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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 53 issue 4 (august 2019) : 447-452,   Doi: 10.18805/IJARe.A-430

Loop Mediated Isothermal Amplification (LAMP) for Nosema bombycis diagnosis by Small subunit Ribosomal RNA (SSU rRNA) gene

S
S. Kampliw
M
M. Monthatong
1Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002 Thailand.
Cite article:- Kampliw S., Monthatong M. (2019). Loop Mediated Isothermal Amplification (LAMP) for Nosema bombycis diagnosis by Small subunit Ribosomal RNA (SSU rRNA) gene. Indian Journal of Agricultural Research. 53(4): 447-452. doi: 10.18805/IJARe.A-430.

ABSTRACT

In present study, Loop Mediated Isothermal Amplification (LAMP) assay was conducted for diagnosis Nosema bombycis, the pebrine disease pathogen in domestic silkworm, Bombyx mori. Nine isolates of N. bombycis were collected from infected silkworms in rearing areas in Khon Kaen province, Thailand. N. bombycis genomic DNAs were extracted by boiling method and used as templates in LAMP and PCR reactions. A LAMP primer set was designed specific to 
N. bombycis small subunit ribosomal RNA (SSU rRNA) gene. The results revealed that the optimal condition was constantly performed at 63oC for 1 hour. The product was directly visualized by naked eye and confirmed with agarose gel eletrophoresis. LAMP assay is more sensitive than traditional PCR, since LAMP was able to detect the least 10 spores/ml while PCR needs 100 spores/ml. In addition, the present novel LAMP primer set was specific only to N. bombycis proven by the negative results when other B. mori pathogen DNAs were tested. In conclusion, the LAMP assay demonstrated a great potential alternative method in diagnosis N. bombycis with high sensitivity, rapidity and accuracy which can apply for pebrine disease surveillance. 

REFERENCES

  1. Bhat, I.A., Buhroo, Z.I., Bhat, M.A. (2017). Microsporidiosis in silkworms with particular reference to mulberry silkworm (Bombyx mori L.). International Journal of Entomology Research. 2(1): 1-9.
  2. Curtis, K.A., Rudolph, D.L., Owen, S.M. (2008). Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP). Journal of Virological Methods. 151: 264-270.
  3. Esvaran, V.G., Gupta, T., Mohanasundaram, A., Ponnuvel, K.M. (2018). Development of isothermal amplification assay for detection of Nosema bombycis infection in silkworm Bombyx mori targeting polar tube protein 1 gene. Invertebrate Survival Journal. 15: 352-361. 
  4. Fu, Z., He, X., Cai, S., Liu, H., He, X, Li, M., Lu, X. (2016). Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae. Journal of Microbiological Methods. 20: 72-78. 
  5. Govindan, R., Narayanaswamy, T.K., Devaiah, M.C. (1997). In: Pebrine Disease of Silkworm. University of Agricultural Sciences GKVK, Bangalore, India.
  6. Hatakeyama, Y. and Hayasaka, S. (2003). A new method of pebrine inspection of silkworm egg using multiprimer PCR. Journal of Invertebrate Pathology. 82: 148-151.
  7. Hema, M., Sudhakara Rao, P., Naseema Begum, A., Rakesh, B. (2011). Susceptibility status of popular silkworm breeds of Bombyx mori L., to infectious flacherie virus. Indian Journal of Animal Research. 45: 109-114.
  8. Kawarabata, T. and Ishihara, R. (1984). Infection and development of Nosema bombycis (Microsporidia: Protozoa) in a cell line of Antheraea eucalypti. Journal of Invertebrate Pathology. 44: 52-64.
  9. Kawakami, Y., Iwano, H., Hatakayama, Y., Inoue, T., Canning, E.U., Ishihara, R. (2001) Use of PCR with the specific primers for discrimination of Nosema bombycis. Journal of Sericultural Science of Japan. 70: 43-48.
  10. Kuboki, N., Inoue, N., Sakurai, T., Cello, D.D., Grab, D.J., Suzuki, H., Sugimoto, C., Igarashi, I. (2003). Loop-mediated isothermal amplification for detection of African Trypanosomes. Journal of Clinical Microbiology. 41: 5517-5524.
  11. Liu, J.P., Cheng, W., Yan, Y.W., Wei, J.Y., Yang, J.L. (2015). Detection of pebrine disease in Bombyx mori eggs with the loop-mediated isothermal amplification (LAMP) method based on EB1 gene. Acta Entomologica Sinica. 58: 846-855. 
  12. Maeda, J., Inoue, M., Nakabayashi, K., Otomo, Y., Shintani, Y., Ohta, M., Okumura, M., Matsuura, N. (2009). Rapid diagnosis of lymph node metastasis in lung cancer with loop-mediated isothermal amplification assay using carcinoembryonic antigen-    mRNA. Lung Cancer. 65: 324-327.
  13. Malone, L.A. and McIvor, C.A. (1995). DNA probe for two microsporidia, Nosema bombycis and Nosema costelytrae. Journal of Invertebrate Pathology. 65: 269-273.
  14. Mori, Y., Nagamine, K., Tomita, N., Notomi, T. (2001). Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and Biophysical Research Communications. 289: 150-154.
  15. Mori, Y. and Notomi, T. (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. Journal of Infection and Chemotherapy. 15: 62-69.
  16. Notomi, T., Okayama, H., Masubuchi, H., Yanekawa, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 28: e63.
  17. Pasteur, L. (1870). Etudes Sur la Maladie Des Vers a Soie. In: Pebrine disease of silkworm, Overseas Technical Cooperation Agency, Japan. 
  18. Singh, G.B., Vijaya Kumari, K.M., Chandrakanth, K.S., Qadri, S.M.H., Das, P.K., Kamble, C.K. (2010). Economics of commercial young age silkworm(Bombyx mori. L.) rearing center. Indian Journal of Agricultural Research. 44: 311-315.
  19. Sirimungkararat, S., Papirom, S., Lawan, M. (2003). Pebrine disease of silkworm, Bombyx mori L. and its epidemic in Khon Kaen province. The Annual Agricultural Seminar for year, Thailand, 27-28 January, 2003.
  20. Sowmyashree, T.S., Sowmya, P., Sudhakara Rao, P., Nataraju, B. (2013). Determination of virulence in the selected bivoltine silkworm breeds through BmNPV inoculation and low temperature induction. Indian Journal of Animal Research. 47: 486-491.
  21. Wei, Y., Shen, Z., Tang, X., Li, X., Li, Q., Yue, Y., Xiao, S., Fu, X. (2014). Detection of Nosema bombycis by FTA cards and loop-    mediated isothermal amplification (LAMP). Current Microbiology. 69: 532-40.
  22. Yeh, H.Y., Shoemaker, C.A., Klesius, P.H. (2005). Evaluation of a loop-mediated Isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella Ictaluri. Journal of Microbiological Methods. 63: 36-44.
     
