Path id multiplex one step rt pcr kit

Rectal swabs, serum and various tissues were tested by a PDCoV M-gene based real-time RT-PCR including viral standards with known infectivity titers for quantification. Briefly, viral RNA was extracted from rectal swabs, serum, and 10% tissue homogenates as previously described (Chen et al., 2014 (link)). Five µl of each template was used in PCR setup in a 25 µl total reaction using Path-ID™ Multiplex One-Step RT-PCR Kit (Life Technologies, Carlsbad, CA) and primers (forward primer 5′-CGACCACATGGCTCCAATTC-3′, reverse primer 5′-CAGCTCTTGCCCATGTAGCTT-3′) and probe (5′-CACACCAGTCGTTAAGCATGGCAAG C-3′). The probe was labeled using the FAM/ZEN/3′Iowa Black detector (Integrated DNA Technologies, Coralville, IA). The RT-PCR was run on an ABI 7500 Fast instrument (Life technologies, Carlsbad, CA) with the following conditions: 1 cycle of 48 °C for 10 min, 1 cycle of 95 °C for 10 min, and 45 cycles of 95 °C for 15 s and 60 °C for 45 s. A PDCoV isolate with known infectivity titer was 10-fold serially diluted for generating a standard curve in each PCR plate. Virus concentration (TCID₅₀/ml) in tested samples was calculated based on the standard curve. The mean cycle threshold (Ct) values were calculated based on PCR positive samples, and the mean virus titers were calculated based on all pigs within the group.

The eluted RNA, primers, and probe were mixed with commercial reagents (Path-ID^® Multiplex One-Step RT-PCR kit, Life Technologies) and the RT-PCR reactions were conducted on an ABI 7500 Fast instrument (Life Technologies) as follows: 48 °C for 10 min, 95 °C for 10 min, 95 °C for 15 s (45 cycles), and 60 °C for 45 s. The results were analyzed using an automatic baseline setting with a threshold at 0.1. Quantification cycle (Cq) values <35 were considered positive for the PDCoV and Cq values <40 were considered positive for the PEDV and TGEV. Data were reported as “adjusted Cq”: