Maxwell blood dna kit

A total of 111 representatives of C. jejuni isolates from chicken meats at regions B, C and E were arbitrarily selected to include highly contaminated sample origin (>3.0 logCFU/g). The bacterial isolates were grown on Mueller–Hinton agar (Becton Dickinson) at 37°C for 20 h under microaerophilic conditions using the AnaeroPack-MicroAero system, followed by centrifugation at 4,000× g for 5 min. Accordingly, we extracted DNA from the bacterial pellets using the Maxwell Blood DNA Kit (Promega, Madison, WI, USA). We measured the concentration and quality of the extracted DNA on a TapeStation 4,150 system (Agilent Technologies, Santa Clara, CA, USA). The samples were stored at −80°C until further use.

After external lysis, DNA was isolated semi-automatically with the Maxwell®16 robotic system and the Maxwell® Blood DNA Kit (Promega, WI, USA), according to the manufacturer’s instructions. The amount of human DNA per sample was determined using the Quantifiler® Trio DNA Quantification Kit (Thermo Fisher Scientific) and a real-time PCR instrument 7500 (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Samples with DNA amounts of less than 0.5 pg/µl were not submitted for subsequent analysis. PCR was conducted for samples with DNA amounts of ≥ 0.5 pg/µl using the PowerPlex® ESX 17 Fast System (Promega, WI, USA) on a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA) in a volume of 12.5 µl (in-house validation). A maximum DNA amount of 300 pg was set per run. Samples ranging from 100 to 300 pg of DNA went through 30 PCR cycles. All samples that contained less than 100 pg of DNA went through 32 PCR cycles. Single DNA fragment analysis was carried out per sample on a 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA) with the GeneMapper® ID-X Software v1.4 (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Positive and negative controls were included at each step.

Three representative chicken cecum samples were selected at each time point, to exclude the samples with maximum and minimum bacterial counts, and subjected to 16S rRNA sequencing analysis. Aliquots of the BPW suspensions (1 ml) were centrifuged at 21,500 × g for 10 min at 4°C. The pellets were then resuspended in 400 μl of homogenization solution containing 2 μl of proteinase K (Promega, Madison, WI, USA). After incubation at 37°C for 10 min, the samples were vortexed for 5 min with Zirconia beads (ZircoPrep Mini; Nippon Genetics, Tokyo, Japan) on a Disruptor Genie instrument (Scientific Industries, Bohemia, NY, USA). After centrifugation at 11,000 × g for 5 min, 100 μl of each supernatant were transferred into 300 μl of lysis buffer (Promega). DNA extraction was then carried out using a Maxwell Blood DNA kit in a Maxwell RSC instrument (Promega). The concentration and quality of the extracted DNA were measured on a Tape Station 4150 system (Agilent Technologies, Santa Clara, CA, USA), and the samples were stored at −80°C until use.