Dataset Information

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

ABSTRACT: To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.

SUBMITTER: Garneret P

PROVIDER: S-EPMC7799764 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

Garneret Pierre P Coz Etienne E Martin Elian E Manuguerra Jean-Claude JC Brient-Litzler Elodie E Enouf Vincent V González Obando Daniel Felipe DF Olivo-Marin Jean-Christophe JC Monti Fabrice F van der Werf Sylvie S Vanhomwegen Jessica J Tabeling Patrick P

PloS one 20210111 1

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point ...[more]

PMID: 33428641

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Project description:In December 2019, a severe acute respiratory syndrome caused by SARS-CoV-2 spread rapidly worldwide. Portable nucleic acid tests of SARS-CoV-2 are critically important for diagnostics. In this study, we used an isothermal amplification method-Multienzyme Isothermal Rapid Amplification (MIRA)-for rapid detection of SARS-CoV-2. We designed the primers and probes in ORF1ab and N gene of SARS-CoV-2. The amplicons could be monitored by lateral flow dipsticks (LFDs). The reaction temperature, time, concentrations of primers and probes, and working volume were optimized. Four commercial swab collection buffers were used to test the amplification efficacy of our assay without RNA extraction. Our assay was able to amplify duplex targets of SARS-CoV-2 in one single reaction using one-step RT-MIRA. The assay worked well in a low volume of 10 μl at 38°C for 20 min. Using three collection buffers without guanidinium, our assay was able to amplify efficaciously without RNA extraction. The 95% limit of detection (LoD) of the RT-MIRA assay was 49.5 (95% CI, 46.8-52.7) copies/ml for ORF1ab gene and 48.8 (95% CI, 46.5-52.6) copies/ml for N gene. There is no cross-reaction with other human respiratory pathogens, such as SARS-CoV, MERS-CoV, influenza A virus, influenza B virus, human adenovirus, respiratory syncytial virus, human parainfluenza virus, and coronavirus 229E in our assay. The precision evaluation revealed that the C50-20% to C50+20% range bounds the C5-C95 interval. This assay also showed high anti-interference ability. The extraction-free RT-MIRA and qPCR detection results of 243 nucleic acid specimens from suspected patients or national references showed a 100.0% (95% confidence interval, 94.2%-100.0%) positive predictive value and a 100.0% (95% confidence interval, 92.7%-100.0%) negative predictive value. Compared with qPCR, the kappa value of the two assays was 1.00 (P < 0.0001). In conclusion, we provide a portable and visualized method for detection of SARS-CoV-2 without RNA extraction, allowing its application in SARS-CoV-2 on-site detection.

Project description:PurposeThe aim of this study is to establish a loop-mediated isothermal amplification (LAMP) method for the rapid detection of vulvovaginal candidiasis (VVC).MethodsWe developed and validated a loop-mediated isothermal amplification (LAMP) method for detecting the most common Candida species associated with VVC, including C. albicans, N. glabratus, C. tropicalis, and C. parapsilosis. We evaluated the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and Kappa value of the LAMP method to detect different Candida species, using the conventional culture method and internal transcribed spacer (ITS) sequencing as gold standards and smear Gram staining and real-time Rolymerase Chain Reaction (PCR) as controls.ResultsA total of 202 cases were enrolled, of which 88 were VVC-positive and 114 were negative. Among the 88 positive patients, the fungal culture and ITS sequencing results showed that 67 cases (76.14%) were associated with C. albicans, 13 (14.77%) with N. glabratus, 5 (5.68%) with C. tropicalis, and 3 (3.41%) with other species. Regarding the overall detection rate, the LAMP method presented sensitivity, specificity, PPV, NPV, and Kappa values of 90.91%, 100%, 100%, 93.4%, and 0.919, respectively. Moreover, the LAMP had a specificity of 100% for C. albicans, N. glabratus, and C. tropicalis, with a sensitivity of 94.03%, 100%, and 80%, respectively. Moreover, the microscopy evaluation had the highest sensitivity, while the real-time PCR was less specific for C. albicans than LAMP. In addition, CHROMagar Candida was inferior to LAMP in detecting non-albicans Candida (NAC) species.ConclusionsBased on the cost-effective, rapid, and inexpensive characteristics of LAMP, coupled with the high sensitivity and specificity of our VVC-associated Candida detection method, we provided a possibility for the point-of-care testing (POCT) of VVC, especially in developing countries and some laboratories with limited resources.

Dataset Information

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

Publications

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

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