key: cord-0919411-2abknqlk authors: Yueying, Wang; Song, Wei; Zhao, Zuguo; Chen, Ping; Liu, Jian; Li, Chende title: The Impacts of Viral Inactivating Methods On Quantitative RT-PCR for COVID-19 date: 2020-05-04 journal: Virus Res DOI: 10.1016/j.virusres.2020.197988 sha: c7850a938b17804028265210b53a1173aae8fe76 doc_id: 919411 cord_uid: 2abknqlk Abstract Objective This paper aims to explore the effect of four virus inactivation methods on the rapid detection results of COVID-19 nucleic acid. Methods Collected samples of nasopharyngeal swabs from 2 patients diagnosed with COVID-19 at the First People's Hospital of Zhaoqing City, each of sample was divided into 5 groups (groupA∼E): A:Non-inactivated raw sample; B:75% ethanol inactivation; C:56 ℃ incubation for 30 min inactivation; D:65 ℃ incubation for 10 min inactivation; E:Pre-inactivation using RNA virus special preservation fluid added into the sampling tube to treated the nasopharyngeal swab sample separately, using real-time fluorescent RT-PCR to detect the N gene of COVID-19 and the ORF1ab gene simultaneously. All the groups are diluted in 1:2, 1:4, 1:8 ratios. The objectives are to compare the effect of the varied inactivation method on CT(Cycle Threshold)results in PCR, conduct correlation and Bland-Altman analysis. Results For the N gene and ORF1ab gene, the CT values of 4 inactivated and Non-inactivated treatment were correlated (P＜0.001). The results of the four treatment methods and specimens without inactivated treatment have shown good consistency. Conclusion The treatment of nasopharyngeal swab specimens using mentioned four inactivated methods had no significant effect on the subsequent detection of the new COVID-19 nucleic acid test. Lab test-persons can flexibly adopt pre-inactivation methods to ensure the accuracy of virus nucleic acid test results, meanwhile guarantee the safety of lab test-persons. inactivation using RNA virus special preservation fluid added into the sampling tube to treated the nasopharyngeal swab sample separately, using real -time fluorescent RT-PCR to detect the N gene of COVID-19 and the ORF1ab gene simultaneously. All the g roups are diluted in 1:2, 1:4, 1:8 ratios. The objectives are to compare the effect of the varied inactivation method on CT(Cycle Threshold)results in PCR, conduct correlation and Bland-Altman analysis. Results For the N gene and ORF1ab gene, the CT values of 4 inactivated and Non-inactivated treatment were correlated (P＜0.001). The results of the four treatment methods and specimens without inactivated treatment have shown good consistency. Conclusion The treatment of nasopharyngeal swab specimens using mentioned four inactivated methods had no significant effect on the subsequent detection of the new COVID-19 nucleic acid test. Lab test-persons can flexibly adopt preinactivation methods to ensure the accuracy of virus nucleic acid test results, meanwhile guarantee the safety of lab test-persons. COVID-19 has infected more than 2,000,000 people all over the world by April 22 nd , including 165,310 deaths, posing a huge threat to the physical and mental health of the general public. At the meantime more than 1000 medical staff were infected in China, resulting in 5 deaths. The First People's Hospital is appointed COVID-19 patients treatment institution in Zhaoqing City. By far it has accepted 20 confirmed cases of new coronary pneumonia patients, out of which 10 cases have been cured and 1 case ended in casualty. According to the COVID-19 Diagnosis and Treatment Plan (6 th Trail Edition), this virus is highly contagious and can spread quickly among population. There is no specific diagnosis and treatment program against the pneumonia caused by COVID-19 (Zhu et al., 2020). Currently, RT-PCR is the most widely adopted clinical screening method for COVID-19 due to its speed, low cost, and convenience in clinical use (Lancet T, 2020 ; National Health Commission of the PRC and National Administration of Traditional Chinese Medicine of the PRC, 2020).Since patient virus samples sent for lab testing are extremely infectious, the experimenter should only carry out the experimental operations when their biosecurity protection measurements are fully equipped, following relevant restrictions and safety guidance. According to documents such as the in-use guidance for the diagnosis and treatment of COVID-19, this virus can be inactivated either at 75% alcohol, 56 degrees incubation for 30 min, or 65 degrees incubation for 10 min according to Chinese expert's consensus(National Health Commission of the People's Republic of China, 2020; Group of experts on medical treatment from Tongji Hospital