Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip
Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pat...
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| Published in | Journal of Veterinary Medical Science Vol. 79; no. 12; pp. 1973 - 1977 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Japan
JAPANESE SOCIETY OF VETERINARY SCIENCE
2017
Japan Science and Technology Agency The Japanese Society of Veterinary Science |
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| Abstract | Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program. |
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| AbstractList | Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program. Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program. Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program. |
| Author | HASHIMOTO, Koji HAYASHI, Tomohito HATA, Eiji INADA, Mika NIKAIDO, Masaru KIKU, Yoshio KAWAI, Kazuhiro ITO, Keiko KATSUDA, Ken TAGAWA, Yuichi |
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| Cites_doi | 10.1292/jvms.14-0159 10.1136/vr.101598 10.1021/ac0715468 10.1079/9781845935504.0033 10.1038/350091a0 10.1093/nar/28.12.e63 10.3168/jds.S0022-0302(00)75126-5 10.1016/j.mimet.2009.02.001 10.1292/jvms.12-0362 10.1051/vetres:2003022 10.1292/jvms.16-0049 10.4172/2155-6210.1000126 |
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| Copyright | 2017 by the Japanese Society of Veterinary Science Copyright Japan Science and Technology Agency Dec 2017 2017 The Japanese Society of Veterinary Science 2017 |
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| References | 9. Ihira, M., Yoshikawa, T., Enomoto, Y., Akimoto, S., Ohashi, M., Suga, S., Nishimura, N., Ozaki, T., Nishiyama, Y., Notomi, T., Ohta, Y. and Asano, Y. 2004. Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification. J. Clin. Microbiol. 42: 140–145. 2. Compton, J. 1991. Nucleic acid sequence-based amplification. Nature 350: 91–92. 12. Keefe, G. P. 1997. Streptococcus agalactiae mastitis: a review. Can. Vet. J. 38: 429–437. 16. Unno, H., Inada, M., Nakamura, A., Hashimoto, M., Ito, K., Hashimoto, K., Nikaido, M., Hayashi, T., Hata, E., Katsuda, K., Kiku, Y., Tagawa, Y. and Kawai, K. 2015. Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens. J. Vet. Med. Sci. 77: 1007–1009. 5. Hayashi, T., Sugita, T., Hata, E., Katsuda, K., Zhang, E., Kiku, Y., Sugawara, K., Ozawa, T., Matsubara, T., Ando, T., Obayashi, T., Ito, T., Yabusaki, T., Kudo, K., Yamamoto, H., Koiwa, M., Oshida, T., Tagawa, Y. and Kawai, K. 2013. Molecular-based identification of yeasts isolated from bovine clinical mastitis in Japan. J. Vet. Med. Sci. 75: 387–390. 3. Fox, L. K. 2012. Mycoplasma mastitis: causes, transmission, and control. Vet. Clin. North Am. Food Anim. Pract. 28: 225–237. 11. Keane, O. M., Budd, K. E., Flynn, J. and McCoy, F. 2013. Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture. Vet. Rec. 173: 268. 14. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28: E63. 18. Watts, J. L., Lowery, D. E., Teel, J. F. and Rossbach, S. 2000. Identification of corynebacterium bovis and other coryneforms isolated from bovine mammary glands. J. Dairy Sci. 83: 2373–2379. 6. Hisaeda, K., Koshiishi, T., Watanabe, M., Miyake, H., Yoshimura, Y. and Isobe, N. 2016. Change in viable bacterial count during preservation of milk derived from dairy cows with subclinical mastitis and its relationship with antimicrobial components in milk. J. Vet. Med. Sci. 78: 1245–1250. 15. Okada, J., Horiuchi, H., Hashimoto, K., Hirosawa, D., Kurosaki, Y., Kawamoto, K., Yasuda, J., Makino, S., Gemma, N. and Nikaido, M. 2012. Mobile Automatic Detection System for Bacillus anthracis using Electrochemical DNA Chip. J Biosens. Bioelectron. 3: 4. 19. Zaini, F., Kanani, A., Falahati, M., Fateh, R., Salimi-Asl, M., Saemi, N., Farahyar, S., Kheirabad, A. K. and Nazeri, M. 2012. Identification of Prototheca zopfii from Bovine Mastitis. Iran. J. Public Health 41: 84–88. 