Pcr purification combo kit

For each species identified in the PCR-RFLP, such as Sarcocystis spp., T. gondii and Neospora spp. a duplicate sample was sent for nucleotide sequencing, from the amplified DNA products of the 18S region. The amplicons were purified using the commercial PureLink ® Quick Gel Extraction Kit and PCR Purification Combo Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's recommendations. Gene sequencing was performed by a specialized company ACTGene - Sequencing Service, Brazil. The samples were sequenced in duplicate, the results obtained were analyzed using the Staden Package software and the similarity with sequences deposited in GenBank determined using the BLAST - Basic Local Alignment Search Tool (NCBI, 2023).

Suspensions of cells showing CPE were processed for RNA extraction with the High Pure Viral RNA Kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. A reverse transcription-polymerase chain reaction (RT-PCR) method was used for NDiV-specific gene (RdRp) detection with the Prime Script RT-PCR Kit (TaKaRa Biotechnology, Dalian, China). The primer sequences were: forward primer PRd1 5′-TCACACAACCGCATGCTACGC-3′ and reverse primer PRd2 5′-GTGGTCACGGCCAGTGGTGT-3′ (targeting a 2394 bp fragment). The polymerase chain reaction (PCR) products were visualized on 2% agarose gels and extracted from the gels using the PCR Purification Combo Kit (Invitrogen). The PCR fragments were sequenced and compared to known RdRp gene sequences of NDiV in the National Center for Biotechnology Information (NCBI) GenBank database by BLASTN.