The Experts below are selected from a list of 14289 Experts worldwide ranked by ideXlab platform
India Leclercq - One of the best experts on this subject based on the ideXlab platform.
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Heat Inactivation of the severe acute respiratory syndrome coronavirus 2
Journal of biosafety and biosecurity, 2021Co-Authors: Christophe Batejat, Quentin Grassin, Jeanclaude Manuguerra, India LeclercqAbstract:Supernatants of cells infected with SARS-CoV-2, and nasopharyngeal and sera samples containing SARS-CoV-2 were subjected to Heat Inactivation for various periods of time, ranging from 30 s to 60 min. Our results showed that SARS-CoV-2 could be inactivated in less than 30 min, 15 min, and 3 min at 56 °C, 65 °C, and 95 °C, respectively. These data could help laboratory workers to improve their protocols by handling the virus in biosafety conditions.
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Heat Inactivation of the severe acute respiratory syndrome coronavirus 2
bioRxiv, 2020Co-Authors: Christophe Batejat, Quentin Grassin, Jeanclaude Manuguerra, India LeclercqAbstract:Abstract Supernatants of cells infected with SARS-CoV-2, nasopharyngeal and sera samples containing SARS-CoV-2 were submitted to Heat Inactivation for various periods of time, ranging from 30 seconds to 60 minutes. Our results showed that SARS-CoV-2 could be inactivated in less than 30 minutes, 15 minutes and 3 minutes at 56°C, 65°C and 95°C respectively. These data could help laboratory workers to improve their protocols with handling of the virus in biosafety conditions.
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Heat Inactivation of the middle east respiratory syndrome coronavirus
Influenza and Other Respiratory Viruses, 2014Co-Authors: Christophe Batejat, India Leclercq, Ana Maria Burguiere, Jeanclaude ManuguerraAbstract:The culture supernatants of the emerging Middle East respiratory syndrome coronavirus (MERS-CoV) were submitted to three temperatures over time and tested for infectivity by TCID50 method on Vero E6 cells. At 56°C, almost 25 minutes were necessary to reduce the initial titre by 4 log10 . Increasing temperature to 65°C had a strong negative effect on viral infectivity as virucidy dropped significantly to 1 minute. On the contrary, no significant decrease in titre was observed after 2 hours at 25°C. These data might be useful in establishing biosafety measures in laboratories against MERS-CoV.
Shirou Mohri - One of the best experts on this subject based on the ideXlab platform.
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eliminating transmissibility of bovine spongiform encephalopathy by dry Heat treatment
Journal of General Virology, 2020Co-Authors: Yuichi Matsuura, Yukiko Ishikawa, Yuichi Murayama, Shirou Mohri, Takashi Yokoyama, Robert A Somerville, Tetsuyuki KitamotoAbstract:Bovine spongiform encephalopathy (BSE) prion is more resistant to Heat Inactivation compared to other prions, but the effect of Heat Inactivation has been reported to differ depending on the BSE-contaminated tissue state or Heating type. We aimed to evaluate the secure level of Inactivation of original BSE transmissibility by dry-Heating. Cattle tissues affected with BSE were subjected to dry-Heat treatment for 20 min at various temperatures ranging from 150 to 1000 °C. To assess the Inactivation effect, we conducted protein misfolding cyclic amplification (PMCA) and follicular dendritic cell (FDC) assays in transgenic mice expressing bovine prion protein genes. Under dry-Heating at 600 °C or higher, BSE cattle tissues lost their transmissibility in transgenic mice. In contrast, transmissibility was detected in the cattle tissues treated at temperatures of 400 °C or lower through the FDC assay combined with PMCA. In this study, we confirmed that transmissibility was eliminated in BSE-affected cattle tissues by dry-Heating at 600 °C or higher.
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quantitative analysis of wet Heat Inactivation in bovine spongiform encephalopathy
Biochemical and Biophysical Research Communications, 2013Co-Authors: Yuichi Matsuura, Yukiko Ishikawa, Xiao Bo, Yuichi Murayama, Takashi Yokoyama, Robert A Somerville, Tetsuyuki Kitamoto, Shirou MohriAbstract:Abstract The bovine spongiform encephalopathy (BSE) agent is resistant to conventional microbial Inactivation procedures and thus threatens the safety of cattle products and by-products. To obtain information necessary to assess BSE Inactivation, we performed quantitative analysis of wet-Heat Inactivation of infectivity in BSE-infected cattle spinal cords. Using a highly sensitive bioassay, we found that infectivity in BSE cattle macerates fell with increase in temperatures from 133 °C to 150 °C and was not detected in the samples subjected to temperatures above 155 °C. In dry cattle tissues, infectivity was detected even at 170 °C. Thus, BSE infectivity reduces with increase in wet-Heat temperatures but is less affected when tissues are dehydrated prior to the wet-Heat treatment. The results of the quantitative protein misfolding cyclic amplification assay also demonstrated that the level of the protease-resistant prion protein fell below the bioassay detection limit by wet-Heat at 155 °C and higher and could help assess BSE Inactivation. Our results show that BSE infectivity is strongly resistant to wet-Heat Inactivation and that it is necessary to pay attention to BSE decontamination in recycled cattle by-products.
