4.7. Quantitative RT-PCR for honey bee and pathogen targets

Detection of specific PCR products can also be made continuously as the PCR proceeds (i.e. in ‘real time’). In this case the cycle number at which the accumulated PCR products reach a fluorescence detection threshold, read after each cycle by laser optics, can be very accurately related to the initial amount of target in the reaction, through the use of exponential algorithms and internal and external quantitation standards (Bustin et al., 2009; 2010). This is the basis for real-time quantitative PCR (qPCR). The great advantage of real-time qPCR, apart from the accurate quantitation of the initial amount of target DNA in the reaction, is that the diagnostic threshold for qualitative detection can be set after the reactions have taken place, or at a number of different levels, from the same data set. This is useful if different diagnostic sensitivities are required for different experimental or reporting purposes, or for quality control management purposes.

There are numerous methods for qPCR in the literature, and this approach has been used for measuring gene activity in honey bees and all of their major parasites and microbial associates. The primary difference in those cases will come in the specific primers used for amplification and in some cases in changes to the chemistry or thermal conditions. One main decision point is between using SYBR green or another non-specific fluorescent marker that measures (amplified) DNA non-discriminately versus reporters that target specific amplified products directly such as TaqMan probes (Applied Biosystems; e.g., Chen et al., 2004). There is considerable debate over the merits of each approach. Assays using Taqman® chemistry and other internal probe methodologies are inherently more specific than those using Sybr chemistry, due to the additional match required in the probe sequence. Therefore, Taqman® assays are more prone to Type II errors (false negative), where a negative result is returned despite the sample being positive (perhaps due to slight modification in the probe region within the sample). Sybr-based assays are more likely to return a Type I error (false positive), due to difficulties in distinguishing between low positive signal at the threshold of detection and non-specific binding. The errors for both methods can be minimized after careful preparatory work.

4.7.5. Multiplex RT-(q)PCR