key: cord-0855714-5oxnzv15 authors: Claas, Eric C.J.; Smit, Pieter W.; van Bussel, Mario J.A.W.M.; Verbakel, Harold; Taouil, Mohammed; Verweij, Jaco J.; Thijsen, Steven F.T. title: A two minute liquid based sample preparation for rapid SARS-CoV2 real-time PCR screening: a multicentre evaluation date: 2020-12-31 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104720 sha: a1d0c1879494d6a55cf1053f14a9d8a9ecd85c07 doc_id: 855714 cord_uid: 5oxnzv15 BACKGROUND: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit components, buffers and even plastics has resulted in suboptimal testing procedures worldwide. Alternative workflows have been implemented to overcome these difficulties. Recently a liquid based sample prep has been launched as solution to overcome limitations in relation to nucleic acid extraction. OBJECTIVE: Multicenter evaluation of the QIAprep& Viral RNA UM kit (QIA P&A) for rapid sample preparation and real-time PCR detection of SARS-CoV-2 in comparison to standardized laboratory testing methods. STUDY DESIGN: Selected samples of the routine diagnostic workflow at Clinical Microbiology Laboratories of four Dutch hospitals have been subjected to the rapid QIA P&A protocol and the results have been compared to routine diagnostic data. RESULTS: Combining results of manual and automated procedures, a total of 377 clinical samples of which 202 had been tested positive with a wide range of C(T) values, showed almost complete concordance in the QIA P&A assay for samples up to C(T) values of 33 with one exception of C(T) 31. Prospectively 60 samples were tested and also showed 100% concordance with 5 positives. The method has been automated by two centres. CONCLUSIONS: Despite an input of only 8 µl of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic. Prospectively 60 samples were tested and also showed 100% concordance with 5 positives. The method has been automated by two centres. Conclusions: Despite an input of only 8µl of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic. J o u r n a l P r e -p r o o f Background The SARS-CoV-2 pandemic has been and still is a challenge for the world. Although the initial identification [1] and subsequent implementation of diagnostic methods [2] was extremely fast, the pandemic could not be contained, in contrast to the 2002-2003 SARS-CoV-1 outbreak [3] . One of the reasons is the efficient spread of SARS-CoV-2, partly because also asymptomatically infected individuals or patients prior clinical symptoms had been found to be contagious [4] The worldwide spread of the virus also resulted in major complications for diagnostic laboratories, partly by insufficient access to reagents and even plastics provided by diagnostic companies. Initially lysis buffers, required for inactivation of the virus and crucial for nucleic acid (NA) extraction from clinical samples, were the main limitation, but later also availability of plastics and even pipet tips became problematic because of the worldwide demand [5] . As a result, most laboratories have spread the risk by investing in implementation of alternative platforms for diagnosing SARS-CoV-2. Recently, a new sample preparation solution was launched by Qiagen, the Qiaprep& Viral RNA UM (QIA P&A) kit, that combined a short, liquid based sample preparation with one-step real-time PCR amplification and detection of SARS-CoV-2, including two internal controls; one synthetic RNA IC and a sampling control amplifying transcripts of human B2M and RNaseP genes. The Research Use Only version of the kit, has been used in combination with the primers and probes developed by the CDC [6] which are included in the SARS-CoV-2 N1+N2 assay (Qiagen) as well as the oligos published by Corman et al [2] . In this study, the performance of the QIA P&A kit was investigated by comparing the result to those of routine diagnostic workflows, that include NA extraction procedures, in four Dutch hospital laboratories. Maasstad hospital managed to automate the QIA P&A procedure using small pipetting volumes of 2µl and 8µl, using a Tecan Fluent 480 pipetting platform (Tecan, Switzerland) . A total of 94 samples were tested using real-time PCR with sarbeco E primers [2] in the QIA P&A and the NeuMoDx™ 96 (Qiagen) as reference assay. The NeuMoDx™ is a fully automated microfluidics based PCR platform [7] . Table 2 shows the results of this panel for both their routine diagnostic procedure and the QIA P&A. Although some variation in the high CT value samples, very comparable results have been obtained. The observed specificity is depending on the primer set used. All assays gave negative results with other circulating human coronaviruses, but the E-gene primers are beta-coronavirus group specific and therefore also detected SARS-CoV-1 (sample 10). Nucleic extraction is a crucial part of molecular diagnostic procedures and results in highly purified DNA or RNA, which may take from 30 minutes up to several hours. This study shows the evaluation of a two-minute, liquid based, sample preparation method for SARS-CoV-2 testing in comparison to different diagnostic workflows at four hospital laboratories in the Netherlands. The overall conclusion is that the QIA P&A procedure provided excellent results, given the small amount of sample that is being used. It should be noted that the sample preparation buffer does not inactivate the SARS-CoV-2, so for proper sample handling a BSL-2 facility is required. This can be overcome by heat inactivation of the sample in transport buffers, for which 50 µl sample at 70°C for 10 minutes would be sufficient according to the manufacturer's instructions. The QIA P&A can handle a broad range of non-fixating buffers and transport media, but is incompatible with inactivating lysis buffers containing detergents or guanidinium thiocyanate. Potentially, the inhibited samples observed in one centre, were performed on swab in lysis buffer. Unfortunately, the original sample tubes were unavailable for reinspection. The QIA P&A can be used in combination with lab developed primer sets for other viruses as well. Early 2021, the company will launch a CE/IVD version of the kit containing specific viral target primers and probes. Altogether the QIA P&A can be considered a valuable addition to the diagnostic laboratory, either as back -up when extraction reagents or plastics are poorly delivered or, when automated, to further increase the diagnostic capacity. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f A Novel Coronavirus from Patients with Pneumonia in China Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin A novel coronavirus associated with severe acute respiratory syndrome Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review Under-allocation: Critical Supply Chain Hurdles Negatively Impact the Ability of Community Hospitals To Perform Repeat SARS US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2. Emerging infectious diseases Development of a fully automated high throughput PCR for the detection of SARS-CoV-2: The need for speed The authors thank Martijn Mostert, Shannon La Grouw, and Dennis Heemskerk for excellent technical assistance. The QIA P&A kit was provided by Qiagen, without further involvement in experimental design and data analysis.