Valeria Levi - One of the best experts on this subject based on the ideXlab platform.
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extraction free protocol combining proteinase k and Heat Inactivation for detection of sars cov 2 by rt qpcr
PLOS ONE, 2021Co-Authors: Valeria Genoud, Martin Stortz, Ariel Waisman, Bruno G Berardino, Paula Verneri, Virginia Dansey, Melina Salvatori, Federico Remes Lenicov, Valeria LeviAbstract:Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by Heat Inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the Heat-Inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.
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extraction free protocol combining proteinase k and Heat Inactivation for detection of sars cov 2 by rt qpcr
medRxiv, 2020Co-Authors: Valeria Genoud, Martin Stortz, Ariel Waisman, Bruno G Berardino, Paula Verneri, Virginia Dansey, Melina Salvatori, Federico Remes Lenicov, Valeria LeviAbstract:Abstract Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by Heat Inactivation (PK+HID method). We demonstrate that PK+HID improves the RT- qPCR performance in comparison to the Heat-Inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2.
Barbara E Miller - One of the best experts on this subject based on the ideXlab platform.
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quantitation of skeletal alkaline phosphatase isoenzyme activity in canine serum
Journal of Bone and Mineral Research, 2009Co-Authors: John R Farley, Susan L Hall, Candace Ritchie, Sandra Herring, Christopher M Orcutt, Barbara E MillerAbstract:Pursuing the hypothesis that quantitation of skeletal alkaline phosphatase (ALP) activity in canine serum would provide an index of the rate of bone formation, we compared three methods for isoenzyme-specific identification of skeletal ALP activity in canine serum: Heat Inactivation, wHeat germ agglutinin (WGA) precipitation, and concanavalin A (ConA) precipitation. ALP isoenzyme activities were extracted from canine bone, intestine, and liver, diluted into Heat-inactivated canine serum (i.e., serum without ALP activity), and used as calibrators of ALP isoenzyme activities. Differential sensitivity to inhibition by 10 mM L-homoarginine was used to distinguish intestinal ALP activity from hepatic and skeletal ALP activities (i.e., 9, 80, and 72% inhibition, respectively). To allow resolution of skeletal ALP activity from hepatic ALP activity, we tested two established methods (Heat Inactivation and WGA precipitation) and a novel method, ConA precipitation. The organ-derived skeletal and hepatic ALP isoenzyme activities were used to compare these three methods with respect to linearity, isoenzyme separation, and precision. All three methods were linear, but the WGA and ConA methods afforded greater isoenzyme separation and precision. The relative extent of isoenzyme separation (i.e., the difference in percentage remaining skeletal and hepatic ALP isoenzyme activities) averaged 23, 40, and 47% remaining ALP activity for the Heat, WGA, and ConA methods, respectively. However, when these methods were applied to the quantitation of skeletal ALP activity in sera from 10 young and 10 adult beagles, the WGA method was found to be unacceptable because most of the results fell outside the range of the WGA assay calibrators (i.e., greater than 100% skeletal ALP activity). The Heat and ConA methods showed that the amount of skeletal ALP activity in the beagle sera decreased with age, both as ALP activity per liter and as percentage of total serum ALP activity (p less than 0.001 for each). Skeletal ALP activity levels determined by ConA were correlated with values determined by Heat Inactivation (r = 0.87, p less than 0.001) but not with WGA-determined levels (r = 0.26). Intestinal ALP activity was detected in only 1 of these 20 sera. We conclude that ConA precipitation can be used for quantitation of skeletal ALP activity in beagle serum.
