effect of heat inactivation on real time reverse transcription pcr of the sars cov 2 CORD-Papers-2021-10-25 (Version 1)

Title: Effect of Heat inactivation on Real-Time Reverse Transcription PCR of the SARS-COV-2 Detection
Abstract: Background: Real-time reverse transcription PCR (rRT-PCR) is commonly used to diagnose SARS-CoV-2 infection. Heat inactivation prior to nucleic acid isolation may allow safe testing, while the effects of heat inactivation on SARS-CoV-2 rRT-PCR detection result need to be determined. Methods: 14 positive nasopharyngeal swab specimens were inactivated at 56{degrees}C for 30min, 56{degrees}C for 60min, 60{degrees}C for 30min, 60{degrees}C for 75min, and 100{degrees}C for 10min, and another 2 positive nasopharyngeal swab specimens were also inactivated at 100{degrees}C for 10min, 100{degrees}C for 30min, 100{degrees}C for 60min, after which the samples were isolated and detected by rRT-PCR. Results: All 14 heat treated samples remained positive. The range of threshold cycle (Ct) values observed when detecting ORF1a/b was 27.228-34.011 in heat-treated samples, while 25.281-34.861 in unheated samples, and the range of threshold cycle (Ct) values observed at the time of detecting N was 25.777-33.351 in heat-treated samples, while 24.1615-35.433 in unheated samples, on basis of which it showed no statistical difference otherwise a good correlation of Ct values between the heat-inactivated samples and the untreated samples. However, the 2 samples inactivated at 100{degrees}C 30min, 100{degrees}C 60min turned into negative. Conclusions: Heat inactivation at 56{degrees}C for 30min, 56{degrees}C for 60min, 60{degrees}C for 30min, 60{degrees}C for 75min, and 100{degrees}C for 10min shall not affect the detection results of Real-Time Reverse Transcription PCR of the SARS-COV2. Furthermore, it is recommended to inactive nasopharyngeal swab specimens 10min at 100{degrees}C before RNA extraction in consideration of efficiency and reliable results. Key Words: SARS-CoV-2, Heat Inactivation, rRT-PCR, Comparison
Published: 5/22/2020
DOI: 10.1101/2020.05.19.20101469
DOI_URL: http://doi.org/10.1101/2020.05.19.20101469
Author Name: liu, y
Author link: https://covid19-data.nist.gov/pid/rest/local/author/liu_y
Author Name: cao, z
Author link: https://covid19-data.nist.gov/pid/rest/local/author/cao_z
Author Name: chen, m
Author link: https://covid19-data.nist.gov/pid/rest/local/author/chen_m
Author Name: zhong, Y
Author link: https://covid19-data.nist.gov/pid/rest/local/author/zhong_y
Author Name: luo, y
Author link: https://covid19-data.nist.gov/pid/rest/local/author/luo_y
Author Name: shi, g
Author link: https://covid19-data.nist.gov/pid/rest/local/author/shi_g
Author Name: Xiang, H
Author link: https://covid19-data.nist.gov/pid/rest/local/author/xiang_h
Author Name: Luo, J
Author link: https://covid19-data.nist.gov/pid/rest/local/author/luo_j
Author Name: Zhou, H
Author link: https://covid19-data.nist.gov/pid/rest/local/author/zhou_h
sha: d94167cbc4f6cfa22018f2fe0ea1bb2c28ae8081
license: medrxiv
source_x: MedRxiv; WHO
source_x_url: https://www.who.int/
url: http://medrxiv.org/cgi/content/short/2020.05.19.20101469v1?rss=1 https://doi.org/10.1101/2020.05.19.20101469
has_full_text: TRUE
Keywords Extracted from Text Content: 25.281-34.861 24.1615-35.433 °C nasopharyngeal swab specimens 25.777-33.351 27.228-34.011 SARS-CoV-2 medRxiv preprint ORF1a/b Heat [16] [17] [18] SARS-CoV-2 RNA blood samples °C SARS-CoV-2 medRxiv preprint 30min Nasopharyngeal swab specimens SARS-CoV-2 heat https://doi.org/10.1101/2020.05.19.20101469 doi samples cells Pastori cell SARS-CoV-2 samples sputum Version8.0 medRxiv preprint ribonucleases dithiothreitol medRxiv nucleocapsid sodium dodecylsarcosine PHEIC tube nasopharyngeal swab specimens 1.418 nasopharyngeal swab samples Fig.1 guanidine isothiocyanate