affiliated of Wuhan Tongji Medical College, 2020; Chinese Society of Laboratory Medicine, 2020). In order to reduce the risk of in-lab infection during the experimental operation, reduce the mental stress of laboratory staff, the high-risk specimen will be inactivated before the lab-test undergoes. However it remains unknown whether the specimen inactivation would affect the subsequent RT-PCR test results or not. Therefore, this study intends to compare 4 different inactivation methods to explore impact of the 4 inactivation methods upon the results of viral nucleic acid PCR detection, and to lower the risk of medical staff in-lab infection while ensuring the accuracy of the test, and eventually to provide new solutions for the laboratory biosecurity environment protection. Specimen Source: 2 confirmed COVID-19 infected cases in Zhaoqing City. Description: 2 patients were hospitalized due to fever, RT-PCR nucleic acid test results showed positive, and confirmed as COVID-19 infected cases by the Guangdong Provincial Center for Disease Control and Prevention. Then the nasopharyngeal swab elution sluice above will be diluted in a ratio of 1:2, 1:4, 1:8 for separated nucleic acid extraction, followed by RT-PCR nucleic acid testing. The thermal cycling conditions used were: an initial reverse transcription step of 50℃for 15min,next to denaturation step of 95℃ for 15min, followed by 45 cycles of 94℃ for 15s, 55℃ for 45s, and a final step of 40℃ for 20s. The Applied Biosystems® 7500 instrument is used in amplification test to determine whether it's negative or positive. If each batch of tests has supplied positive and negative control, and the results are within controlled range, the batch test is valid, on the contrary, invalid. All tests are repeated for 3 times,and test-person will use the average cycle threshold for calculation. Statistical analysis software SPSS 19.0 was used in this particular research. Correlation between different specimens CT values were analyzed using Pearson product-moment correlation coefficient analysis, and the graphics were illustrated by Graphpad 5.0. Furthermore, The Bland-Altman plots was drawn using MedCalc software, as well as the the consistency of CT values analysis. P<0.05 is considered as statistically significant. The results of the RT-PCR were found to be similar to those of the Non-inactive specimens after the detection of specimens with different concentrations, as shown in Table 1 . Using Pearson product-moment correlation coefficient analysis, the results showed that the CT values between the four inactivated and non-inactivated treatments were correlated with both N gene and ORF1ab gene (P＜0.001)( Table 2) The scatter diagram will be drawn using the Bland-Altman method, for which the difference For the ORF1ab gene, the mean of the CT value difference of 75% 30min ethanol inactivated and non-inactivated is among is 1.5, 95%CI(-3.1,6.1); 56℃ 30min inactivated difference among means of CT value is 1.4, 95%CI(-3.4,6.2); 65℃ 10min inactivated difference among means of CT value is -1.67, 95%CI(-2.76,-0.58);RNA Virus-specific storage fluid in inactivated difference among means of CT value is 0, 95%CI(-2.4,2.3), All points are distributed within the 95% trust interval. Recent studies have found that the COVID-19 is mainly spread through droplets, body contact, respiratory aerosols and daily contacts in an infected environment. Fever, Fatigue, dry cough, etc. are the first symptoms to some patients, while others start with diarrhea as the first symptoms, in some cases, patients are symptom-free, regarded as asymptomatic infection. A small number of patients may suffer acute respiratory distress syndrome, heavy metabolic acidosis and blood clotting dysfunction, even death (Huang, 2020 J o u r n a l P r e -p r o o f A Novel Coronavirus from Patients with Pneumonia in China Emerging understandings of 2019-nCoV National Health Commission (NHC) of the PRC and National Administration of Traditional Chinese Medicine of the PRC. Guidance for Corona Virus Disease 2019: Prevention, Control, Diagnosis and Management National Health Commission of the People's Republic of China. Handbook of Prevention and Treatment of the Pneumonia Caused by the 2019-nCoV Group of experts on medical treatment from Tongji Hospital affiliated of Wuhan Tongji Medical College. Guidelines for diagnosis and treatment of Corona Virus Disease The Experts' Consensus on Nucleic Acid Detection of 2019-nCoV Clinical features of patients infected with 2019 novel coronavirus in Wuhan Ping Chen: Validation.Jian Liu: Validation.Chende Li: Supervision, Conceptualization. The authors declare no conflict of interest.