Smartphone-assisted point-of-care testing of nucleic acids based on hybridization chain reaction, magnetic beads, and gold nanorods etching

Colorimetric biosensors are simple but effective tools that are gaining popularity due to their ability to provide low-cost, rapid, and accurate detection for viruses like the Novel coronavirus, Influenza A, and Dengue virus, especially in point-of-care testing (POCT) and visual detection. In this study, a smartphone-assisted nucleic acid POCT was built using hybridization chain reaction (HCR), magnetic beads (MBs), and oxidized 3,3′,5,5′-tetramethylbenzidine (TMB2+)-mediated etching of gold nanorods (GNRs). The application of HCR without enzyme isothermal characteristics and MBs with easy separation, can quickly amplify nucleic acid signal and remove other reaction components. The blue shift of longitudinal localized surface plasmon resonance (LSPR) based on GNRs showed significant differences in etching color for different concentrations of target nucleic acid, which convert the signal into a visually semi-quantitative colorimetric result, achieving quantitative analysis with the color recognition software built into smartphones. This strategy, which only takes 40 min to detect and is two-thirds less time than the PCR, was successfully applied for the detection of the Dengue target sequence with a detection limit of 1.25 nM and exhibited excellent specificity for distinguishing single-base mutations, indicating broad application prospects in clinical laboratory diagnosis and enriching the research of nucleic acid POCT.

A Rolling Circle-Amplified G-Quadruplex/Hemin DNAzyme for Chemiluminescence Immunoassay of the SARS-CoV-2 Protein.

Sensitive detection of the SARS-CoV-2 protein remains a great research interest in clinical screening and diagnosis owing to the coronavirus epidemic. Here, an ultrasensitive chemiluminescence (CL) imaging strategy was developed through proximity hybridization to trigger the formation of a rolling circle-amplified G-quadruplex/hemin DNAzyme for the detection of the SARS-CoV-2 protein. The target protein was first recognized by a pair of DNA-antibody conjugates, Ab-1 and Ab-2, to form a proximity-ligated complex, Ab-1/SARS-CoV-2/Ab-2, which contained a DNA sequence complemental to block DNA and thus induced a strand displacement reaction to release the primer from a block/primer complex. The released primer then triggered a rolling circle amplification to form abundant DNAzyme units in the presence of hemin, which produced a strong chemiluminescent signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide. The proposed assay showed a detectable concentration range over 5 orders of magnitude with the detection limit down to 6.46 fg/mL. The excellent selectivity, simple procedure, acceptable accuracy, and intrinsic high throughput of the imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis.

A retrospective analysis of 12,400 SARS-CoV-2 RNA tests in patients with COVID-19 in Wuhan.

ABSTRACT: The outbreak and widely spread of coronavirus disease 2019 (COVID-19) has become a global public health concern. COVID-19 has caused an unprecedented and profound impact on the whole world, and the prevention and control of COVID-19 is a global public health challenge remains to be solved. The retrospective analysis of the large scale tests of SARS-CoV-2 RNA may indicate some important information of this pandemic. We selected 12400 SARS-CoV-2 tests detected in Wuhan in the first semester of 2020 and made a systematic analysis of them, in order to find some beneficial clue for the consistent prevention and control of COVID-19.SARS-CoV-2 RNA was detected in suspected COVID-19 patients with real-time fluorescence quantitative PCR (RT-qPCR). The patients' features including gender, age, type of specimen, source of patients, and the dynamic changes of the clinical symptoms were recorded and statistically analyzed. Quantitative and qualitive statistical analysis were carried out after laboratory detection.The positive rate of SARS-CoV-2 was 33.02% in 12,400 suspected patients' specimens in Wuhan at the first months of COVID-19 epidemics. SARS-CoV-2 RT-qPCR test of nasopharyngeal swabs might produce 4.79% (594/12400) presumptive results. The positive rate of SARS-CoV-2 RNA was significantly different between gender, age, type of specimen, source of patients, respectively (P < .05). The median window period from the occurrence of clinical symptom or close contact with COVID-19 patient to the first detection of positive PCR was 2 days (interquartile range, 1-4 days). The median interval time from the first SARS-CoV-2 positive to the turning negative was 14 days (interquartile range, 8-19.25 days).This study reveals the comprehensive characteristics of the SARS-CoV-2 RNA detection from multiple perspectives, and it provides important clues and may also supply useful suggestions for future work of the prevention and treatment of COVID-19.

The prognostic value of general laboratory testing in patients with COVID-19.

BACKGROUND: Lymphocyte count (LYM) of peripheral blood and some indices of general biochemical analysis had diagnostic and prognostic value for coronavirus disease 2019 (COVID-19), and the value of other remaining indices is rare. METHODS: A total of 94 patients with COVID-19 were enrolled at Renmin Hospital of Wuhan University. According to the severity of COVID-19, the patients were divided into three groups (moderate 49, severe 35, and critical 10), and 40 healthy cases were enrolled in the same period as healthy controls. The diagnostic and prognostic value of indices in peripheral blood cell count and general biochemical analysis was analyzed. RESULTS: Compared with healthy cases, the value differences in peripheral blood analysis in patients with COVID-19 were statistically significant (p < 0.01), the differences in LYM, neutrophil count (Neu), platelet count (PLT), and white blood cell count (WBC) were statistically significant among different severity of COVID-19 (p < 0.05). Compared with healthy cases, the differences in general biochemical results in patients with COVID-19 were statistically significant (p < 0.01), the value differences in direct bilirubin (DBIL), low-density lipoprotein cholesterol (LDL-Ch), and nitrogen (urea) were statistically significant among different severity of COVID-19 (p < 0.05). Neutrophil/lymphocyte ratio (NLR) had higher sensitivity and specificity for COVID-19 diagnosis. CONCLUSIONS: Some indices of peripheral blood cell count and general biochemical analysis were valuable in discriminating COVID-19 and predicting severity and adverse outcome of patients with COVID-19. For clinician, it is better to use more economical and easy-to-get indices to diagnose and predict the prognosis of COVID-19.

