## 4 Discussion and Conclusions
(2018) [41] developed a real-time triplex PCR for the qualitative detection of European and American folulbrood in honeybee using primers and a probe designed on the sequence of the tnp60 gene of _P. larvae_. [PERSON] et al. (2021) [44] described a novel real-time PCR assay based on the use of a TaqMan(r) probe detecting the chromosomal metalloproteinase gene, a highly conserved single-copy target, for the presence and reliable assessment of _P. larvae_ spores in live samples. The authors used for the first time digital-PCR technology for absolute quantification of _P. larvae_ DNA present in the samples, even in minimal quantities. Digital-PCR technology has the advantage of using the same detection chemistry as qPCR. It allows absolute quantification, without the need for a standard curve. Although digital-PCR is a very promising technology, its use is not yet widespread on a large scale in diagnostic laboratories, like traditional real-time PCR machines. Here, we describe the use of a new TaqMan(r) probe-based real-time PCR assay for the detection and quantification of the bacterial spores. To our knowledge, this is the first description of a TaqMan(r) assay targeting the multiple-copy chromosomal and highly specific 165 rRNA gene of _P. larvae_[2,47]. The choice of the 165 rRNA gene as a target for this new TaqMan(r) probe-based real-time PCR assay was suggested by the proven robustness of this gene for the detection of _P. larvae_, as previously described [2,29,30]. Furthermore, the presence in multiple-copies (eight copies) of 165 rRNA gene target in the genome of _P. larvae_ increases the possibility for the detection of the pathogen in the samples under examination. This aspect is useful and to keep in mind for the development of _P. larvae_ diagnostic tests, since a few spores (<10) are enough to cause the arising of the disease. The new probe-based qPCR method, described in the present work, has been validated for the detection and quantification of the bacterial spores in powdered sugar samples from colonies with different histories of AFB's presence, but it could also be used for the examination of other types of specimens. The examination of a powdered sugar sample by qPCR can be an effective and useful way for the nondestructive quantitative evaluation of _P. larvae_ infection in honeybee colonies, as has been previously described [28]. The results obtained here showed that a significant difference was found between the culture and the qPCR used in favor of the second, which also found a higher level of infection than the culture method did. This is beneficial for the rapid detection of hives infected with _P. larvae_, and it is important for controlling the spread and reducing the impact of AFB. In addition, the results obtained were closely concordant with the clinical-epidemiological conditions of the colonies. Futures research directions will be focused on to the improvement of qPCR for absolute quantification of _P. larvae_ spores. A synthetic _P. larvae_ DNA standard could be designed and produced as recombinant plasmid, containing the _P. larvae_ target region, according to the available 165 rRNA gene sequences described (Table S2). The synthetic standard will be calibrated using digital-PCR, as previously descried [44], for a reliable and more accurate quantification of _P. larvae_ spores in powdered sugar samples, honey, and other live specimens. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12199895/s1](https://www.mdpi.com/article/10.3390/app12199895/s1)]([https://www.mdpi.com/article/10.3390/app12199895/s1](https://www.mdpi.com/article/10.3390/app12199895/s1)), Table S1: Primers and probe of the TaqMan(r)Real-Time PCR used in the present work; Table S2: Sequences of _P. larvae_ 165 rRNA gene from GenBank; Table S3: 165 rRNA gene sequences of Bacterial contaminants from GenBank; Table S4: Details of the results obtained by both methods. Conceptualization, S.B., E.C. ([PERSON]) and E.C. ([PERSON]); methodology, S.B. and E.C.