Extracted Text Content in Record: First 5000 Characters:Background: Real-time reverse transcription PCR (rRT-PCR) is commonly used to diagnose SARS-CoV-2 infection. Heat inactivation prior to nucleic acid isolation may allow safe testing, while the effects of heat inactivation on SARS-CoV-2 rRT-PCR detection result need to be determined. Methods: 14 positive nasopharyngeal swab specimens were inactivated at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min, and another 2 positive nasopharyngeal swab specimens were also inactivated at 100°C for 10min, 100°C for 30min, 100°C for 60min, after which the samples were isolated and detected by rRT-PCR. : All 14 heat treated samples remained positive. The range of threshold cycle (Ct) values observed when detecting ORF1a/b was 27.228-34.011 in heat-treated samples, while 25.281-34.861 in unheated samples, and the range of threshold cycle (Ct) values observed at the time of detecting N was 25.777-33.351 in heat-treated samples, while 24.1615-35.433 in unheated samples, on basis of which it showed no All rights reserved. No reuse allowed without permission. : medRxiv preprint statistical difference otherwise a good correlation of Ct values between the heatinactivated samples and the untreated samples. However, the 2 samples inactivated at 100°C 30min, 100°C 60min turned into negative. Conclusions: Heat inactivation at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min shall not affect the detection results of Real-Time Reverse Transcription PCR of the SARS-COV2. Furthermore, it is recommended to inactive nasopharyngeal swab specimens 10min at 100°C before RNA extraction in consideration of efficiency and reliable results. Notwithstanding the foregoing, different protein concentrations may have protective effects and may affect the inactivation efficiency, due to which, it is well-advised to prolong the heating time or increase the temperature to completely inactivate viruses [12] [13] [14] [15] . However, because viral RNA would easily be degraded by ribonucleases, which affects the detection result of rRT-PCR of virus. Systematic research on the heat inactivation efficacies of rRT-PCR remains to be studied and confirmed. we evaluated the efficacy of SARS-CoV-2 heat inactivation in nasopharyngeal swab specimens in our study, where we performed different commonly used heat condition to inactive the SARS-CoV-2, i.e, 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, 100°C for 10min, 100°C for 30min and 100°C for 60min. And then we evaluated and compared the ct values of heat treated and unheated samples in nasopharyngeal swab specimens. Nasopharyngeal swab specimens were obtained according to CDC guidelines and collected in a separate sterile tube containing 1 ml of viral transport medium (including guanidine isothiocyanate, sodium dodecylsarcosine, and dithiothreitol). This study was approved by the ethics commission of Zhujiang Hospital. The 14 samples were submitted to three temperatures with different durations. Which included: unheated, 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min. While the other 2 samples were submitted to three conditions of 100°C for 10min, 100°C for 30min and 100°C for 60min. RNA was extracted and tested by real-time RT-PCR using the kit (bioperfectus technologies) according to the manufacturer's instructions. Tests were performed in biosafety level 2 facilities at the Zhuzhou Center for Disease Control and Prevention. The sample would be considered to be laboratory-confirmed, if two targets (open reading frame 1a or 1b, nucleocapsid protein) are tested positive by specific real-time RT-PCR. And a cycle threshold value (Ct-value) less than 36 shall be defined as positive, with a Ct-value of 40 or more as negative. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 22, 2020. . Quantitative results were expressed as mean±SD, and groups were compared by ttest. P values <0.05 was considered statistically significant. Graphpad Prism software (Version8.0) was used for statistical analysis. The Spearman correlation coefficient was used to analyze the correlation between CT values of different treated samples, and the Bland-Altman plot was used to analyze the consistency of CT values. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 22, 2020. . https://doi.org/10.1101/2020.05.19.20101469 doi: medRxiv preprint 30min and the non-inactivated samples, -0.1625 to 1.921 between the inactivated samples at 60°C for 60min and the non-inactivated samples, -2.082 to 1.418 between the inactivated samples at 100°C for 10min and non-inactivated samples.
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