Nucleospin blood mini kit

Genomic DNA of S. suis and other bacterial strains were extracted using a heat-lysis method with some modification [45 (link)]. Briefly, a loopful of the bacterial colonies were suspended into 30 µL lysis buffer (a mixture of 0.25% (vol/vol) sodium dodecyl sulphate (SDS), 0.05 M sodium hydroxide (NaOH) and distilled water), mixed by vortex and heated at 95 °C for 15 min, followed by centrifugation at 12,000× g for 5 min. Finally, the supernatant was transferred into a new tube, and we added 200 µL of sterile distilled water. The extracted genomic DNA was kept at −20 °C until used.
Genomic DNA from hemoculture was extracted using a NucleoSpin^® Blood Mini kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. To remove PCR inhibitors, genomic DNA extracted from hemoculture were diluted to 1:10 with sterile distilled water prior to use as a template for PCR. Cerebrospinal fluid (CSF) was centrifuged, and the pellet was further processed to obtain genomic DNA using the heat-lysis method.

Genomic DNA (NA12878, NA06891, NA07537, and NA20241, representing cells with diverse FMR1 alleles) was purchased from the Coriell Institute for Medical Research. All the other samples were isolated from the whole blood of unrelated healthy donors (Blood Center, Verona Hospital) following informed written consent. Venous blood samples were collected in EDTA tubes, de-identified immediately after collection, and stored at −80°C until use. The study was approved by the Ethics Committee for Clinical Research of Verona and Rovigo Provinces and all the investigations were conducted according to the Declaration of Helsinki. Genomic DNA was extracted using the Genomic Tip 100/G kit (Qiagen, Hilden, Germany), Nanobind CBB Big DNA Kit (Circulomics, Baltimore, MD, United States), NucleoSpin Blood Mini kit (Macherey-Nagel, Düren, Germany), or the Miller’s protocol (Miller et al., 1988 (link)). All protocols were carried out according to the manufacturer’s instructions, and for the Circulomics kit, we used either the HMW or ultra-HMW protocol. The different DNA extraction methods were tested on samples from distinct donors, an aspect that may represent a weakness of the study.

For the LAMP assay, a simple boiling technique was used in this study to extract genomic DNA [16 (link), 17 (link)]. Briefly, 50 µL of EDTA treated whole blood was added into a 1.5 ml-microcentrifuge tube containing 150 µL of nuclease-free distilled water, boiled for 5 min at 95 °C and centrifuged for 3 min at 2046×g. For each LAMP reaction, 100 µL of the clear supernatant was transferred to a new clean tube and 5.5 µL of the supernatant was used immediately. Based on the observation, the supernatant containing gDNA could be kept at − 80 °C for not more than 7 days without compromising stability.
For nested PCR, DNA extraction was done from frozen EDTA blood samples using a NucleoSpin^® Blood mini kit (Macherey–Nagel GmbH, Germany) according to the manufacturer’s protocol. For nested PCR analysis, 5 µL of the eluted DNA was used directly.