The impacts of viral inactivating methods on quantitative RT-PCR for COVID-19.

This paper aims to explore the effect of four virus inactivation methods on the rapid detection results of COVID-19 nucleic acid. 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. 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. 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.

Wang Y ，Song W ，Zhao Z ，Chen P ，Liu J ，Li C ... -  《-》

Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads.

Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

Pan Y ，Long L ，Zhang D ，Yuan T ，Cui S ，Yang P ，Wang Q ，Ren S ... -  《-》

SARS-CoV-2 and RT-PCR in asymptomatic patients: Results of a cohort of workers at El Dorado International Airport in Bogotá, 2020.

Malagón-Rojas J ，Gómez-Rendón C ，Parra EL ，Almentero J ，Palma R ，López R ，Toloza-Pérez YG ，Rubio V ，Bedoya JF ，López-Díaz F ，Franco-Muñoz C ，Reales-González J ，Mercado-Reyes M ... -  《-》

Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits.

The severe shortage of nucleic acid extraction kits during the current COVID-19 pandemic represents a key limiting factor in testing capacity. This study compared the results of SARS-CoV-2 RT-PCR using different simple nucleic acid extraction methods on nasopharyngeal and saliva specimens. Fifty nasopharyngeal swab and saliva specimens previously tested positive for SARS-CoV-2 were retrieved. Three different methods of nucleic acid extraction were compared. The first method involves incubating the specimen with proteinase K, and then heat treatment at 98 °C for 5 min (PKH); the second method involves heat treatment at 98 °C for 5 min without proteinase K pre-incubation (heat only); the third method involves no pre-processing steps (direct). The products from all 3 methods were tested by SARS-CoV-2 RT-PCR. PKH had significantly higher positive rate in SARS-CoV-2 RT-PCR (80 %) than those of heat only (58 %; P = 0.001) or direct (56 %; P = 0.002). The median Ct value was significantly earlier for PKH (median Ct: 37.0, IQR 31.7-40) than that of heat only (median Ct: 40, IQR 36.2-41; P < 0.0001) and direct (median Ct, 37.5; IQR 33.9-41.0; P = 0.0049). Subgroup analysis showed that PKH had higher detection rate, lower limit of detection and earlier Ct values than the other two groups for both NPS and saliva specimens. PKH pre-processing resulted in the highest detection rate of SARS-CoV-2 by RT-PCR, and represents an alternative method for nucleic acid extraction when commercial extraction kits are not available.

Chu AW ，Chan WM ，Ip JD ，Yip CC ，Chan JF ，Yuen KY ，To KK ... -  《-》

Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing.

Al-Saud H ，Al-Romaih K ，Bakheet R ，Mahmoud L ，Al-Harbi N ，Alshareef I ，Judia SB ，Aharbi L ，Alzayed A ，Jabaan A ，Alhadrami H ，Albarrag A ，Azhar EI ，Al-Mozaini MA ... -  《-》