Qiaquick dna purification kit

The positive PCR products were purified using the QIAquick DNA purification kit™ Qiagen, Germany [21 (link), 22 (link)]. 5 μl aliquot of the product was mixed with 1 μl of 6× loading dye and loaded onto a 2% agarose with 2 μl ethidium bromide gel alongside a 100 bp ladder for gel electrophoresis and ultraviolet visualization using 4% agarose-Tris-Borate-EDTA 10×. The presence of the expected 160 bp amplicon was considered positive for HPV DNA PCR [7 ].

The total RNA was isolated from the cultural medium of infected cells using the RNeasy minikit (Qiagen, Hilden, Germany), reverse–transcribed by the SuperScript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with random hexanucleotide primers (Syntol, Russia), amplified by PCR to produce 8 overlapping DNA fragments (primers are available upon request), and purified with QiaQuick DNA purification kit (Qiagen, Hilden, Germany). For sequencing, an ABI 3130 Genetic Analyzer was used.

From the 11 SOPs received by CPC, we listed 4 main methods of DNA extraction used: 1) Automatized DNA extraction using the Maxwell Instrument (Promega), used by 2 laboratories: 2) Commercial kits; Gentra Puregene kit (as stated in the Buruli ulcer Manual for Health Care Providers) and QIAamp DNA mini kit with an enzymatic lysis step (Qiagen), used by 4 laboratories: 3) Alkaline lysis method using “in-house” buffers [8 (link)] and a commercial kit [9 (link)] (GenoLyse, Hain LifeScience) used by 4 other laboratories, and 4) The Modified Boom method [10 (link)] used by a single laboratory.
The first two methods have been discarded as too expensive, too long and with an enzymatic lysis step not adapted to lyse mycobacterial cell wall. We decided to focus on the alkaline lysis procedure, which is known to be a robust, rapid and inexpensive method based on lysis of bacterial cell walls with sodium hydroxide (NaOH) and heat. This method was or was not associated with a purification step using a QIAquick DNA purification kit (Qiagen) based on a silica membrane transfer to eliminate any inhibitors for qPCR assay. We chose to compare the alkaline lysis procedure from the “in house” SOP and the commercial kit with and without the purification step. Evaluation of the DNA extraction methods was performed using four swabs and four FNA samples with known concentration of M. ulcerans.

Peritoneal fluid showing a positive PCR result was then purified using QIAquick DNA Purification Kit (Qiagen). Sequencing was then performed in accordance with the Applied Biosystem Genetic Analyzer protocol. Homology analysis was conducted with a positive sample at https://blast.ncbi.nlm.nih.gov/Blast.cgi. Phylogenetic analysis of FIPV Surabaya isolate was performed using reference sequences from GenBank: FCoV (MT444152), FIPV (DQ010921), and SARS-CoV-2, including MT135041 (China), MT135042 (China), MT135044 (China), MN994467 (USA), MT152824 (USA), MT163717 (USA), MT198652 (Spain), MT198653 (Spain), MT039888 (USA), MT020781 (Finland), MT192765 (USA), LC529905 (Japan), LC529905 (Japan), MT066156 (Italy), MT007544 (Australia), and MT039890 (Republic of Korea).

Sequencing libraries were enriched for mitochondrial DNA using hybridisation capture enrichment15 (link). Two micrograms of each library were required for hybridisation capture. To obtain this amount of template library, four 1 μl aliquots of each stock library were PCR amplified in 100 μl reaction volume each with reactions again being terminated after reaching PCR amplification plateau. The four amplicons from each library were then pooled, purified using a Qiagen Minelute kit, and eluted in 20 μl of 0.1xTE. DNA extracted from modern Vicugna pacos (alpaca) samples was used to produce the capture bait as described in Maricic et al.15 (link). In brief, modern samples were extracted using a Qiagen DNeasy extraction kit, complete mitochondrial genomes were amplified by long range PCR as two separate fragments using primers based on the Vicugna pacos mitochondrial genome NCBI Reference Sequence: NC_002504.1 (Supplementary Table S2), purified using Qiagen QIAquick DNA purification kit and quantified using a thermo fisher scientific nanodrop 2000c spectrophotometer. Long range PCR products were pooled in 750ng equimolar amounts and fragmented through sonication. Biotinylated adapters were ligated to the sonicated mitochondrial genome fragments. Adapter ligated fragments were then bound to streptavidin coated beads to be used as bait in the hybridisation capture.