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Early and simultaneous detection of Nosema bombycis (Microsporidia: Nosematidae), nucleopolyhedrovirus (Baculoviridae), and densovirus (Parvoviridae) by multiplex real-time polymerase chain reaction in Bombyx mori (Lepidoptera: Bombycidae)

Published online by Cambridge University Press:  07 February 2017

Shan Wu ,
Yong-Qiang He ,
Xing-Meng Lu ,
Xiao-Feng Zhang ,
Jiang-Bing Shuai ,
Hui-Zhen Yu ,
Chen-Chen Jin  and
Hong-Fei Mo
Shan Wu
Affiliation:
Zhejing Academy of Science and Technology for Inspection and Quarantine, 126 Fuchun Road, Hangzhou 310016, P.R. China
Yong-Qiang He*
Affiliation:
Zhejing Academy of Science and Technology for Inspection and Quarantine, 126 Fuchun Road, Hangzhou 310016, P.R. China
Xing-Meng Lu
Affiliation:
Institute of Sericulture and Apiculture, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, P.R. China
Xiao-Feng Zhang
Affiliation:
Zhejing Academy of Science and Technology for Inspection and Quarantine, 126 Fuchun Road, Hangzhou 310016, P.R. China
Jiang-Bing Shuai
Affiliation:
Zhejing Academy of Science and Technology for Inspection and Quarantine, 126 Fuchun Road, Hangzhou 310016, P.R. China
Hui-Zhen Yu
Affiliation:
Zhejing Academy of Science and Technology for Inspection and Quarantine, 126 Fuchun Road, Hangzhou 310016, P.R. China
Chen-Chen Jin
Affiliation:
Zhejing Academy of Science and Technology for Inspection and Quarantine, 126 Fuchun Road, Hangzhou 310016, P.R. China
Hong-Fei Mo
Affiliation:
Zhejing Academy of Science and Technology for Inspection and Quarantine, 126 Fuchun Road, Hangzhou 310016, P.R. China
*
1Corresponding author (e-mail: [email protected])
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Abstract

An effective multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of three major pathogens, Nosema bombycis Nägeli (Microsporidia: Nosematidae), Bombyx mori nucleopolyhedrovirus (Baculoviridae: genus Alphabaculovirus) (NPV), and Bombyx mori densovirus (Parvoviridae: genus Iteravirus) (DNV), in silkworms (Bombyx mori (Linnaeus); Lepidoptera: Bombycidae) was developed in this study. Polymerase chain reaction and real-time PCR tests and basic local alignment search tool searches revealed that the primers and probes used in this study had high specificities for their target species. The ability of each primer/probe set to detect pure pathogen DNA was determined using a plasmid dilution panel, in which under optimal conditions the multiplex real-time PCR assay showed high efficiency in the detection of three mixed target plasmids with a detection limit of 8.5×103 copies for N. bombycis and Bombyx mori NPV (BmNPV) and 8.5×104 copies for Bombyx mori DNV (BmDNV). When the ability to detect these three pathogens was examined in artificially inoculated silkworms, our method presented a number of advantages over traditional microscopy, including specificity, sensitivity, and high-throughput capabilities. Under the optimal volume ratio for the three primer/probe sets (3:2:2=N. bombycis:BmNPV:BmDNV), the multiplex real-time PCR assay showed early detection of BmNPV and BmDNV by day 1 post inoculation using DNA templates of the three pathogens in various combinations from individually infected silkworms; the early detection of N. bombycis was possible by day 3 post inoculation using the DNA isolated from the midgut of N. bombycis-infected silkworms.

Type
Techniques
Information
The Canadian Entomologist , Volume 149 , Issue 2 , April 2017 , pp. 265 - 275
Copyright
© Entomological Society of Canada 2017 

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Footnotes

Subject editor: Susan Bjornson

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