Sybr qpcr mix reagent kit

Real-time PCR was performed in triplicate to determine the viral load in the heart, liver, spleen, intestine, lungs, and bronchus samples that had been collected, using the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) with SYBR green fluorescence detection. Total RNA samples were extracted from the aforementioned tissues using TRIzol (Gibco) reagent according to the manufacturer’s instructions. Total RNA was reverse transcribed into complementary DNA (cDNA) using Moloney murine leukemia virus (M-MLV), reverse transcriptase, and Oligo(dT)₁₈ Primers (Takara, Dalian, China). The cDNA was prepared for real-time qRT-PCR using a SYBR^® qPCR Mix Reagent Kit (Takara). Real-time quantification PCR was performed to determine the absolute copy numbers of CDV in each tissue. A standard curve was generated by plotting the threshold values against the serially diluted plasmid DNA encoding the CDV H protein fragment. The primer pair, H5 and H6, was based on inserted gene H. A negative control reaction, lacking template DNA, was also as performed with using the above protocol.

Real-Time quantitative RT-PCR (qRT-PCR) was used to determine the viral load in intestinal tissues using the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) with SYBR green fluorescent dye. Prior to the assay, a standard curve was established for the absolute quantification of the virus. A standard plasmid with an initial concentration of 1 × 10¹⁰ copies/μL was subjected to a tenfold dilution, and each dilution was repeated five times for qRT-PCR. Using the logarithm of the dilution of the plasmid standard as the x-axis and the corresponding Ct value (cycle threshold) as the y-axis, a quantitative standard curve corresponding to the plasmid copy number and Ct value was constructed. Total RNA was extracted from 0.1 g of intestine samples and reverse-transcribed into complementary DNA (cDNA) according to the manufacturer’s instructions. The cDNA was diluted to the same level and prepared for qRT-PCR using a SYBR® qPCR Mix Reagent Kit (Takara) to test the PEDV copy number in the intestine samples.