ABSTRACT
BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.
Subject(s)
Bacterial Proteins/metabolism , Betacoronavirus/genetics , CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endodeoxyribonucleases/metabolism , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Limit of Detection , Nasal Cavity/virology , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , Reference Standards , SARS-CoV-2 , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
BACKGROUND: The outbreak of a new coronavirus, COVID-19, which was earliest reported in Wuhan, China, is now transmitting throughout the world. The aim of this study was to articulate the clinical characteristics of COVID-19 and to reveal possible factors that may affect the persistent time of positive SARS-CoV-2 nucleic acid test, so as to identify which patients may deteriorate or have poor prognoses as early as possible. METHODS: Retrospective cohort study was carried out on 47 patients with confirmed COVID-19 infection admitted to XinYu People's Hospital of JiangXi Province. Epidemiological, demographic, clinical, laboratorial, management, treatment, and outcome data were also collected and analyzed. RESULTS: In this study, patients were divided into two groups based on whether their SARS-CoV-2 nucleic acid tests in respiratory specimens turn negative within (Group Rapid or Group R) or over (Group Slow or Group S) a week. There was no significant difference in age, sex, travel or exposure history, and smoking history between the two groups. Forty-two patients had been observed with comorbidities. Similar clinical manifestations, for instance fever, cough, sputum, and fatigue, have been observed among patients in both groups, except that patients in Group S were obviously more likely to get fatigue than patients in Group R. Both groups had shown decrease in white blood cell or lymphocyte counts. Chest X-ray or computed tomography scan showed unilateral or bilateral infiltrates. High proportion in both groups has used nasal cannula (89.47% vs. 85.71%) to inhale oxygen. 10.53% of Group S have applied high-flow nasal cannula, while Group R used none. The current treatment is mainly antibiotics, antiviral, and traditional Chinese medicine, while a couple of patients has used methylprednisolone. Only 1 patient out of both groups got even worse despite this active treatment. CONCLUSION: Clinical characteristics of COVID-19 include the exposure history and typical systemic symptoms such as fever, cough, fatigue, decreased WBC and lymphocyte counts, and infiltration in both lower lobes on CT imaging. Among them, fatigue appears to be an important factor that affects the duration of positive SARS-CoV-2 nucleic acid test in respiratory specimens.