Magmax pathogen rna dna kit

Two identical series of n = 13 oviduct homogenates, either negative or spiked with different loads of G1 or Y280 viruses, were analyzed in parallel with a territorial laboratory of the IZSVe (SCT1—Buttapietra, Verona). Nucleic acids were isolated with the MagMAX Pathogen RNA/DNA Kit (Applied Biosystems, Waltham, MA, USA) on a KingFisher Flex Processor (Thermo Fisher Scientific, Waltham, MA, USA) (sample volume 200 μL; protocol “low-cell-content samples”). For each sample aliquot, rRT-PCR was run in duplicate. Reproducibility was assessed also by participating in the OFFLU proficiency testing program 2021 for avian influenza A virus, H5 and H7 subtyping [63 ] organized by the Australian National Science Agency—Commonwealth Scientific and Industrial Research Organisation (CSIRO). Nucleic acids’s extraction was performed both with automatic and manual systems (i.e., MagMAX Pathogen RNA/DNA Kit, Applied Biosystems, Waltham, MA, USA and QIAamp Viral RNA Mini Kit, Qiagen, Hilden, Germany). Although H9 subtyping was not part of the assessable parameters of the exercise, the organization provided decoding for H9N2 samples included in the panel to allow self-assessment.

All experiments were conducted under biosafety level (BSL)-2 conditions. The swab-containing tubes were swirled, and the supernatants were collected after centrifugation. AIV RNAs were extracted using the MagMAX™ Pathogen RNA/DNA Kit (Applied Biosystems, Foster City, CA, USA) with the Magmax-96 Express instrument (Applied Biosystems). After extraction, the samples were screened for the presence of AIVs by real-time reverse transcription PCR (qRT-PCR) with primers and probes (WHO, 2009) specific to the matrix gene using a 7500 real-time PCR instrument (Applied Biosystems). The positive samples were transcribed into cDNA using the Uni12 primer (5ʹ-AGC AAA AGC AGG-3ʹ) and PrimeScript™ II 1st Strand cDNA synthesis kit (Takara, Japan). AIV subtypes were determined using primers specific to HA and NA genes [19 (link),22 (link)], and the eight segments of the H10–H12 AIV isolates were amplified using universal primers [23 (link)]. The PCR reactions consisted of 1 μL of cDNA, 1 μL each of forward and reverse primers, 12.5 μL of Taq HS Perfect Mix (Takara, Shiga, Japan) and 10.5 μL of RNAse-free water, with a final volume of 25 μL. All PCR products were sequenced by Sangon Biotech Co, Ltd (Shanghai, China) using a BigDye termination kit on an ABI 3730 sequence analyzer (Applied Biosystems, Foster City, CA, USA).

DNA was extracted from 200 µL of serum or 300 µL of OF samples, by using the MagMAX™ Pathogen RNA/DNA Kit (Applied Biosystems) following the manufacturer’s instructions. The DNA obtained was suspended in 90 µL of elution solution.
To quantify the PCV2 DNA in serum and OF samples, a real-time qPCR assay (LSI VetMAX™ Porcine Circovirus Type 2 Quantification, Life Technologies) was performed. Each extraction and qPCR plate included negative controls (diethylpyrocarbonate (DEPC)-treated water) and each sample reaction had an internal positive control (IPC) to monitor DNA extraction and amplification procedures. Viral concentrations were expressed as the mean log₁₀ PCV2 genome copies/mL. Area under the curve (AUC) of viral load in serum samples from 2 to 25 weeks of age was calculated according to the trapezoidal method as previously described [23 (link)].