184.108.40.206. External standard for viral target quantification
- Extract RNA (Qiagen RNeasy® Mini Kit and QiaShredder®, according to manufacturer´s protocol) of bees with an RNA target (in this example DWV).
- Generate an external standard by amplifying a DWV genomic fragment of 1520 bp via RT-PCR, using the primers Fstd (5´-GGACCATCCTTCCAGTCTACGAT-3´) and Bstd (5´-CTGTAGGTTGTGCTCCTGATGAAGA-3´) and the one-step RT-PCR kit from Qiagen.
- This fragment contains the 354 bp fragment, which can be amplified by the primer pair F1/B1 (Genersch, 2005), for quantification.
- Quantify the number of PCR-fragments via photometric analysis at 260 nm wavelength (Nanodrop, section 3.2.1).
- Prepare a dilution series from
the initial concentration through three orders (10-fold dilutions) of