomenon ( 8 ) due to potentially large variability between clinical samples, low-viral-titer samples, and even disrupted binding of RT-qPCR primers due to variation in the viral genome ( 18 ). In this study, we used Bst DNA polymerase isolated in-house for the developed RT-LAMP assay, which was demonstrated to have higher polymerization activity than the commercial Bst DNA polymerase ( 19 ) and ensured the high sensitivity of this RT-LAMP method. Based on these findings, we propose that the RT-LAMP assay can detect viral RNA not only in samples testing positive by RT-qPCR but also in inconclusive samples.…”

Section: Discussionmentioning

confidence: 99%

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Deng

et al. 2020

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals. IMPORTANCE We developed a visual and rapid reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.

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Section: Discussionmentioning

confidence: 99%

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Deng

et al. 2020

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“…Bst LF DNA polymerase was purified following Li et. al 2017 protocol ( Milligan et al, 2018 ; Ma et al, 2016 ). The collected protein fractions were quantified, and aliquots were flash frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2

et al. 2020

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“…Analysis and imaging procedures were conducted in a separate room to avoid potential contamination. Individual 25 μL RT-LAMP reactions were conducted in tubes containing 5 μL of RNA template, 0.8 μM F3/B3 primers, 0.4 μM LF/LB primers, 1.6 μM FIP/BIP primers, 6 mM MgSO 4 (Sigma, USA), 1.6 mM dNTPs (Sangon Biotech Co., Ltd., China), 2.5 μL 10× Buffer (200 mM Tris-HCl, 100 mM (NH4) 2 SO 4 , 100 mM KCl, 20 mM MgSO 4 , 1% Triton-100, pH8.8), 0.8 M Betaine (Sigma, USA), 8 U of Bst DNA polymerase mix (Optimized internally [ 40 ]), 5 U Reverse Transcriptase AMV (Takara Biotechnology (Dalian) Co., Ltd., China), 0.12 mM HNB (Macklin Biochemical Co., Ltd., China) for optimizing visualization. The final volume was adjusted to 25 μL with DEPC H 2 O.…”

Section: Methodsmentioning

confidence: 99%

A novel One-pot rapid diagnostic technology for COVID-19

Wang

et al. 2021

Analytica Chimica Acta

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Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 minutes with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per μL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.

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Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I fromGeobacillus stearothermophilusby Site-Directed Mutagenesis at the Active Site

Cited by 28 publications

References 30 publications

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2

A novel One-pot rapid diagnostic technology for COVID-19

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