# Nosemosis in Honeybees: A Review Guide on Biology and Diagnostic Methods
## 5 Diagnostic Methods
### Molecular Diagnostics  
#### 5.3.1 Endpoint PCR  
As conventional microscopy is rather imperfect in distinguishing _Nosema_ species, and electron microscopy requires a lot of work, a polymerase chain reaction (PCR) method was developed as a very specific tool to distinguish between the species (Table 2). Later on, a real-time PCR (qPCR) method was developed as a quantitative molecular tool. In research, mainly multiplex-PCR, PCR-RFLP, and qPCR are used. The first attempt of PCR reaction to distinguish _Nosema_ species was in 1996, and the product of this reaction was sequenced and placed in Gene Bank (accession number U26533) [68, 32]. The BEEBOOK is a practical manual of standard methods for the honey bee. The goal of this project is to standardize honey bee research methods around the world so that test results from different laboratories are comparable and reliable. The fact that all the methods described in the BEEBOOK have been validated by leading honeybee experts is the reason we used it in the review. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Method** & **Result** & **Equipment Required** & **Reagents** & **Primers (If Applicable)** & **Advantages** & **Disadvantages** & **References** \\ \hline \multirow{4}{*}{\begin{tabular}{c} Immuno- \\ diffusion \\ \end{tabular} } & \multirow{4}{*}{Qualitative} & \multirow{4}{*}{Microplates} & \multirow{4}{*}{\begin{tabular}{c} ELISA kit for rabbit \\ primary antibodies \\ \end{tabular} } & \multirow{4}{*}{n/a} & \multirow{4}{*}{\begin{tabular}{c} Fast, simple, \\ low cost \\ \end{tabular} } & \multirow{4}{*}{Only detects _N. ceranae_} & \multirow{4}{*}{[59, 60, 61]} \\ \cline{3-4}  & & & & TMB & & \\ \cline{3-4}  & & & & Stop solution & & \\ \hline \multirow{4}{*}{Fluorescence} & \multirow{4}{*}{Qualitative} & \multirow{4}{*}{\begin{tabular}{c} Fluorescence \\ microscope \\ \end{tabular} } & \multirow{4}{*}{\begin{tabular}{c} Pipes \\ \end{tabular} } & \multirow{4}{*}{n/a} & \multirow{4}{*}{\begin{tabular}{c} Reliable, \\ distinguishes \\ dead/living \\ \end{tabular} } & \multirow{4}{*}{\begin{tabular}{c} No species \\ termination \\ \end{tabular} } & \multirow{4}{*}{[62, 63, 64]} \\ \cline{3-4}  & & & DAPI & & & \\ \hline \multirow{4}{*}{\begin{tabular}{c} Chromato- \\ graphy \\ \end{tabular} } & \multirow{4}{*}{Qualitative} & \multirow{4}{*}{\begin{tabular}{c} Dryer (Speedvac) \\ \end{tabular} } & \multirow{4}{*}{\begin{tabular}{c} Methanol-ethyl \\ acetate \\ \end{tabular} } & \multirow{4}{*}{n/a} & \multirow{4}{*}{\begin{tabular}{c} Reliable, \\ useful in \\ research \\ \end{tabular} } & \multirow{4}{*}{\
\begin{tabular}{c} Expensive, \\ equipment \\ needed \\ \end{tabular} } & \multirow{4}{*}{[65, 66]} \\ \cline{3-4}  & & Gas & &
\begin{tabular}{c} Methoxylamine \\ hydrochloride \\ \end{tabular} & & & & \\ \cline{3-4}  & & & solution & & & \\ \cline{3-4}  & & MSTFA & & & & \\ \hline \hline \end{tabular}  
* \({}^{1}\) Preparation of DNA the same as for end-point PCR. \({}^{2}\) PCR amplification the same as for end-point PCR.  
\end{table} 
Table 2: _Cont._The first step in all polymerase chain reaction methods is DNA extraction from insect tissues, the best from all bees in a sample (homogenate). [PERSON] et al., in BEEBOOK [44], recommended using 30 bees and mixing with 15 mL double-distilled water (ddH\({}_{2}\)O) (0.5 mL/bee) in a one-to-one ratio. 100 \(\upmu\)L of the homogenate is centrifuged for 3 min at 16,100 g. The obtained pellet contains microsporidia and other cellular material. The pellet should then be frozen in liquid nitrogen and crushed to open spore walls. Alternatively, another method can be used. It is based on spore germination rather than mechanically breaking their walls, but it takes significantly more time [5]. Usually, to carry on DNA extraction commercial kits, such as DNeasy(r) Plant Mini kit (Qiagen) [44] or High Pure PCR template preparation kit (Roche Diagnostic) [47; 5], are used.