The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93 %, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.
medRxiv preprint BACKGROUND: The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 140 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnostics of SARS-CoV-2. However, the positive rate of RT-qPCR assay of pharyngeal swab samples are reported to vary from 30~60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. METHODS:We established a reverse transcription digital PCR (RT-dPCR) protocol to detect SARS-CoV-2 on 194 clinical pharyngeal swab samples, including 103 suspected patients, 75 close contacts and 16 supposed convalescents.
RESULTS:The limit of blanks (LoBs) of the RT-dPCR assays were ~1.6, ~1.6 and ~0.8 copies/reaction for ORF 1ab, N and E genes, respectively. The limit of detection (LoD) was 2 copies/reaction. For the 103 fever suspected patients, the sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For close contacts, the suspect rate was greatly decreased from 21% down to 1%. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 90%, 100% and 93 %, respectively. In addition, quantification of the viral load for convalescents by RT-dPCR showed that a longer observation period was needed in the hospital for elderly patients.
The papain-like cysteine protease CEP1 is involved in clearing cellular contents during programmed cell death and regulating secondary wall thickening during xylem development.
4-Coumarate/coenzyme A ligases (4CLs) play key roles in the biosynthesis of plant secondary compounds and act at the divergence point from general phenylpropanoid metabolism to several major branch pathways, such as lignins and flavonoids. In this study, five 4CL genes in Populus tomentosa were cloned and characterized. Quantitative polymerase chain reaction (qPCR) and promoter-β-glucuronidase analysis showed that the expression of Pto4CLs is significantly divergent and certainly overlapping. The five Pto4CL recombinant proteins also have different substrate specificities and catalytic turnover rates. Pto4CL4 has the highest substrate affinity and catalytic turnover rate compared with other Pto4CL proteins. All five Pto4CL recombinant proteins had no detectable activity towards sinapate. Deletion of V338 residues in Pto4CL mutants resulted in activity towards sinapate. The low accumulation of sinapate and high proportion of S unit lignin implies that the ability of 4CL to catalyze the activation of sinapate is not necessary for lignin biosynthesis. Overexpression of the five Pto4CLs in transgenic tobacco using the 35S promoter revealed significantly increased lignin contents and decreased hydroxycinnamate derivative contents in phloem, xylem, root, and leaf tissues. An increased content of naringenin in Pto4CL4 transgenic tobacco roots was also observed. Our results suggest that the substrate specificity and divergent and overlapping expression patterns of Pto4CL isoforms may play important roles in regulating the lignin and flavonoid biosynthesis.
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