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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
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: hyq@ziq.gov.cn)

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
Copyright
© Entomological Society of Canada 2017 

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Footnotes

Subject editor: Susan Bjornson

References

Aldras, A.M., Orenstein, J.M., Kotler, D.P., Shadduck, J.A., and Didier, E.S. 1994. Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies. Journal of Clinical Microbiology, 32: 608612.Google ScholarPubMed
Burgher-MacLellan, K.L., Williams, G.R., Shutler, D., MacKenzie, K., and Rogers, R.E.L. 2010. Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera . The Canadian Entomologist, 142: 271283.CrossRefGoogle Scholar
Cai, S.F., He, X.Y., He, X.K., Qiu, H.H., Li, G.C., He, Y.Q., et al. 2011. A protocol for fast and efficient preparation of genomic DNA and total proteins of Nosema bombycis . Science of Sericulture, 37: 10191024. [In Chinese with abstract in English].Google Scholar
Chaivisuthangkura, P., Srisuk, C., Rukpratanporn, S., Longyant, S., Sridulyakul, P., and Sithigorngul, P. 2009. Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification. Journal of Virological Methods, 162: 188193.CrossRefGoogle ScholarPubMed
Chen, Y. 2008. Analysis of oral infection and helicase gene of the nucleopolyhddroviruses isolated from Philosamia cynthia vicini and Antheraea pernyi. M.Sc. dissertation. Jiangsu University, Zhenjiang, China. Pp. 4–5. [In Chinese with abstract in English].Google Scholar
Du, J., Zhang, C., Fu, J., Chen, Z., and Xiao, Q. 2012. Preparation of a monoclonal antibody against polyhedrin of Ectropis obliqua nucleopolyhedrovirus. Chinese Journal of Biotechnology, 28: 7685. (in Chinese with abstract in English).Google ScholarPubMed
Fredricks, D.N., Smith, C., and Meier, A. 2005. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. Journal of Clinical Microbiology, 43: 51225128.CrossRefGoogle ScholarPubMed
Hatakeyama, Y. and Hayasaka, S. 2001. Specific detection and amplification of microsporidia DNA fragments using multiprimer PCR. The Journal of Sericulture Science of Japan, 70: 4348.Google Scholar
Hatakeyama, Y. and Hayasaka, S. 2003. A new method of pebrine inspection of silkworm egg using multiprimer PCR. Journal of Invertebrate Pathology, 82: 148151.CrossRefGoogle ScholarPubMed
Huang, X., Pan, Z., Zhu, F., Lin, Q., Luo, M., Shi, M., et al. 2011. Infectiousness of insect microsporidia to Bombyx mori and its propagation. Journal of Southern Agriculture, 42: 664667. [In Chinese with abstract in English].Google Scholar
Kawakami, Y., Iwano, H., Hatakeyama, Y., Inoue, T., and Canning, E.U. 2001. Use of PCR with the specific primers for discrimination of Nosema bombycis . The Journal of Sericulture Science of Japan, 70: 4348.Google Scholar
Nimitphak, T., Meemetta, W., Arunrut, N., Senapin, S., and Kiatpathomchai, W. 2010. Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateral-flow dipstick. Molecular and Cellular Probes, 24: 15.CrossRefGoogle ScholarPubMed
Pan, Z., Gong, C., Zheng, X., Guo, R., and Shen, W. 2011. Application of pebrine detection by PCR infected Bombyx mori eggs. Advanced Materials Research, 175–176: 812.CrossRefGoogle Scholar
Panichareon, B., Khawsak, P., Deesukon, W., and Sukhumsirichart, W. 2011. Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp. Journal of Virological Methods, 178: 1621.CrossRefGoogle ScholarPubMed
Pegels, N., López-Calleja, I., García, T., Martín, R., and González, I. 2013. Detection of rabbit and hare processed material in compound feeds by TaqMan real-time PCR. Food Additives & Contaminants: Part A, 30: 771779.CrossRefGoogle ScholarPubMed
Ravikumar, G., Raje Urs, S., Vijaya Prakash, N.B., Rao, C.G., and Vardhana, K.V. 2011. Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms. Journal of Invertebrate Pathology, 107: 193197.CrossRefGoogle ScholarPubMed
Shamim, M., Ghosh, D., Baig, M., Nataraju, B., Datta, R.K., and Gupta, S.K. 1997. Production of monoclonal antibodies against Nosema bombycis and their utility for detection of pebrine infection in Bombyx mori L. Journal of Immunoassay, 18: 357370.CrossRefGoogle ScholarPubMed
Thorne, C.M., Otvos, I.S., Conder, N., and Levin, D.B. 2007. Development of a dipstick immunoassay to detect nucleopolyhedroviruses in Douglas-fir tussock moth larvae. Journal of Virological Methods, 146: 188195.CrossRefGoogle ScholarPubMed
Wangman, P., Longyant, S., Chaivisuthangkura, P., Sridulyakul, P., Rukpratanporn, S., and Sithigorngul, P. 2012. Penaeus monodon nucleopolyhedrovirus detection using an immunochromatographic strip test. Journal of Virological Methods, 183: 210214.CrossRefGoogle ScholarPubMed
Wangman, P., Longyant, S., Chaivisuthangkura, P., Sridulyakul, P., Rukpratanporn, S., Sithigorngul, P., et al. 2009. Simple immunoblot and immunohistochemical detection of Penaeus stylirostris densovirus using monoclonal antibodies to viral capsid protein expressed heterologously. Journal of Virological Methods, 162: 126132.Google Scholar
Xie, L. 2007. Establishing proteomic platform of 2-d electrophoresis and mass spectrum analyses of Nosema bombycis. M.Sc. dissertation. Southwest University, Chongqing, China. Pp. 17–18. [In Chinese with abstract in English].Google Scholar
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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)
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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)
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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)
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