Taq dna polymerase

There were outbreaks of suspected ND in a racing pigeon loft in Gansu Province, China, on September 2020. The brains, livers and kidneys of dead pigeons were collected and homogenized. Then, the samples were centrifuged after freeze–thawing 3 times to obtain the supernatants. BHK-21 cells in 6-well plates were infected with the supernatants to observe cytopathic effects (CPEs), including syncytium formation. Total RNA was extracted from the sample supernatants of infected BHK-21 cells and allantoic fluids of 9-day-old embryonated chicken eggs by TRI Gene Reagent (GenStar, China) according to the manufacturer's instructions. The DNA was synthesized using a StarScript II First-strand cDNA Synthesis Kit (GenStar, China). The detection genes were amplified from the cDNA by PCR utilizing Taq DNA Polymerase (GenStar, China), and the primers were designed according to the conserved sequence of PPMV-1. The sequence of the forward primer was 5’-ATGGGCYCCAGAYCTTCTAC-3’, and the reverse primer was 5’-CTGCCACTGCTAGTTGTGATAATCC-3’ (designed in the current study). The program was as follows: 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, an extension at 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR products were visualized by 1% agarose gel electrophoresis and sequenced.

Fresh fungal mycelia grown on PDA for 7 d at 25 °C were scraped from the colony margin and used for genomic DNA extraction using a modified CTAB protocol as described in Guo et al. (2000) . PCR amplification and sequencing of the large subunit (LSU) rDNA using the primer pair LR0R/LR5 and the 5.8S nuclear ribosomal gene with the two flanking transcribed spacers (ITS) using primer pair ITS1/ITS4 was performed (Vilgalys & Hester 1990 (link), White et al. 1990 ). Part of the translation elongation factor 1-alpha (TEF1-α) was amplified and sequenced using primer pair EF1-728F (Carbone & Kohn 1999 ) and EF-2 (O’Donnell et al. 1998 ). Bt-2a and Bt-2b (Glass & Donaldson 1995 (link)) were used for the Beta-tubulin fragment (TUB2). PCR was performed in a 25 μL reaction containing 18.95 μL double distilled water, 2.5 μL 10 × PCR buffer, 0.3 μL dNTP mix (2.5 mM), 1 μL per primer (10 mM), 1 μL DNA template, 0.25 μL Taq DNA polymerase (Genstar). Amplification conditions for ITS, LSU and TEF1-α followed Crous et al. (2013) (link) and for TUB2, Lee et al. (2004) . Purification and sequencing of PCR amplicons were carried out at the SinoGenoMax Company, Beijing. DNA sequences were generated with upper surface and reverse primers to obtain consensus sequences analysed with MEGA v. 6.0 (Tamura et al. 2013 (link)).