Onestep rt pcr kit

The whole genome of the ALSV strain Harz Mountain was obtained by using next-generation sequencing (MW094149.1; MW094151.1; MW094148.1; MW094150.1). Based on these sequences of each segment, primers targeting the NS5-like gene were designed for the screening of the samples. The primers used for the detection of ALSV included the forward primer (5′-ATAATCCAGTACCTCCCAGCCG-3′) (nt position 1704) and the reverse primer (5′-CCCCGATGAAACCTGTCCTCTG-3′) (nt position 2039), which target a 334 bp region of the NS5 gene of ALSV. The samples were analyzed using the Qiagen One-Step RT-PCR kit according to the manufacturer’s instructions. Sanger sequencing of positive PCR products was conducted using the forward primer (Eurofins Scientific, Luxemburg). In addition, the positive pools were with primers targeting a 447 bp fragment of the capsid/membrane gene of ALSV using the forward primer (5′-GATGAGGCTAGGGACTTGTTCC-3′) (nt position 1201) and the reverse primer (5′-GTCAGCAGCATCCTAGCCACAT-3′) (nt position 1649) using and the Qiagen One-Step RT-PCR kit, and the positive samples were sequenced, as described above.
For the quantification of viral particles by Sybr RT-qPCR, the Luna^® Universal One-Step RT-qPCR Kit (New England Biolabs, Ipswich, MA, USA) was used with the NS5 primer pair. Therefore, a standard curve (10⁶–10³ RNA copies) based on the NS5-like gene was prepared.

RNA from zebrafish larvae or zebrafish tissues was isolated with TRI reagent (Sigma-Aldrich) RT-PCR was performed with the OneStep RT-PCR Kit (Qiagen).
For RT-PCR analysis of Oli-Neu cells, total RNA was purified from a fraction of the transfected cells with the RNeasy Mini Kit (Qiagen) and mRNA levels of β-actin, Dendra2 and G6pd were assessed by RT-PCR using the OneStep RT-PCR Kit (Qiagen).

Single cells were prepared using a protocol adapted from Kim et al. [28 ]. The cells were coated with CD14 Dynabeads and resuspended in PBS containing 2% BSA (~100 cells/ml). Under a microscope, single cells were aspirated with 0.5 μl medium and transferred into 3.5 μl resuspension buffer (5x RT buffer, OneStep RT PCR Kit, Qiagen). The reaction tubes were kept at -80°C until the next step. RT-PCR followed the protocol from Kim et al. [28 ] using the OneStep RT-PCR Kit (Qiagen) and specific reverse primers for IgM, IgG, Igκ and Igλ. Each reaction was then split into four aliquots and PCRs using specific forward and reverse primers for either IgM, IgG, Igκ or Igλ were performed following the protocols described above.

Our first approach to RVA detection used two established real-time RT-PCR protocols, allowing the detection of a wide range of mammalian RVAs (Pang et al. 2004 (link); Otto et al. 2015 (link)). In a second approach, primers were delineated from a set of available mammalian and avian RVA sequences for the VP1-encoding genome segment, along with a previously determined corresponding sequence of an RVA from a common shrew (GenBank no. MN307986). The resulting primers generating a 387 base-pair (bp) fragment are shown in Supplementary Data S2. Conventional RT-PCR was performed using the QIAGEN One Step RT-PCR Kit (Qiagen). The temperature profile included reverse transcription at 42°C for 30 min, enzyme activation at 95°C for 15 min, followed by forty cycles with denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and elongation at 74°C for 40 s, followed by a final elongation at 74°C for 5 min. PCR products were analysed on ethidium bromide-stained agarose gels.