During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the ...Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.
Production and maintenance of virus‐free planting materials is pivotal for the control of viral diseases. The present study attempted to test exogenous application of melatonin for eradication of ...apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple cultivar Gala. Exogenous application of 15 μm melatonin to the shoot proliferation medium significantly increased the number of shoots and shoot length. The level of endogenous indole‐3‐acetic acid (IAA) was the highest in the shoots proliferating on the shoot proliferation medium containing 15 μm melatonin. Shoot regrowth levels were significantly higher in shoot tips of the virus‐infected shoots cultured for 4 weeks on this medium than the control. In addition, culture of shoot tips of the virus‐infected in vitro shoots proliferated for 4 weeks on this medium resulted in 95% of shoots being virus‐free, while no virus‐free shoots were obtained in shoot tips of the virus‐infected shoots cultured without melatonin. Analyses by microtissue direct RT‐PCR and RT‐qPCR showed that ASGV concentration decreased in shoot tips of the virus‐infected shoots proliferating on the medium containing 15 μm melatonin for 4 weeks. Virus localization showed that exogenous application of melatonin enlarged the virus‐free area in the virus‐infected shoot tips. These data provide explanations as to why exogenous application of melatonin can efficiently eradicate ASGV. Exogenous application of melatonin provides an alternative means for plant virus eradication and has the potential to produce virus‐free plants.
To face the new Covid-19 pandemic, the need for early and accurate diagnosis of the disease among suspected cases quickly became obvious for effective management, and for better control of the spread ...of the disease in the population. Since the beginning of this disease epidemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), reverse transcriptase polymerase chain reaction (RT-PCR) has routinely been used to confirm diagnosis. However, several authors have pointed out the poor performance of this technique, particularly in terms of sensitivity.1,2 This article is protected by copyright. All rights reserved.
In this study, we collected a total of 610 hospitalized patients from Wuhan between February 2, 2020, and February 17, 2020. We reported a potentially high false negative rate of real‐time ...reverse‐transcriptase polymerase chain reaction (RT‐PCR) testing for SARS‐CoV‐2 in the 610 hospitalized patients clinically diagnosed with COVID‐19 during the 2019 outbreak. We also found that the RT‐PCR results from several tests at different points were variable from the same patients during the course of diagnosis and treatment of these patients. Our results indicate that in addition to the emphasis on RT‐PCR testing, clinical indicators such as computed tomography images should also be used not only for diagnosis and treatment but also for isolation, recovery/discharge, and transferring for hospitalized patients clinically diagnosed with COVID‐19 during the current epidemic. These results suggested the urgent needs for the standard of procedures of sampling from different anatomic sites, sample transportation, optimization of RT‐PCR, serology diagnosis/screening for SARS‐CoV‐2 infection, and distinct diagnosis from other respiratory diseases such as fluenza infections as well.
Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA‐PRRSV2), highly pathogenic (HP‐PRRSV2) and NADC30‐like (NL‐PRRSV2)) are ...currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real‐time RT‐PCR assays using TaqMan‐MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real‐time RT‐PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real‐time RT‐PCR assays were further validated by comparing with a universal PRRSV conventional RT‐PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real‐time RT‐PCR assays and the conventional RT‐PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV‐positive, including 2 samples that were determined to be co‐infected with NL‐PRRSV2 and HP‐PRRSV2 isolates by the quadruplex real‐time RT‐PCR assay. ORF5 sequencing confirmed the real‐time RT‐PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA‐PRRSV2, NL‐PRRSV2 and HP‐PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.
•Viral respiratory infections represent a serious risk for morbidity and mortality, especially in immunosuppressed patient cohorts.•We present an automated high-throughput PCR multiplex-panel testing ...16 different respiratory viruses in 3 reactions.•The assay panel allows for efficient, scalable screening in at risk patient populations.
: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform.
Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1–4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays.
Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4–7 log-steps with pooled standard differentials (SD) ranging between 0.18–0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13–0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80–100 % range. Negative agreement varied between 96.3–100 %.
Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.
Severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease‐2019 (COVID‐19). ...Here, we examined the duration of clearance of SARS‐CoV‐2 in respiratory samples from 207 infected cases by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR). A substantial proportion of COVID‐19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS‐CoV‐2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID‐19, irrespective of their Ct values, cleared SARS‐CoV‐2 RNA within 14 days of the initial detection. Longitudinal assessment of RT‐PCR test results in individuals requiring 15–30 days to clear SARS‐CoV‐2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow‐up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID‐19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS‐CoV‐2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.
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