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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 55 issue 8 (august 2021) : 956-959,   Doi: 10.18805/IJAR.B-4173

Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea

S
Sanjeev Kumar
J
Jagan Mohanarao Gali
T
T.K. Dutta
P
P. Roychoudhury
P
P.K. Subudhi
1Department of Veterinary Microbiology, Central Agricultural University-Imphal, Selesih, Aizawl-796 015, Mizoram, India.
Cite article:- Kumar Sanjeev, Gali Mohanarao Jagan, Dutta T.K., Roychoudhury P., Subudhi P.K. (2021). Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea. Indian Journal of Animal Research. 55(8): 956-959. doi: 10.18805/IJAR.B-4173.

ABSTRACT

Background: Diarroeagenic Escherichia coli (DEC) including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene is considered as marker for EPEC and EHEC. Laboratory diagnosis of such infections is carried out by traditional bacteriological techniques and by conventional PCR assays. Those techniques often provide false negative result and at the same time are costly as well as difficult to perform in the field level. The loop-mediated isothermal amplification (LAMP) is a new generation DNA amplification assay is developed for detection of eaeA gene in E. coli isolated from diarrhoeic piglets.
Methods: Samples were collected from diarrhoeic piglets for isolation and identification of E. coli. eaeA gene was detected by conventional PCR using specific primers in all the isolates. LAMP assay was standardized for detection of eaeA gene. Analytical sensitivity of LAMP was evaluated using 10 fold serially diluted E. coli genomic DNA. The specificity of the LAMP assay was determined by evaluating the cross reactivity with 19 other enteric and non-enteric bacterial species. Standardized LAMP was applied for detection of eaeA gene in the field isolates.
Result: A total of 37 (24.67%) isolates were recorded as positive for eaeA gene by conventional PCR, while 49 (32.67%) isolates were recorded as positive for eaeA gene by LAMP assay. The LAMP assay was 10 times more sensitive than conventional PCR. LAMP assay was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR, which can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.

REFERENCES

  1. Ali, S.A., Kaur, G., Boby, N., Sabarinath, T., Solanki, K., Pal, D., Chaudhuri, P. (2017). Rapid and visual detection of Leptospira in urine by LigB-LAMP assay with pre-addition of dye. Molecular and Cellular Probes. 36: 29-35.
  2. Blanco, M., Blanco, J.E., Dahbi, G., Alonso, M.P., Mora, A., Coira, M.A., Madrid, C., Juárez, A., Bernardez, M.I., González, E.A., Blanco, J. (2006). Identification of two new intimin types in atypical enteropathogenic Escherichia coli. International Microbiology. 9: 103-110.
  3. Chen, H.D. and Frankel, G. (2005). Enteropathogenic Escherichia coli: unravelling pathogenesis. FEMS Microbiology Reviews. 29: 83-98.
  4. Dhanashree, B. and Mallya, P.S. (2008). Detection of shiga-toxigenic Escherichia coli (STEC) in diarrhoeagenic stool & meat samples in Mangalore, India. Indian Journal of Medical Research. 128: 271-276.
  5. Drolet, R., Fairbrother, J.M., Harel, J., Helie, P. (1994). Attaching and effacing and enterotoxigenic Escherichia coli associated with enteric colibacillosis in the dog. Canadian Journal of Veterinary Research. 58: 87-93.
  6. Dutta, T.K., Vangchhia, B.L., Roychoudhury, P., Ralte, R.L., Subudhi, P.K. (2019). Rapid visual detection of diarrhoea-associated eaeA gene using polymerase spiral reaction (PSR) from clinical samples. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases. 40: 89-93.
  7. García-Meniño, I., García, V., Mora, A., Díaz-Jiménez, D., Flament-    Simon, S.C., Alonso, M.P., Blanco, J.E., Blanco, M., Blanco, J. (2018). Swine enteric colibacillosis in Spain: pathogenic potential of mcr-1 ST10 and ST131 E. coli isolates. Frontiers in Microbiology. 9: 2659-2667.
  8. Ghosh, P.K. and Ali, A. (2010). Isolation of atypical enteropathogenic Escherichia coli from children with and without diarrhoea in Delhi and the National Capital Region, India. Journal of Medical Microbiology. 59: 1156-1162.
  9. Kim, J.H. and Oh, S.W. (2019). Development of a filtration-based LAMP–LFA method as sensitive and rapid detection of E. coli O157: H7. Journal of Food Science and Technology. 56: 2576-2583.
  10. Liu, W., Huang, S., Liu, N., Dong, D., Yang, Z., Tang, Y., Ma, W., He, X., Ao, D., Xu, Y., Zou, D. (2017). Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. Scientific Reports. 7: 1-9.
  11. Luppi, A. (2017). Swine enteric colibacillosis: diagnosis, therapy and antimicrobial resistance. Porcine Health Management. 3: 16-29.
  12. Mori, Y., Kanda, H., Notomi, T. (2013). Loop-mediated isothermal amplification (LAMP): recent progress in research and development. Journal of Infection and Chemotherapy. 19: 404-411.
  13. Notomi, T., Mori, Y., Tomita, N., Kanda, H. (2015). Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of Microbiology. 53: 1-5.
  14. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 28: e63.
  15. Paton, A.W. and Paton, J.C. (1998). Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfb O111, and rfb O157. Journal of Clinical Microbiology. 36: 598-602.
  16. Priya, G.B., Agrawal, R.K., Milton, A.A.P., Mishra, M., Mendiratta, S.K., Agarwal, R.K., Luke, A., Singh, B.R., Kumar, D. (2018). Development and evaluation of isothermal amplification assay for the rapid and sensitive detection of Clostridium perfringens from chevon. Anaerobe. 54: 178-187.
  17. Ramezani, R., Kardoost, P. Z., Ghorbanmehr, N., and Mirshafiee, H. (2018). Rapid and simple detection of Escherichia coli by Loop-mediated isothermal amplification assay in urine specimens. Avicenna J Med Biotechnol. 10: 269-272.
  18. Tomita, N., Mori, Y., Kanda, H., Notomi, T. (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocol. 3: 877-882.
  19. Turk, J., Maddox, C., Fales, W., Ostlund, E., Miller, M., Johnson, G., Pace, L., Turnquist, S., Kreeger, J. (1998). Examination for heat-labile, heat-stable, and Shiga-like toxins and for the eaeA gene in Escherichia coli isolates obtained from dogs dying with diarrhea: 122 cases (1992–1996). Journal of American Veterinary Medical Association. 212: 1735-1736.
  20. Yan, M., Xu, L., Jiang, H., Zhou, Z., Zhou, S. and Zhang, L. (2017). PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state. Microbial Pathogenesis. 105:245-250.
  21. Zhu, C., Harel, J., Jacques, M., Desautels, C., Donnenberg, M.S., Beaudry, M., Fairbrother, J. M. (1994). Virulence properties and attaching-effacing activity of Escherichia coli O45 from swine postweaning diarrhea. Infection and Immunity. 62: 4153-4159. 
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Indian Journal of Animal Research
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    Research Article