Necessity for detection of SARS-CoV-2 RNA in multiple types of specimens for the discharge of the patients with COVID-19.

BACKGROUND: The SARS-CoV-2 RNA was detected positive again after discharged from hospital in some COVID-19 patients, with or without clinical symptoms such as fever or dry cough. METHODS: 1008 severe COVID-19 patients, with SARS-CoV-2 RNA positive detected with the mixed specimen of nasopharyngeal swab and oropharyngeal swab by real-time fluorescence quantitative PCR (RT-qPCR), were selected to monitor SARS-CoV-2 RNA with the 12 types of specimens by RT-qPCR during hospitalization. All of 20 discharged cases with COVID-19 were selected to detect SARS-CoV-2 RNA in isolation period with 7 types of specimens by RT-qPCR before releasing the isolation period. RESULTS: Of the enrolled 1008 severe patients, the nasopharyngeal swab specimens showed the highest positive rate of SARS-CoV-2 RNA (71.06%), followed by alveolar lavage fluid (66.67%), oropharyngeal swab (30.77%), sputum (28.53%), urine (16.30%), blood (12.5%), stool (12.21%), anal swab (11.22%) and corneal secretion (2.99%), and SARS-CoV-2 RNA couldn't be detected in other types of specimen in this study. Of the 20 discharged cases during the isolation period, the positive rate of SARS-CoV-2 RNA was 30% (6/20): 2 cases were positive in sputum at the eighth and ninth day after discharge, respectively, 1 case was positive in nasopharynx swab at the sixth day after discharge, 1 case was positive in anal swab at the eighth day after discharge, and 1 case was positive in 3 specimens (nasopharynx swab, oropharynx swab and sputum) simultaneously at the fourth day after discharge, and no positive SARS-CoV-2 RNA was detected in other specimens including stool, urine and blood at the discharged patients. CONCLUSIONS: SARS-CoV-2 RNA should be detected in multiple specimens, such as nasopharynx swab, oropharynx swab, sputum, and if necessary, stool and anal swab specimens should be performed simultaneously at discharge when the patients were considered for clinical cure and before releasing the isolation period.

Proposal for standardization of 2019-nCoV nucleic acid detection in clinical laboratories

In December, the outbreak of a novel coronavirus (2019-nCoV) in Wuhan, China, has attracted extensive global attention. On January 20, 2020, the Chinese health authorities upgraded the coronavirus to a Class B infectious disease in the Law of the People's Republic of China on the Prevention and Treatment of Infectious Diseases, and considered it as Class A infectious diseases in disease control and prevention. On January 18, 2020, the 2019-nCoV nucleic acid detection test was listed as the diagnostic criteria in the "guidelines for diagnosis and treatment of pneumonia due to 2019-nCoV (Trial Version 2)". Therefore, standardizing the operation process of the 2019-nCoV nucleic acid detection in clinical laboratories has become a top priority. It is of paramount importance to establish standard protocols for detection of the 2019-nCoV nucleic acids in clinical laboratories to improve the reliability of the results and ensure the biosafety of laboratory personnel.

The diagnostic and prognostic role of myocardial injury biomarkers in hospitalized patients with COVID-19.

OBJECTIVE: The aim of this study was to systematically and comprehensively evaluate the diagnostic and prognostic value of myocardial injury biomarkers in COVID-19 patients. METHODS: This is a retrospective cohort study of confirmed COVID-19 patients that were admitted to the Renmin Hospital of Wuhan University from January 30, 2020 to February 15, 2020. RESULTS: Receiver operating characteristic (ROC) curve analysis demonstrated that cTnI-ultra had the highest area under the curve (AUC) at 0.855, with a sensitivity of 67.3% and a specificity of 88.7% for the prediction of in-hospital mortality. Patients with higher troponin I-ultra (cTnI-ultra), creatinine kinase-myocardial band (CK-MB), and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were associated with higher mortality, compared to those who lower levels. The multivariable cox regression indicated that age (hazard ratio (HR) 3.450, 95% confidence interval (CI) 1.627-7.314, P = 0.001), coronary heart disease (HR 1.855, 95% CI 1.006-3.421; P = 0.048), elevated cTnI-ultra (HR 3.083, 95% CI 1.616-5.883, P = 0.001), elevated CK-MB (HR 2.907, 95% CI 1.233-6.854; P = 0.015), and elevated NT-proBNP (HR 5.776, 95% CI 2.272-14.682; P < 0.001) were associated with in-hospital mortality. CONCLUSIONS: cTnI-ultra might be the best predictor of in-hospital mortality among myocardial injury biomarkers. Elevated cTnI-ultra, CK-MB, and NT-proBNP were independent biomarkers of the mortality in COVID-19 patients.