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    Research Article

    volume 53 issue 4 (august 2019) : 447-452,   Doi: 10.18805/IJARe.A-430

    Loop Mediated Isothermal Amplification (LAMP) for Nosema bombycis diagnosis by Small subunit Ribosomal RNA (SSU rRNA) gene

    S
    S. Kampliw
    M
    M. Monthatong
    1Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, 40002 Thailand.
    Cite article:- Kampliw S., Monthatong M. (2019). Loop Mediated Isothermal Amplification (LAMP) for Nosema bombycis diagnosis by Small subunit Ribosomal RNA (SSU rRNA) gene. Indian Journal of Agricultural Research. 53(4): 447-452. doi: 10.18805/IJARe.A-430.

    ABSTRACT

    In present study, Loop Mediated Isothermal Amplification (LAMP) assay was conducted for diagnosis Nosema bombycis, the pebrine disease pathogen in domestic silkworm, Bombyx mori. Nine isolates of N. bombycis were collected from infected silkworms in rearing areas in Khon Kaen province, Thailand. N. bombycis genomic DNAs were extracted by boiling method and used as templates in LAMP and PCR reactions. A LAMP primer set was designed specific to 
    N. bombycis small subunit ribosomal RNA (SSU rRNA) gene. The results revealed that the optimal condition was constantly performed at 63oC for 1 hour. The product was directly visualized by naked eye and confirmed with agarose gel eletrophoresis. LAMP assay is more sensitive than traditional PCR, since LAMP was able to detect the least 10 spores/ml while PCR needs 100 spores/ml. In addition, the present novel LAMP primer set was specific only to N. bombycis proven by the negative results when other B. mori pathogen DNAs were tested. In conclusion, the LAMP assay demonstrated a great potential alternative method in diagnosis N. bombycis with high sensitivity, rapidity and accuracy which can apply for pebrine disease surveillance. 