    volume 55 issue 8 (august 2021) : 956-959,   Doi: 10.18805/IJAR.B-4173

    Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea

    S
    Sanjeev Kumar
    J
    Jagan Mohanarao Gali
    T
    T.K. Dutta
    P
    P. Roychoudhury
    P
    P.K. Subudhi
    1Department of Veterinary Microbiology, Central Agricultural University-Imphal, Selesih, Aizawl-796 015, Mizoram, India.
    Cite article:- Kumar Sanjeev, Gali Mohanarao Jagan, Dutta T.K., Roychoudhury P., Subudhi P.K. (2021). Development of Isothermal LAMP Assay for Detection of Intimin Gene (eae) in E. coli Associated with Piglet Diarrhea. Indian Journal of Animal Research. 55(8): 956-959. doi: 10.18805/IJAR.B-4173.

    ABSTRACT

    Background: Diarroeagenic Escherichia coli (DEC) including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene is considered as marker for EPEC and EHEC. Laboratory diagnosis of such infections is carried out by traditional bacteriological techniques and by conventional PCR assays. Those techniques often provide false negative result and at the same time are costly as well as difficult to perform in the field level. The loop-mediated isothermal amplification (LAMP) is a new generation DNA amplification assay is developed for detection of eaeA gene in E. coli isolated from diarrhoeic piglets.
    Methods: Samples were collected from diarrhoeic piglets for isolation and identification of E. coli. eaeA gene was detected by conventional PCR using specific primers in all the isolates. LAMP assay was standardized for detection of eaeA gene. Analytical sensitivity of LAMP was evaluated using 10 fold serially diluted E. coli genomic DNA. The specificity of the LAMP assay was determined by evaluating the cross reactivity with 19 other enteric and non-enteric bacterial species. Standardized LAMP was applied for detection of eaeA gene in the field isolates.
    Result: A total of 37 (24.67%) isolates were recorded as positive for eaeA gene by conventional PCR, while 49 (32.67%) isolates were recorded as positive for eaeA gene by LAMP assay. The LAMP assay was 10 times more sensitive than conventional PCR. LAMP assay was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR, which can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.

