## 1 Introduction
larvae_ ERIC II AFB cases clinical diagnosis is more laborious, with the risk of missing them, especially in the colonies with asymptomatic infections. AFB generally occurs in clinical form when a certain level of _P. larvae_ spore contamination is reached in honeybee colonies [20]. A relatively large number of spores can be present over several seasons in some colonies without evidence of clinical signs of the disease [21; 22; 23; 24]. Subclinical infections can lead to relapses of clinical forms in apiaries and promote the horizontal spread of AFB from one hive to another [25]. The presence of _P. larvae_ spores and their quantification in hive materials allows for the identification of the contamination levels of the colonies, mostly in those with atypical symptoms or with asymptomatic infections; in these cases, it is decisive for reducing the impact of AFB. The search for the _P. larvae_ spores load in adult bees and hive materials such as honey, wax, and wax debris is described in standard methods, based on microbiological and biomolecular techniques [2; 26; 27]. Recently, a powdered sugar examination was described by [PERSON] et al. (2022) [28] as an easy-to-use, nondestructive, and practical material for the assessment of _P. larvae_ infection levels in honeybee colonies. Many molecular methods based on conventional PCR [29; 30; 31; 32; 33; 34; 35; 36] or on real-time PCR [37; 38; 39; 40; 41; 42; 43] have been previously described for the presence and/or quantification of _P. larvae_ infection levels in different hive materials. Quantitative PCR assay (qPCR) is a time- and cost-effective choice with respective to the quantification of _P. larvae_ spores and overcomes the limitations of the traditional methods. Among qPCR methods described in the literature, to our knowledge, only three have been used for the assessment of _P. larvae_ spores in hive debris and/or honey samples. Two assays are designed to target the 16S rRNA gene and use SyBr technology, which suffers from important disadvantages [39; 42]. The 16S rRNA gene is a useful target for molecular diagnostic purposes for two main reasons: (a) for being highly conserved among bacterial pathogens, including _P. larvae_ strains, although single-nucleotide polymorphisms are known in the sequences of _P. larvae_ of different geographic origin and/or genotype; and (b) for being present in eight copies per genome of _P. larvae_, allowing for increased sensitivity of diagnostic assays. These features are considered for the development of diagnostic assays for many bacterial pathogens, including the _P. larvae_. The third assay is a new TaqMan(r) probe-based real-time qPCR protocol whose target is the single-copy chromosomal metalloproteinase gene. It was developed for the detection and quantification of spores in hive debris and honey samples [44]. The TaqMan(r) probe-based real-time qPCR protocol overcomes the limitation of the qPCR based on the use of SyBr technology, not requiring post-PCR processing step, such as the analysis of the melting curves [45]. TaqMan(r) probes, also known as \"fluorogenic 5' nuclease chemistry\", are widely used in many diagnostic real-time PCR assays. Specificity is much higher with the use of specific probes besides primers [45]. The availability of these fluorogenic probes enabled the development of a real-time method for detecting only specific amplification products. Specific hybridization between probe and target is required to generate fluorescent signal. The TaqMan(r) probe can be labeled with many different fluorophores useful in multiplex assays: probes can be labeled with distinguishable reporter dyes, which allows amplification and detection of two or more distinct sequences in one reaction tube. Post-PCR processing is eliminated, which reduces assay labor and material costs. The aim of the present work was to describe a new quantitative TaqMan(r) probe-based real-time PCR (qPCR) assay, targeting the 16S rRNA gene of _P. larvae_, in comparison to the culture. The two methods were employed for the assessment of _P.