Atl lysis buffer

Total DNA from high passage C. burnetii strains (n>30x times) propagated in cell-free culture was isolated by phenol-chloroform method [32 (link)] after overnight incubation with ATL lysis buffer and proteinase K (QIAGEN, Hilden, Germany). Whole genome sequencing was performed using 250 bp paired-end sequencing libraries (Nextera TAG-mentation sequencing kits, Epicentre, Madison, USA) on an Illumina MiSeq sequencer. High quality paired end reads were mapped against the reference NM RSA493 genome available from the NCBI database (Accession number: AE016828.2) using Bowtie2 alignment tool [33 (link)]. The average coverage and standard deviation (excluding repetitive regions) were obtained for the whole genome using a sliding-window approach and were used to calculate the coverage of genes involved in LPS synthesis (CBU0676 to CBU0706). A coverage below the minus 2 SD interval was considered as a significantly deleted gene. Apart from genome analysis, the protein profiles of low and high passaged 602 strain were also analysed by SDS-PAGE using the whole cell lysates, as described previously [34 (link)]. The method was basically the same except for staining of proteins with SYPRO Orange dye (Molecular probes) and visualising the protein profiles with a Typhoon Trio imager and ImageQuant 5.2 software (Molecular Dynamics).

DNAs from samples were extracted by two different methods. First, a standardized silica-based DNA extraction kit (DNAeasy Blood & Tissue Kit, QIAGEN GmbH, Hilden, Germany) was used for the purification of total DNA, as instructed by the manufacturer. A total of 5 µL was used as a template in the RPA reaction. Second, the same clinical samples were incubated with 200 µL QIAGEN ATL lysis buffer at 70 °C for 20 min. Then, 1 µL of the processed sample was diluted in 9 µL nuclease-free water, and 1 µL of the mix was used as a template.

Prior to RNA extraction, 200 µL of VTM supplemented samples were inactivated by mixing with 200 µL of ATL Lysis buffer (Qiagen). RNA extractions were performed on 200 µL of inactivated samples and experiments were carried out using three different processes, according to their availability at University Hospital: (1) Extraction on the Alinity m system (Abbott) using the Alinity m Sample Prep Kit 2, Alinity m Lysis Solution, and Alinity m Diluent Solution (Abbott) followed by RT-qPCR on the Alinity m system using the proprietary Alinity m SARS-CoV-2 AMP Kit targeting RdRp and N genes; (2) Automatic extraction on a Starlet platform (Hamilton) using the Starmag 96 Universal kit (Seegene) followed by RT-qPCR targeting the RdRp, E and N viral genes on a CFX 96 (Biorad) using the Allplex 2019-nCov assay kit (Seegene); (3) Extraction on MGISP-NE32 (MGI) was carried out using the MGIEasy Nucleic Acid Extraction Kit (MGI) followed by RT-qPCR on a LightCycler480 (Roche) using either Real time fluorescent RT-PCR (RdRp probe) (BGI) or the RT-PCR Argene SARS-CoV2 R-gene kit (N and RdRp Probes) (BioMérieux).

RNA testing involved elution from the DBS by lysing a 6 mm spot for 2 h at 56°C with 20 µl of proteinase K and 300 µl of ATL lysis buffer (Qiagen products: 19133 and 19076). The entire eluate was extracted on the Qiagen Qiasymphony platform using the Qiasymphony DSP Virus/Pathogen mini kit (Qiagen product: 937036) and ‘cell‐free V6/7 DSP default IC’ protocol. Bacteriophage MS2 was added as the internal control.