    REFERENCES

    1. Ali, S.A., Kaur, G., Boby, N., Sabarinath, T., Solanki, K., Pal, D., Chaudhuri, P. (2017). Rapid and visual detection of Leptospira in urine by LigB-LAMP assay with pre-addition of dye. Molecular and Cellular Probes. 36: 29-35.
    2. Blanco, M., Blanco, J.E., Dahbi, G., Alonso, M.P., Mora, A., Coira, M.A., Madrid, C., Juárez, A., Bernardez, M.I., González, E.A., Blanco, J. (2006). Identification of two new intimin types in atypical enteropathogenic Escherichia coli. International Microbiology. 9: 103-110.
    3. Chen, H.D. and Frankel, G. (2005). Enteropathogenic Escherichia coli: unravelling pathogenesis. FEMS Microbiology Reviews. 29: 83-98.
    4. Dhanashree, B. and Mallya, P.S. (2008). Detection of shiga-toxigenic Escherichia coli (STEC) in diarrhoeagenic stool & meat samples in Mangalore, India. Indian Journal of Medical Research. 128: 271-276.
    5. Drolet, R., Fairbrother, J.M., Harel, J., Helie, P. (1994). Attaching and effacing and enterotoxigenic Escherichia coli associated with enteric colibacillosis in the dog. Canadian Journal of Veterinary Research. 58: 87-93.
    6. Dutta, T.K., Vangchhia, B.L., Roychoudhury, P., Ralte, R.L., Subudhi, P.K. (2019). Rapid visual detection of diarrhoea-associated eaeA gene using polymerase spiral reaction (PSR) from clinical samples. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases. 40: 89-93.
    7. García-Meniño, I., García, V., Mora, A., Díaz-Jiménez, D., Flament-    Simon, S.C., Alonso, M.P., Blanco, J.E., Blanco, M., Blanco, J. (2018). Swine enteric colibacillosis in Spain: pathogenic potential of mcr-1 ST10 and ST131 E. coli isolates. Frontiers in Microbiology. 9: 2659-2667.
    8. Ghosh, P.K. and Ali, A. (2010). Isolation of atypical enteropathogenic Escherichia coli from children with and without diarrhoea in Delhi and the National Capital Region, India. Journal of Medical Microbiology. 59: 1156-1162.
    9. Kim, J.H. and Oh, S.W. (2019). Development of a filtration-based LAMP–LFA method as sensitive and rapid detection of E. coli O157: H7. Journal of Food Science and Technology. 56: 2576-2583.
    10. Liu, W., Huang, S., Liu, N., Dong, D., Yang, Z., Tang, Y., Ma, W., He, X., Ao, D., Xu, Y., Zou, D. (2017). Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. Scientific Reports. 7: 1-9.
    11. Luppi, A. (2017). Swine enteric colibacillosis: diagnosis, therapy and antimicrobial resistance. Porcine Health Management. 3: 16-29.
    12. Mori, Y., Kanda, H., Notomi, T. (2013). Loop-mediated isothermal amplification (LAMP): recent progress in research and development. Journal of Infection and Chemotherapy. 19: 404-411.
    13. Notomi, T., Mori, Y., Tomita, N., Kanda, H. (2015). Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. Journal of Microbiology. 53: 1-5.
    14. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 28: e63.
    15. Paton, A.W. and Paton, J.C. (1998). Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfb O111, and rfb O157. Journal of Clinical Microbiology. 36: 598-602.
    16. Priya, G.B., Agrawal, R.K., Milton, A.A.P., Mishra, M., Mendiratta, S.K., Agarwal, R.K., Luke, A., Singh, B.R., Kumar, D. (2018). Development and evaluation of isothermal amplification assay for the rapid and sensitive detection of Clostridium perfringens from chevon. Anaerobe. 54: 178-187.
    17. Ramezani, R., Kardoost, P. Z., Ghorbanmehr, N., and Mirshafiee, H. (2018). Rapid and simple detection of Escherichia coli by Loop-mediated isothermal amplification assay in urine specimens. Avicenna J Med Biotechnol. 10: 269-272.
    18. Tomita, N., Mori, Y., Kanda, H., Notomi, T. (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocol. 3: 877-882.
    19. Turk, J., Maddox, C., Fales, W., Ostlund, E., Miller, M., Johnson, G., Pace, L., Turnquist, S., Kreeger, J. (1998). Examination for heat-labile, heat-stable, and Shiga-like toxins and for the eaeA gene in Escherichia coli isolates obtained from dogs dying with diarrhea: 122 cases (1992–1996). Journal of American Veterinary Medical Association. 212: 1735-1736.
    20. Yan, M., Xu, L., Jiang, H., Zhou, Z., Zhou, S. and Zhang, L. (2017). PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state. Microbial Pathogenesis. 105:245-250.
    21. Zhu, C., Harel, J., Jacques, M., Desautels, C., Donnenberg, M.S., Beaudry, M., Fairbrother, J. M. (1994). Virulence properties and attaching-effacing activity of Escherichia coli O45 from swine postweaning diarrhea. Infection and Immunity. 62: 4153-4159. 
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