Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed. To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials. Different SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene. The protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 μl of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing. Direct RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.

Lübke N ，Senff T ，Scherger S ，Hauka S ，Andrée M ，Adams O ，Timm J ，Walker A ... -  《-》

Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA.

Hasan MR ，Mirza F ，Al-Hail H ，Sundararaju S ，Xaba T ，Iqbal M ，Alhussain H ，Yassine HM ，Perez-Lopez A ，Tang P ... -  《PLoS One》

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 ... -  《-》

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 ... -  《-》