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J Invertebr Pathol. 2019 Jun;164:59-65. doi: 10.1016/j.jip.2019.04.004. Epub 2019 Apr 27.

Development of a nucleic acid lateral flow strip for rapid, visual detection of Nosema bombycis in silkworm eggs.

Author information

1
State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, PR China; Chongqing Key Laboratory of Microsporidia Infection and Control, Southwest University, Beibei, Chongqing 400715, PR China; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, College of Biotechnology, Southwest University, Chongqing 400715, PR China.
2
State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, PR China; Chongqing Key Laboratory of Microsporidia Infection and Control, Southwest University, Beibei, Chongqing 400715, PR China; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, College of Biotechnology, Southwest University, Chongqing 400715, PR China; Laboratory of Animal Biology, Chongqing Normal University, Chongqing 400047, PR China. Electronic address: zyzhou@swu.edu.cn.
3
State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, PR China; Chongqing Key Laboratory of Microsporidia Infection and Control, Southwest University, Beibei, Chongqing 400715, PR China; Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, College of Biotechnology, Southwest University, Chongqing 400715, PR China. Electronic address: gqpan@swu.edu.cn.

Abstract

Nosema bombycis, the pathogen of silkworm pébrine, causes enormous economic losses to sericulture. As such, quarantine of commercial silkworm eggs represents an important safeguard to the silkworm industry. Here, we established a user-friendly detection system based on a nucleic acid lateral flow strip (NAFLS) that combines polymerase chain reaction (PCR) and a colloidal gold strip. PCR primers were designed based on the sequence of LSU rDNA of N. bombycis and has favourable specificity for common microsporidian isolates in silkworms. The forward and reverse primers were labeled on the 5' end with biotin and carboxyfluorescein (FAM), respectively. Genomic DNA was extracted from egg samples and was used as a template for PCR, followed by subsequent detection by NALFS. The detection limit of purified N. bombycis genomic DNA was 1 pg, 100× more sensitive than that of agarose gel electrophoresis (AGE). Furthermore, the sensitivity of detection of simulated "infected" silkworm eggs was 10-100× higher than that of AGE. NALFS detected infection in 27 of 29 samples of silkworm eggs oviposited by female moths infected in lab; ≥2% infected eggs per batch are detected as positive, while ≥40% infected eggs per batch are required for detection by AGE. Collectively, NALFS is easy to use and has great potential for widespread use in the detection of N. bombycis in silkworm egg production.

KEYWORDS:

Detection; Microsporidia; Nosema bombycis; Nucleic acid lateral flow strip; Silkworm eggs

PMID:
31039370
DOI:
10.1016/j.jip.2019.04.004

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