8.3.2. Quantitative PCR, qPCR
Procedure (after Budge et al., 2010):
- Genomic DNA is amplified in a 25 µl
- 1 x buffer A (Applied Biosystems),
- 0.025 U/μl AmpliTaq Gold,
- 0.2 mM each dNTP,
- 5.5 mM MgCl2,
- 300 nM of each primer,
- 100 nM probe,
- 10 μl of nucleic acid extract.
- PCR reactions are carried out in duplicate
or triplicate wells and plates cycled using generic system conditions:
- 95ºC for 10 min,
- 40 cycles of 60ºC for 1 min,
- 95ºC for 15 sec.
- Quantification of M. plutonius in each sample can be achieved using the standard curve method (Anon, 1997) with assay EFBFor/EFBRev2/EFBProbe (M. plutonius 16S; Budge et al., 2010; Table 2) as the target and assay AJ307465-955F/1016R/975T (A. mellifera 18S; Budge et al., 2010) as the reference assay.
- As fluorescence increases in the presence of the target, the change in fluorescence (DRn) enters an exponential phase. The quantification cycle (Cq) is defined as midway through the exponential phase of this amplification curve (Bustin et al., 2009). It is often required to manually move the threshold of measurement manually to intercept midway through the exponential phase of the amplification curve and obtain an appropriate Cq.
- To account for variation in extraction efficiency between samples, the result can be expressed as a ratio of the number of M. plutonius and A. mellifera cells.