Development of a Probe-Based quantitative PCR assay for detecting Nosema bombycis and its application in inspection...

Pébrine, a severe disease in sericulture, is caused by the microsporidium Nosema bombycis. As the pathogen can be transmitted through silkworm eggs, it is the only legally regulated quarantine target in silkworm egg production. Traditional detection methods rely on microscopic examination of female moths (female moth microscopy, FMM) and newly hatched larvae (Confirmatory microscopy), which are labor-intensive and prone to subjective interpretation. To address this, we developed a probe-based quantitative PCR (qPCR) method targeting the small-subunit ribosomal RNA (SSU rRNA) gene of N. bombycis. This method achieved a detection limit of 15 fg for N. bombycis genomic DNA and specifically identified microsporidia infecting silkworms, including N. bombycis and Vairimorpha necatrix BM, without cross-reactivity with other silkworm pathogens. With qPCR, N. bombycis can be detected in the midgut at 2 days post-infection (2 dpi), in the hemolymph at 3 dpi, and at 6 dpi in feces. The established qPCR was evaluated across 3616 samples of grandparent female moths, 2643 samples of parent female moths, and 1022 samples of first-generation hybrid (F₁) newly hatched larvae. Comparing results with traditional microscopic inspection, concordance rates for positive samples were 93.82 %, 96.48 %, and 91.18 %, respectively, demonstrating a high level of agreement between the two methods; qPCR was able to detect 43.51 %, 58.95 %, and 2.13 % of suspicious samples, respectively, despite microscopic negative results, underscoring the superior sensitivity of qPCR over microscopic examination. Furthermore, qPCR enabled the direct detection of N. bombycis in F₁ eggs, preventing the unnecessary elimination of 21.21 % of uninfected eggs that were misclassified by FMM. This highly sensitive and efficient method detects the pathogen 13-14 days earlier than traditional confirmatory microscopy in F₁ newly hatched larvae without incubation and natural mortality. In conclusion, the probe-based qPCR method developed in this study exhibits high specificity, sensitivity, and accuracy, confirming its suitability for the detection of N. bombycis in silkworm egg production systems. This work offers solid data basis for qPCR application in the quarantine and inspection of N. bombycis, paving the way for more efficient and reliable molecular detection in sericulture.