4.10.2.3. External standard for viral target quantification

  1. Extract RNA (Qiagen RNeasy® Mini Kit and QiaShredder®, according to manufacturer´s protocol) of bees with an RNA target (in this example DWV).
  2. 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.
  3. This fragment contains the 354 bp fragment, which can be amplified by the primer pair F1/B1 (Genersch, 2005), for quantification.
  4. Quantify the number of PCR-fragments via photometric analysis at 260 nm wavelength (Nanodrop, section 3.2.1).
  5. Prepare a dilution series from the initial concentration through three orders (10-fold dilutions) of concentrated solutions.

This set of fixed dilutions will be used to ensure that PCR efficiency is maintained and to identify the precise predicted copy number for a particular Cq threshold.

 

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