tetano
Editor, Senior Moderator
J Clin Microbiol
. 2020 May 28;JCM.00958-20.
doi: 10.1128/JCM.00958-20. Online ahead of print.
Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number
Hailong Chen[SUP] 1 [/SUP], Rui Wu[SUP] 1 [/SUP], Yuan Xing[SUP] 2 [/SUP], Quanli Du[SUP] 1 [/SUP], Zerun Xue[SUP] 1 [/SUP], Yanli Xi[SUP] 1 [/SUP], Yujie Yang[SUP] 1 [/SUP], Yangni Deng[SUP] 1 [/SUP], Yuewen Han[SUP] 1 [/SUP], Kaixin Li[SUP] 1 [/SUP], Yang Luan[SUP] 1 [/SUP], Yalan Zhang[SUP] 1 [/SUP], Xiaoguang Wei[SUP] 1 [/SUP], Tongtong Yu[SUP] 1 [/SUP], Hao Li[SUP] 1 [/SUP], Lingxiang Zhu[SUP] 3 [/SUP], Shengshi Su[SUP] 4 [/SUP], Hao Lian[SUP] 5 [/SUP], Linping Lu[SUP] 5 [/SUP], Chianru Tan[SUP] 4 [/SUP], Haichao Zheng[SUP] 1 [/SUP], Baozhong Chen[SUP] 1 [/SUP], Pengbo Yu[SUP] 6 [/SUP], Yong Guo[SUP] 7 [/SUP], Chaofeng Ma[SUP] 8 [/SUP]
Affiliations
Abstract
The outbreak of COVID-19 has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed with three different methods: (1) incubation with TRIzol? LS Reagent for 10 min at room temperature, (2) heating in a waterbath at 56?C for 30 min, and (3) high-temperature treatment, including 121?C autoclaving for 20 min, 100?C boiling for 20 min, and 80?C heating for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of N gene and 39.85% of ORF 1ab. For samples treated at 56?C for 30 min, the copy number of N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. Viral RNA copy number dropped by 50-66% after 80?C heating for 20 min. Nearly no viral RNA was detected after autoclaving at 121?C or boiling at 100?C for 20 min. These results indicated that inactivation reduced the quantity of detectable viral RNA and may cause false negative results especially in weakly positive cases. Thus, TRIzol is recommended for sample inactivation in comparison to heat inactivation as Trizol has the least effect on RNA copy number among the tested methods.
. 2020 May 28;JCM.00958-20.
doi: 10.1128/JCM.00958-20. Online ahead of print.
Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number
Hailong Chen[SUP] 1 [/SUP], Rui Wu[SUP] 1 [/SUP], Yuan Xing[SUP] 2 [/SUP], Quanli Du[SUP] 1 [/SUP], Zerun Xue[SUP] 1 [/SUP], Yanli Xi[SUP] 1 [/SUP], Yujie Yang[SUP] 1 [/SUP], Yangni Deng[SUP] 1 [/SUP], Yuewen Han[SUP] 1 [/SUP], Kaixin Li[SUP] 1 [/SUP], Yang Luan[SUP] 1 [/SUP], Yalan Zhang[SUP] 1 [/SUP], Xiaoguang Wei[SUP] 1 [/SUP], Tongtong Yu[SUP] 1 [/SUP], Hao Li[SUP] 1 [/SUP], Lingxiang Zhu[SUP] 3 [/SUP], Shengshi Su[SUP] 4 [/SUP], Hao Lian[SUP] 5 [/SUP], Linping Lu[SUP] 5 [/SUP], Chianru Tan[SUP] 4 [/SUP], Haichao Zheng[SUP] 1 [/SUP], Baozhong Chen[SUP] 1 [/SUP], Pengbo Yu[SUP] 6 [/SUP], Yong Guo[SUP] 7 [/SUP], Chaofeng Ma[SUP] 8 [/SUP]
Affiliations
- PMID: 32467359
- DOI: 10.1128/JCM.00958-20
Abstract
The outbreak of COVID-19 has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed with three different methods: (1) incubation with TRIzol? LS Reagent for 10 min at room temperature, (2) heating in a waterbath at 56?C for 30 min, and (3) high-temperature treatment, including 121?C autoclaving for 20 min, 100?C boiling for 20 min, and 80?C heating for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of N gene and 39.85% of ORF 1ab. For samples treated at 56?C for 30 min, the copy number of N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. Viral RNA copy number dropped by 50-66% after 80?C heating for 20 min. Nearly no viral RNA was detected after autoclaving at 121?C or boiling at 100?C for 20 min. These results indicated that inactivation reduced the quantity of detectable viral RNA and may cause false negative results especially in weakly positive cases. Thus, TRIzol is recommended for sample inactivation in comparison to heat inactivation as Trizol has the least effect on RNA copy number among the tested methods.