Infinite 200 nanoquant

The limit of detection (LOD) of the multiplex RT-qPCR assays was determined by testing six replicates of 10-fold serial dilutions of RVA-, RVB-, RVC- and RVH-positive samples at a known genomic copy titer, starting with an initial concentration of 10⁴ to a final concentration of 10 gene copies/reaction. The LOD was calculated as the lowest RNA concentration that could be detected in all six replicates. The specificity of the two assays was determined by a RT-qPCR analysis of a panel of 15 isolates of porcine viruses and bacteria that are causative of enteritis in swine. VP6 RVA-, RVB-, RVC- and RVH-specific gene fragments were cloned, in vitro transcribed and quantified by a spectrophotometer (Infinite^® 200 NanoQuant, Tecan, Männedorf, Switzerland). Serial dilutions of such transcripts (10⁷ to 10³ genomic copies for reaction) were run on the RVA-B and RVC-H RT-qPCR assays to generate a standard curve and determine the efficiency of the reactions and the linear correlation index (R²).

To evaluate the diagnostic performance of the PCR, nested PCR, and one-tube nested real-time PCR methods, a total of 127 field samples, including 37 lung tissues, 30 blood samples, 30 serums, and 30 feces samples, were provided by the Optipharm Animal Disease Diagnostic Center, which was commissioned from January to December 2019. In addition, 10 organs (lung, liver, pancreas, spleen, kidney, brain, heart, small intestine, nasal concha, and tonsil) of six pigs were analyzed to determine the infection rate of PCMV for each organ by one-tube nested real-time PCR assay. According to the manufacturer's recommendation, DNA was extracted from 200 μL of serum or 20 mg of organ tissue homogenate using a commercial automated system (Miracle-AutoXT Automated Nucleic Acid Extraction System, Intronbio, Seongnam, Republic of Korea). To avoid cross-contamination, all samples were individually processed and stored at −20°C. The content and purity of the extracted DNA was analyzed by measuring the absorbance at 260 and 280 nm by a spectrophotometer (Infinite 200 NanoQuant; Tecan, Switzerland).

Liver tissue was homogenized on a Tissuelyzer II (Qiagen, Hilden, Germany) and suspended in Ribozol^™ (Amresco Inc., OH, USA) phenol reagent. Chloroform was added and the samples centrifuged, thus separating the proteinaceous organic phase and the aqueous nucleic acids containing interphase. The aqueous phase was mixed with an equivalent volume isopropanol. Finally, samples were washed three times in 70% ethanol/diethylpyrocarbonate (DEPC)-water, dried over laminar flow and the RNA resuspended in DEPC-water.
The final RNA concentrations were determined using an Infinite^® 200 Nanoquant (Tecan, Männedorf, Switzerland) and normalized to 1000 ng/μL. We synthesized complementary deoxyribonucleic acids (cDNA) with SuperScript^® Reverse Transcriptase (Thermo Fischer) on a MyCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol.

To evaluate the diagnostic performance of the multiplex real-time PCR and PCR-REBA methods, a total of 180 samples suspected to be infected with PCVAD including 109 tissues and 71 bloods were provided by the Optipharm Animal Disease Diagnostic Center, which was commissioned from January to December, 2019. DNA was extracted from 200 μL of serum or 20 mg of organ tissue homogenate using a commercial automated system (Miracle-AutoXT Automated Nucleic Acid Extraction System, intronbio, Seongnam, Republic of Korea) according to the manufacturer's recommendations. To avoid cross contamination, all the samples were processed individually and stored at −20°C. The content and purity of the extracted DNA were assayed by measuring absorbance at 260 and 280 nm using an Infinite 200 NanoQuant (Tecan, Switzerland) spectrophotometer.

Genomic DNA was extracted from whole blood using QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA concentration and purity were determined using Infinite® 200 NanoQuant (Tecan, Switzerland). DNA pellet was dissolved in 100 μL of TE buffer (approximately 20 ng/μL DNA concentration) and was stored at −20 °C until use.
The multiplex PCR method was developed in accordance with QIAGEN^® Multiplex PCR Handbook (20 ) using QIAGEN^® Multiplex PCR Plus Kit. A uniplex PCR method was first conducted to determine the specificity, functionality and annealing temperature of each primer set.

GSH was determined using a quantification kit for oxidized and reduced glutathione (Sigma-Aldrich, Darmstadt, Germany) according to manufacturer´s instructions. Activity of enzymes involved in the pentose phosphate pathway and oxidative defense: G6PD, GLD, and GPx was determined according to the methods recommended by the International Committee for Standardization in Haematology [60 (link)], as we previously described [61 (link),62 (link)]. Leukocyte- and platelet-free erythrocyte lysates were used, and the absorbance was measured by spectrophotometer (Infinite 200 Nanoquant; Tecan, Männedorf, Switzerland). All chemicals and purified enzymes were purchased from Sigma-Aldrich (Darmstadt, Germany).