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quantification of skeletal alkaline phosphatase in osteoporotic serum by wHeat germ agglutinin precipitation Heat Inactivation and a two site immunoradiometric assay
Clinical Chemistry, 1994Co-Authors: John R Farley, Susan L Hall, Barbara E Miller, Daniel Ilacas, Christopher Orcutt, Craig S Hill, David J BaylinkAbstract:Three methods for quantifying skeletal alkaline phosphatase (ALP; EC 3.1.3.1) activity/immunoactivity in serum--Heat Inactivation, wHeat germ agglutinin (WGA) precipitation, and an immunoradiometric assay--were tested for recovery and specificity and applied to 81 sera collected from 14 postmenopausal osteoporotic subjects. The Heat-Inactivation and WGA precipitation assays showed relative recoveries of 91-100% and 16-32%, respectively, for skeletal ALP with complete specificity (no cross-reactivity with hepatic or intestinal ALP); the IRMA showed a relative recovery of 86-100% and 11-14% cross-reactivity with hepatic ALP. There was a closer correlation between the Heat-Inactivation assay and IRMA (r = 0.833) than between the WGA precipitation assay and IRMA (r = 0.673) or between the Heat-Inactivation and WGA-precipitation assays (r = 0.568). The WGA precipitation assay failed to detect skeletal ALP in three serum samples that contained significant amounts as determined by the Heat-Inactivation assay and the IRMA.
Jeffrey I Cohen - One of the best experts on this subject based on the ideXlab platform.
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sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019
The Journal of Infectious Diseases, 2020Co-Authors: Peter D Burbelo, Francis X Riedo, Chihiro Morishima, Stephen A Rawlings, Davey M Smith, Sanchita Das, Jeffrey R Strich, Daniel S Chertow, Richard T Davey, Jeffrey I CohenAbstract:BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is associated with respiratory-related disease and death. Assays to detect virus-specific antibodies are important to understand the prevalence of infection and the course of the immune response. METHODS: Quantitative measurements of plasma or serum antibodies to the nucleocapsid and spike proteins were analyzed using luciferase immunoprecipitation system assays in 100 cross-sectional or longitudinal samples from patients with SARS-CoV-2 infection. A subset of samples was tested both with and without Heat Inactivation. RESULTS: At >14 days after symptom onset, antibodies against SARS-CoV-2 nucleocapsid protein showed 100% sensitivity and 100% specificity, whereas antibodies to spike protein were detected with 91% sensitivity and 100% specificity. Neither antibody levels nor the rate of seropositivity were significantly reduced by Heat Inactivation of samples. Analysis of daily samples from 6 patients with COVID-19 showed anti-nucleocapsid and spike protein antibodies appearing between days 8 and 14 after initial symptoms. Immunocompromised patients generally had a delayed antibody response to SARS-CoV-2, compared with immunocompetent patients. CONCLUSIONS: Antibody to the nucleocapsid protein of SARS-CoV-2 is more sensitive than spike protein antibody for detecting early infection. Analyzing Heat-inactivated samples with a luciferase immunoprecipitation system assay is a safe and sensitive method for detecting SARS-CoV-2 antibodies.
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detection of nucleocapsid antibody to sars cov 2 is more sensitive than antibody to spike protein in covid 19 patients
medRxiv, 2020Co-Authors: Peter D Burbelo, Francis X Riedo, Chihiro Morishima, Stephen A Rawlings, Davey M Smith, Sanchita Das, Jeffrey R Strich, Daniel S Chertow, Richard T Davey, Jeffrey I CohenAbstract:Background: SARS-CoV-2, the cause of coronavirus disease 2019 (COVID-19), is associated with respiratory-related morbidity and mortality. Assays to detect virus-specific antibodies are important to understand the prevalence of infection and the course of the immune response. Methodology: Quantitative measurements of plasma or serum antibodies by luciferase immunoprecipitation assay systems (LIPS) to the nucleocapsid and spike proteins were analyzed in 100 cross-sectional or longitudinal samples from SARS-CoV-2-infected patients. A subset of samples was tested with and without Heat Inactivation. Results: Fifteen or more days after symptom onset, antibodies against SARS-CoV-2 nucleocapsid protein showed 100% sensitivity and 100% specificity, while antibodies to spike protein were detected with 91% sensitivity and 100% specificity. Neither antibody levels nor the rate of seropositivity were significantly reduced by Heat Inactivation of samples. Analysis of daily samples from six patients with COVID-19 showed anti-nucleocapsid and spike antibodies appearing between day 8 to day 14 after initial symptoms. Immunocompromised patients generally had a delayed antibody response to SARS-CoV-2 compared to immunocompetent patients. Conclusions: Antibody to the nucleocapsid protein of SARS-CoV-2 is more sensitive than spike protein antibody for detecting early infection. Analyzing Heat-inactivated samples by LIPS is a safe and sensitive method for detecting SARS-CoV-2 antibodies.
