title: Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae.
abstract: Pebrine disease is the only mandatory quarantine item in sericultural production due to its destructive consequences. So far, the mother moth microscopic examination method established by Pasteur (1870) remains the only detection method for screening for the causative agent Nosema bombycis (N. bombycis). Because pebrine is a horizontal and vertical transmission disease, it is better to inspect silkworm eggs and newly hatched larvae to investigate the infection rate, vertical transmission rate and spore load of the progenies. There is a rising demand for a more direct, effective and accurate detection approach in the sericultural industry. Here, we developed a molecular detection approach based on real-time quantitative PCR (qPCR) for pebrine inspection in single silkworm eggs and newly hatched larvae. Targeting the small-subunit rRNA gene of N. bombycis, this assay showed high sensitivity and reproducibility. Ten spores in a whole sample or 0.1 spore DNA (1 spore DNA represents the DNA content of one N. bombycis spore) in a reaction system was estimated as the detection limit of the isolation and real-time qPCR procedure. Silkworm egg tissues impact the detection sensitivity but are not significant in single silkworm egg detection. Of 400 samples produced by infected moths, 167 and 195 were scored positive by light microscopy and real-time qPCR analysis, respectively. With higher accuracy and the potential capability of high-throughput screening, this method is anticipated to be adaptable for pebrine inspection and surveillance in the sericultural industry. In addition, this method can be applied to ecology studies of N. bombycis-silkworm interactions due to its quantitative function.
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Zhangwuke
authors.initials: Z
authors.lastname: Fu
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xiangkang
authors.initials: X
authors.lastname: He
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Shunfeng
authors.initials: S
authors.lastname: Cai
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Han
authors.initials: H
authors.lastname: Liu
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xinyi
authors.initials: X
authors.lastname: He
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Mingqian
authors.initials: M
authors.lastname: Li
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China. Electronic address: xmlu@zju.edu.cn.
authors.forename: Xingmeng
authors.initials: X
authors.lastname: Lu
mesh_terms: D000818:Animals; D012831:Bombyx / Q000382:microbiology*; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D005260:Female; D020459:Genes, rRNA; D007218:Industrial Microbiology / Q000379:methods; D007814:Larva / Q000382:microbiology; D008297:Male; D016818:Nosema / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D013172:Spores, Fungal / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal
keywords: Light microscopy; Nosema bombycis; Pebrine inspection; Real-time qPCR; Transovarial transmission