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High Ct-values falling in the grey zone are frequently encountered in SARS-CoV-2 detection by real-time reverse transcription PCR (rRT-PCR) and have brought urgent challenges in diagnosis of samples with low viral load. Based on the single-stranded DNA reporter trans-cleavage activity by Cas12a upon target DNA recognition, we create a Specific Enhancer for detection of PCR-amplified Nucleic Acids (SENA) to confirm SARS-CoV-2 detection through specifically targeting its rRT-PCR amplicons. SENA is highly sensitive, with its limit of detection being at least 2 copies/reaction lower than that of the corresponding rRT-PCR, and highly specific, which identifies both false-negative and false-positive cases in clinic applications. SENA provides effective confirmation for nucleic acid amplification-based molecular diagnosis, and may immediately eliminate the uncertainty problems of rRT-PCR in SARS-CoV-2 clinic detection. One Sentence SummaryCRISPR-Cas12a-based COVID-19 diagnosis.
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BackgroundThe SARS-CoV-2 outbreak urgently requires 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 in qualified laboratories and point-of-care settings. MethodsPatients with suspected COVID-19 and close contacts were recruited from two hospitals between Jan 26 and April 8, 2020. Respiratory samples were collected and tested using the RT-LAMP assays, and the results were compared with those obtained by RT-qPCR. Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. The RT-LAMP assay was also tested on an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. ResultsSamples 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: 88.89% and 99.00%; positive and negative predictive values: 94.74% and 97.78%, respectively) and diagnostically useful (positive and negative likelihood ratios: 88.89 and 0.11, respectively). RT-LAMP showed an increased sensitivity (88.89% vs 81.48%) and high consistency (kappa 0.92) compared with 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 result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. ConclusionThe developed RT-LAMP assay offers rapid, sensitive and straightforward detection of SARS-CoV-2 infection and could aid the expansion of COVID-19 testing in the public domain and hospitals.
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In order to provide a theoretical basis for better understanding the function and properties of proteins, we proposed a simple and effective feature extraction method for protein sequences to determine the subcellular localization of proteins. First, we introduced sparse coding combined with the information of amino acid composition to extract the feature values of protein sequences. Then the multilayer pooling integration was performed according to different sizes of dictionaries. Finally, the extracted feature values were sent into the support vector machine to test the effectiveness of our model. The success rates in data set ZD98, CH317 and Gram1253 were 95.9%, 93.4% and 94.7%, respectively as verified by the Jackknife test. Experiments showed that our method based on multilayer sparse coding can remarkably improve the accuracy of the prediction of protein subcellular localization.