    REFERENCES

    1. Bhat, I.A., Buhroo, Z.I., Bhat, M.A. (2017). Microsporidiosis in silkworms with particular reference to mulberry silkworm (Bombyx mori L.). International Journal of Entomology Research. 2(1): 1-9.
    2. Curtis, K.A., Rudolph, D.L., Owen, S.M. (2008). Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP). Journal of Virological Methods. 151: 264-270.
    3. Esvaran, V.G., Gupta, T., Mohanasundaram, A., Ponnuvel, K.M. (2018). Development of isothermal amplification assay for detection of Nosema bombycis infection in silkworm Bombyx mori targeting polar tube protein 1 gene. Invertebrate Survival Journal. 15: 352-361. 
    4. Fu, Z., He, X., Cai, S., Liu, H., He, X, Li, M., Lu, X. (2016). Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae. Journal of Microbiological Methods. 20: 72-78. 
    5. Govindan, R., Narayanaswamy, T.K., Devaiah, M.C. (1997). In: Pebrine Disease of Silkworm. University of Agricultural Sciences GKVK, Bangalore, India.
    6. Hatakeyama, Y. and Hayasaka, S. (2003). A new method of pebrine inspection of silkworm egg using multiprimer PCR. Journal of Invertebrate Pathology. 82: 148-151.
    7. Hema, M., Sudhakara Rao, P., Naseema Begum, A., Rakesh, B. (2011). Susceptibility status of popular silkworm breeds of Bombyx mori L., to infectious flacherie virus. Indian Journal of Animal Research. 45: 109-114.
    8. Kawarabata, T. and Ishihara, R. (1984). Infection and development of Nosema bombycis (Microsporidia: Protozoa) in a cell line of Antheraea eucalypti. Journal of Invertebrate Pathology. 44: 52-64.
    9. Kawakami, Y., Iwano, H., Hatakayama, Y., Inoue, T., Canning, E.U., Ishihara, R. (2001) Use of PCR with the specific primers for discrimination of Nosema bombycis. Journal of Sericultural Science of Japan. 70: 43-48.
    10. Kuboki, N., Inoue, N., Sakurai, T., Cello, D.D., Grab, D.J., Suzuki, H., Sugimoto, C., Igarashi, I. (2003). Loop-mediated isothermal amplification for detection of African Trypanosomes. Journal of Clinical Microbiology. 41: 5517-5524.
    11. Liu, J.P., Cheng, W., Yan, Y.W., Wei, J.Y., Yang, J.L. (2015). Detection of pebrine disease in Bombyx mori eggs with the loop-mediated isothermal amplification (LAMP) method based on EB1 gene. Acta Entomologica Sinica. 58: 846-855. 
    12. Maeda, J., Inoue, M., Nakabayashi, K., Otomo, Y., Shintani, Y., Ohta, M., Okumura, M., Matsuura, N. (2009). Rapid diagnosis of lymph node metastasis in lung cancer with loop-mediated isothermal amplification assay using carcinoembryonic antigen-    mRNA. Lung Cancer. 65: 324-327.
    13. Malone, L.A. and McIvor, C.A. (1995). DNA probe for two microsporidia, Nosema bombycis and Nosema costelytrae. Journal of Invertebrate Pathology. 65: 269-273.
    14. Mori, Y., Nagamine, K., Tomita, N., Notomi, T. (2001). Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and Biophysical Research Communications. 289: 150-154.
    15. Mori, Y. and Notomi, T. (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. Journal of Infection and Chemotherapy. 15: 62-69.
    16. Notomi, T., Okayama, H., Masubuchi, H., Yanekawa, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 28: e63.
    17. Pasteur, L. (1870). Etudes Sur la Maladie Des Vers a Soie. In: Pebrine disease of silkworm, Overseas Technical Cooperation Agency, Japan. 
    18. Singh, G.B., Vijaya Kumari, K.M., Chandrakanth, K.S., Qadri, S.M.H., Das, P.K., Kamble, C.K. (2010). Economics of commercial young age silkworm(Bombyx mori. L.) rearing center. Indian Journal of Agricultural Research. 44: 311-315.
    19. Sirimungkararat, S., Papirom, S., Lawan, M. (2003). Pebrine disease of silkworm, Bombyx mori L. and its epidemic in Khon Kaen province. The Annual Agricultural Seminar for year, Thailand, 27-28 January, 2003.
    20. Sowmyashree, T.S., Sowmya, P., Sudhakara Rao, P., Nataraju, B. (2013). Determination of virulence in the selected bivoltine silkworm breeds through BmNPV inoculation and low temperature induction. Indian Journal of Animal Research. 47: 486-491.
    21. Wei, Y., Shen, Z., Tang, X., Li, X., Li, Q., Yue, Y., Xiao, S., Fu, X. (2014). Detection of Nosema bombycis by FTA cards and loop-    mediated isothermal amplification (LAMP). Current Microbiology. 69: 532-40.
    22. Yeh, H.Y., Shoemaker, C.A., Klesius, P.H. (2005). Evaluation of a loop-mediated Isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella Ictaluri. Journal of Microbiological Methods. 63: 36-44.
       
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