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Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae
Zhangwuke Fu1, Xiangkang He1, Shunfeng Cai1
1Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
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.
Insights
A new real-time quantitative PCR (qPCR) method detects pebrine disease in silkworm eggs and larvae. This molecular approach offers higher accuracy and high-throughput screening for Nosema bombycis (N. bombycis) surveillance in sericulture.
Area of Science:
- Sericulture
- Molecular Biology
- Parasitology
Background:
- Pebrine disease, caused by Nosema bombycis (N. bombycis), is a major threat to silk production.
- Current detection relies on outdated microscopic methods, necessitating more accurate and efficient diagnostics.
Purpose of the Study:
- To develop a novel molecular detection approach for N. bombycis in silkworm eggs and larvae.
- To establish a sensitive, reproducible, and high-throughput method for pebrine inspection.
Main Methods:
- Development of a real-time quantitative PCR (qPCR) assay targeting the N. bombycis small-subunit rRNA gene.
- Application of the qPCR assay for detecting N. bombycis in single silkworm eggs and newly hatched larvae.
Main Results:
- The qPCR assay demonstrated high sensitivity and reproducibility, with a detection limit estimated at 10 spores per sample.
- Compared to light microscopy (167/400 positive), qPCR identified more infected samples (195/400 positive) from infected moth progenies.
- Silkworm egg tissue presence had a minor impact on detection sensitivity in single-egg analysis.
Conclusions:
- The developed real-time qPCR method provides a more accurate and efficient alternative for pebrine inspection and surveillance in the sericultural industry.
- This quantitative assay is suitable for high-throughput screening and ecological studies of N. bombycis-silkworm interactions.
