Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

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  • ABSTRACT

    Beetles Protaetia brevitarsis seulensis Kolbe (Coleoptera: Cetoniidae) and Allomyrina dichotoma Linn . (Coleoptera: Scarabaeidae) are widely used in traditional medicine, and the number of insect-rearing farms is increasing in South Korea. The purpose of this study was to establish a multiplex PCR-based assay for rapid simultaneous detection of multiple pathogens causing insect diseases. Six insect parasites such as fungi Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae), bacteria Bacillus thuringiensis Berliner (Bacillales: Bacillaceae), Pseudomonas aeruginosa Migula (Pseudomonadales: Pseudomonadaceae), and Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae), and Oryctes rhinoceros nudivirus were chosen based on the severity and incidence rate of insect diseases in South Korea. Pathogen-specific primers were designed and successfully applied for simultaneous detection of multiple infectious agents in farm-bred insects P. b. seulensis and A. dichotoma using multiplex PCR and high resolution capillary electrophoresis. Our results indicate that multiplex PCR is an effective and time-saving method for simultaneous detection of multiple infections in insects, and the QIAxcel capillary electrophoresis system is useful for quantitative evaluation of the individual impact of each infectious agent on the severity of insect disease. The approach designed in this study can be utilized for rapid and accurate diagnostics of infection in insect farms.


  • KEYWORD

    Applied entomology , Allomyrina dichotoma , Protaetia brevitarsis seulensis , Insect pathogens , Multiplex PCR , Capillary electrophoresis

