At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR ...Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the "bulk" manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3' end of the N3 probe and the 3' end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the "bulk" material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.
The ongoing global novel coronavirus pneumonia COVID‐19 outbreak has engendered numerous cases of infection and death. COVID‐19 diagnosis relies upon nucleic acid detection; however, currently ...recommended methods exhibit high false‐negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long‐read, real‐time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS‐CoV‐2 and other respiratory viruses simultaneously within 6–10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS‐CoV‐2 reaches 100%. Parallel testing with approved real‐time reverse transcription‐polymerase chain reaction kits for SARS‐CoV‐2 and NTS using 61 nucleic acid samples from suspected COVID‐19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS‐CoV‐2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID‐19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.
A detection technology, nanopore targeted sequencing (NTS), for the accurate and comprehensive detection of SARS‐CoV‐2 and other respiratory viruses within 6–10 h is developed, which is suitable for the identification of suspected cases and used as a supplementary technique for the SARS‐CoV‐2 test. NTS can also monitor mutations in the virus and the type of virus.
The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In ...2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.
Coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become pandemic, with substantial mortality.
To evaluate the ...pathologic changes of organ systems and the clinicopathologic basis for severe and fatal outcomes.
Prospective autopsy study.
Single pathology department.
11 deceased patients with COVID-19 (10 of whom were selected at random for autopsy).
Systematic macroscopic, histopathologic, and viral analysis (SARS-CoV-2 on real-time polymerase chain reaction assay), with correlation of pathologic and clinical features, including comorbidities, comedication, and laboratory values.
Patients' age ranged from 66 to 91 years (mean, 80.5 years; 8 men, 3 women). Ten of the 11 patients received prophylactic anticoagulant therapy; venous thromboembolism was not clinically suspected antemortem in any of the patients. Both lungs showed various stages of diffuse alveolar damage (DAD), including edema, hyaline membranes, and proliferation of pneumocytes and fibroblasts. Thrombosis of small and mid-sized pulmonary arteries was found in various degrees in all 11 patients and was associated with infarction in 8 patients and bronchopneumonia in 6 patients. Kupffer cell proliferation was seen in all patients, and chronic hepatic congestion in 8 patients. Other changes in the liver included hepatic steatosis, portal fibrosis, lymphocytic infiltrates and ductular proliferation, lobular cholestasis, and acute liver cell necrosis, together with central vein thrombosis. Additional frequent findings included renal proximal tubular injury, focal pancreatitis, adrenocortical hyperplasia, and lymphocyte depletion of spleen and lymph nodes. Viral RNA was detectable in pharyngeal, bronchial, and colonic mucosa but not bile.
The sample was small.
COVID-19 predominantly involves the lungs, causing DAD and leading to acute respiratory insufficiency. Death may be caused by the thrombosis observed in segmental and subsegmental pulmonary arterial vessels despite the use of prophylactic anticoagulation. Studies are needed to further understand the thrombotic complications of COVID-19, together with the roles for strict thrombosis prophylaxis, laboratory and imaging studies, and early anticoagulant therapy for suspected pulmonary arterial thrombosis or thromboembolism.
None.
The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing ...instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if â¥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.
The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the ...outbreak is more widespread than initially thought, and international spread through travellers does already occur.
We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.
Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.
The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project.
The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
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► Analysis of the parameters responsible for rapid microPCR is performed. ► Progress and requirement of completely integrated microPCR systems are discussed. ► Current status of ...microLAMP systems is presented. ► Advantages of microLAMP over microPCR towards rapid and POC diagnostics are discussed.
Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10–20min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics.
Several studies have demonstrated the advantages of environmental surveillance through the monitoring of sewage for the assessment of viruses circulating in a given community (wastewater-based ...epidemiology, WBE).
During the COVID-19 public health emergency, many reports have described the presence of SARS-CoV-2 RNA in stools from COVID-19 patients, and a few studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide. Italy is among the world's worst-affected countries in the COVID-19 pandemic, but so far there are no studies assessing the presence of SARS-CoV-2 in Italian wastewaters. To this aim, twelve influent sewage samples, collected between February and April 2020 from Wastewater Treatment Plants in Milan and Rome, were tested adapting, for concentration, the standard WHO procedure for Poliovirus surveillance. Molecular analysis was undertaken with three nested protocols, including a newly designed SARS-CoV-2 specific primer set.
SARS-CoV-2 RNA detection was accomplished in volumes of 250 ml of wastewaters collected in areas of high (Milan) and low (Rome) epidemic circulation, according to clinical data. Overall, 6 out of 12 samples were positive. One of the positive results was obtained in a Milan wastewater sample collected a few days after the first notified Italian case of autochthonous SARS-CoV-2.
The study confirms that WBE has the potential to be applied to SARS-CoV-2 as a sensitive tool to study spatial and temporal trends of virus circulation in the population.
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•Detection of SARS-CoV-2 in wastewater in Italy is described for the first time.•Use of the PEG/dextran concentration method for SARS-CoV-2 is reported.•A novel nested PCR assay specific for SARS-CoV-2 was designed.•Wastewater-based epidemiology can be applied for COVID-19 surveillance.
•A multiplex RT-qPCR assay for three regulated RNA viruses of citrus was developed.•A large number of diverse virus isolates was used to design the RT-qPCR assay.•Multi and singleplex RT-qPCR ...detection of different virus isolates was comparable.•One and two step multiplex RT-qPCR assays produced comparable Cq values.•The multiplex RT-qPCR assay streamlines virus detection in citrus programs.
A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory “Section 3701: Citrus Nursery Stock Pest Cleanliness Program”. Adopting a compatible multiplex RT-qPCR testing protocol for these viruses as well as other RNA and DNA regulated pathogens will provide a valuable alternative tool for virus detection and efficient program implementation.
As a city famous for tourism, the public healthcare system of Macau SAR has been under great pressure during the outbreak of the Coronavirus Disease 2019 (COVID-19). In this study, we report clinical ...and microbiological features of ten COVID-19 patients enrolled in the Centro Hospitalar Conde de São Januário (CHCSJ) between January 21 to February 16, 2020. Clinical samples from all patients including nasopharyngeal swab (NPS)/sputum, urine, and feces were collected for serial virus RNA testing by standard qRT-PCR assay. In total, seven were imported cases and three were local cases. The median duration from Macau arrival to admission in imported cases was 3 days. Four patients required oxygen therapy but none of them needed machinal ventilation. No fatal cases were noted. The most common symptoms were fever (80%) and diarrhea (80%). In the "Severe" group, there was significantly more elderly patients (p=0.045), higher lactate dehydrogenase levels (p=0.002), and elevated C-Reactive protein levels compared to the "Mild to Moderate" group (p<0.001). There were positive SARS-CoV-2 RNA signals in all patients' NPS and stool specimens but negative in all urine specimens. Based on our data on SARS-CoV-2 RNA shedding in stool and the possibility of a lag in viral detection in NPS specimens, the assessment of both fecal and respiratory specimen is recommended to enhance diagnostic sensitivity, and also to aid discharge decision before the role of viral RNA shedding in stool is clarified.
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