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  • [Fig. 1.] Geographical distribution of insect farms breeding P. b. seulensis and A. dichotoma in South Korea.
    Geographical distribution of insect farms breeding P. b. seulensis and A. dichotoma in South Korea.
  • [Table 1.] Incidence date, place, and rate of Protaetia brevitarsis seulensis and Allomyrina dichotoma infections with main insect pathogens All larva samples were collected from insect farms in Korea from February 2013 to July 2014 (in total, P. b. seulensis and A. dichotoma were tested for the infection with 28 insect pathogens, including six detected pathogens).
    Incidence date, place, and rate of Protaetia brevitarsis seulensis and Allomyrina dichotoma infections with main insect pathogens All larva samples were collected from insect farms in Korea from February 2013 to July 2014 (in total, P. b. seulensis and A. dichotoma were tested for the infection with 28 insect pathogens, including six detected pathogens).
  • [Table 2.] Target genes and primers
    Target genes and primers
  • [Fig. 2.] The sensitivity of insect pathogen detection by PCR. (A) Single PCR-based detection using corresponding parasite-specific primers. Lane 1: M. anisopliae genomic (g)DNA; lane 2: S. marcescens gDNA; lane 3: P. aeruginosa gDNA, lane 4: B. bassiana gDNA, lane 5: B. thuringiensis gDNA, lane 6: O. rhinoceros virus gDNA. (B) Multiplex PCR-based detection. Lane 7: gDNA mix of O. rhinoceros virus, B. thuringiensis, B. bassiana, P. aeruginosa, S. marcescens, and M. anisopliae analyzed with the combination of primer pairs specific for each pathogen.
    The sensitivity of insect pathogen detection by PCR. (A) Single PCR-based detection using corresponding parasite-specific primers. Lane 1: M. anisopliae genomic (g)DNA; lane 2: S. marcescens gDNA; lane 3: P. aeruginosa gDNA, lane 4: B. bassiana gDNA, lane 5: B. thuringiensis gDNA, lane 6: O. rhinoceros virus gDNA. (B) Multiplex PCR-based detection. Lane 7: gDNA mix of O. rhinoceros virus, B. thuringiensis, B. bassiana, P. aeruginosa, S. marcescens, and M. anisopliae analyzed with the combination of primer pairs specific for each pathogen.
  • [Fig. 3.] Multiplex PCR detection of genomic DNA from six insect pathogens by using the combination of pathogen-specific primer pairs. Lane 1: genomic (g)DNA mix of six insect pathogens; M: molecular weight markers; lane 2: M. anisopliae (M.a.) gDNA; lane 3: S. marcescens (S.m.) gDNA, lane 4: P. aeruginosa (P.a.) gDNA; lane 5: B. bassiana (B.b.) gDNA; lane 6: B. thuringiensis (B.t.) gDNA; lane 7: O. rhinoceros virus (O.r.) gDNA; lane 8: B. thuringiensis, P. aeruginosa, and S. marcescens gDNA; lane 9: B. bassiana and M. anisopliae gDNA; lane 10: O. rhinoceros virus, B. thuringiensis, P. aeruginosa, and S. marcescens gDNA; lane 11: O. rhinoceros virus, B. bassiana, and M. anisopliae gDNA.
    Multiplex PCR detection of genomic DNA from six insect pathogens by using the combination of pathogen-specific primer pairs. Lane 1: genomic (g)DNA mix of six insect pathogens; M: molecular weight markers; lane 2: M. anisopliae (M.a.) gDNA; lane 3: S. marcescens (S.m.) gDNA, lane 4: P. aeruginosa (P.a.) gDNA; lane 5: B. bassiana (B.b.) gDNA; lane 6: B. thuringiensis (B.t.) gDNA; lane 7: O. rhinoceros virus (O.r.) gDNA; lane 8: B. thuringiensis, P. aeruginosa, and S. marcescens gDNA; lane 9: B. bassiana and M. anisopliae gDNA; lane 10: O. rhinoceros virus, B. thuringiensis, P. aeruginosa, and S. marcescens gDNA; lane 11: O. rhinoceros virus, B. bassiana, and M. anisopliae gDNA.
  • [Fig. 4.] Application of multiplex PCR for the detection of insect pathogens in P. b. seulensis and A. dichotoma obtained from insect farms in Korea. (A) P. b. seulensis-isolated gDNA. Lanes 1?3: diseased P. b. seulensis from Hwoengsung; lanes 4?6: diseased P. b. seulensis from Yangju; lane 7: healthy P. b. seulensis; M: molecular weight markers; lane 8: gDNA mix of six insect pathogens (B. bassiana, M. anisopliae, B. thuringiensis, P. aeruginosa, S. marcescens, and O. rhinoceros virus). (B) A. dichotoma-isolated gDNA. Lanes 1?4: diseased A. dichotoma from Youngdong; lane 5: healthy A. dichotoma.
    Application of multiplex PCR for the detection of insect pathogens in P. b. seulensis and A. dichotoma obtained from insect farms in Korea. (A) P. b. seulensis-isolated gDNA. Lanes 1?3: diseased P. b. seulensis from Hwoengsung; lanes 4?6: diseased P. b. seulensis from Yangju; lane 7: healthy P. b. seulensis; M: molecular weight markers; lane 8: gDNA mix of six insect pathogens (B. bassiana, M. anisopliae, B. thuringiensis, P. aeruginosa, S. marcescens, and O. rhinoceros virus). (B) A. dichotoma-isolated gDNA. Lanes 1?4: diseased A. dichotoma from Youngdong; lane 5: healthy A. dichotoma.
  • [Fig. 5.] Specificity of the primers to six insect pathogens analyzed by capillary electrophoresis (QIAxcel). (A) Gel image. M: molecular weight markers; PC: positive control (six primer pairs and gDNA of six pathogens); NC: negative control (six primer pairs and gDNA of healthy P. b. seulensis); lanes 1?4: diseased A. dichotoma from Youngdong; lanes 5?48: diseased P. b. seulensis from Hwoengsung (5?9), Yangju (10?15), Suwon (16?18), Jeonju (19?33), Cheongju (34?44), and Jeju (45?48). (B?E) Electropherograms of negative control (normal P. b. seulensis) (B), diseased A. dichotoma from Youngdong (C), and diseased P. b. seulensis from Yangju (D) and Suwon (E).
    Specificity of the primers to six insect pathogens analyzed by capillary electrophoresis (QIAxcel). (A) Gel image. M: molecular weight markers; PC: positive control (six primer pairs and gDNA of six pathogens); NC: negative control (six primer pairs and gDNA of healthy P. b. seulensis); lanes 1?4: diseased A. dichotoma from Youngdong; lanes 5?48: diseased P. b. seulensis from Hwoengsung (5?9), Yangju (10?15), Suwon (16?18), Jeonju (19?33), Cheongju (34?44), and Jeju (45?48). (B?E) Electropherograms of negative control (normal P. b. seulensis) (B), diseased A. dichotoma from Youngdong (C), and diseased P. b. seulensis from Yangju (D) and Suwon (E).