17. Walker, G. T., Fraiser, M. S., Schram, J. L., Little, M. C., Nadeau, J. G. and Malinowski, D. P. 1992. Strand displacement amplification--an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20: 1691–1696. 10. Iwamoto, T., Sonobe, T. and Hayashi, K. 2003. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J. Clin. Microbiol. 41: 2616–2622. 1. Blowey, R. and Edmondson, P. 2010. The Mastitis Organisms, pp. 33–59. In: Mastitis control in dairy herds, 2nd ed., CAB International, Oxfordshire, U.K. 7. Hogan, J. and Larry Smith, K. 2003. Coliform mastitis. Vet. Res. 34: 507–519. 8. Hogan, J. S., Gonzalez, R. N., Harmon, R. J., Nickerson, S. C. and Oliver, S. P. 6. Pankey, J. W. and Smith, K. L. 1999. Laboratory Handbook on Bovine Mastitis, Revised ed. National Mastitis Council, Madison. 13. Nakamura, N., Ito, K., Takahashi, M., Hashimoto, K., Kawamoto, M., Yamanaka, M., Taniguchi, A., Kamatani, N. and Gemma, N. 2007. Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip. Anal. Chem. 79: 9484–9493. 4. Gao, H., Lei, Z., Jia, J., Wang, S., Chen, Y., Sun, M. and Liang, C. 2009. Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat. J. Microbiol. Methods 77: 198–201. 11 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 9 10 |
| References_xml | – reference: 8. Hogan, J. S., Gonzalez, R. N., Harmon, R. J., Nickerson, S. C. and Oliver, S. P. 6. Pankey, J. W. and Smith, K. L. 1999. Laboratory Handbook on Bovine Mastitis, Revised ed. National Mastitis Council, Madison. – reference: 17. Walker, G. T., Fraiser, M. S., Schram, J. L., Little, M. C., Nadeau, J. G. and Malinowski, D. P. 1992. Strand displacement amplification--an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20: 1691–1696. – reference: 6. Hisaeda, K., Koshiishi, T., Watanabe, M., Miyake, H., Yoshimura, Y. and Isobe, N. 2016. Change in viable bacterial count during preservation of milk derived from dairy cows with subclinical mastitis and its relationship with antimicrobial components in milk. J. Vet. Med. Sci. 78: 1245–1250. – reference: 19. Zaini, F., Kanani, A., Falahati, M., Fateh, R., Salimi-Asl, M., Saemi, N., Farahyar, S., Kheirabad, A. K. and Nazeri, M. 2012. Identification of Prototheca zopfii from Bovine Mastitis. Iran. J. Public Health 41: 84–88. – reference: 9. Ihira, M., Yoshikawa, T., Enomoto, Y., Akimoto, S., Ohashi, M., Suga, S., Nishimura, N., Ozaki, T., Nishiyama, Y., Notomi, T., Ohta, Y. and Asano, Y. 2004. Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification. J. Clin. Microbiol. 42: 140–145. – reference: 18. Watts, J. L., Lowery, D. E., Teel, J. F. and Rossbach, S. 2000. Identification of corynebacterium bovis and other coryneforms isolated from bovine mammary glands. J. Dairy Sci. 83: 2373–2379. – reference: 4. Gao, H., Lei, Z., Jia, J., Wang, S., Chen, Y., Sun, M. and Liang, C. 2009. Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat. J. Microbiol. Methods 77: 198–201. – reference: 13. Nakamura, N., Ito, K., Takahashi, M., Hashimoto, K., Kawamoto, M., Yamanaka, M., Taniguchi, A., Kamatani, N. and Gemma, N. 2007. Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip. Anal. Chem. 79: 9484–9493. – reference: 2. Compton, J. 1991. Nucleic acid sequence-based amplification. Nature 350: 91–92. – reference: 10. Iwamoto, T., Sonobe, T. and Hayashi, K. 2003. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J. Clin. Microbiol. 41: 2616–2622. – reference: 7. Hogan, J. and Larry Smith, K. 2003. Coliform mastitis. Vet. Res. 34: 507–519. – reference: 11. Keane, O. M., Budd, K. E., Flynn, J. and McCoy, F. 2013. Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture. Vet. Rec. 173: 268. – reference: 5. Hayashi, T., Sugita, T., Hata, E., Katsuda, K., Zhang, E., Kiku, Y., Sugawara, K., Ozawa, T., Matsubara, T., Ando, T., Obayashi, T., Ito, T., Yabusaki, T., Kudo, K., Yamamoto, H., Koiwa, M., Oshida, T., Tagawa, Y. and Kawai, K. 2013. Molecular-based identification of yeasts isolated from bovine clinical mastitis in Japan. J. Vet. Med. Sci. 75: 387–390. – reference: 1. Blowey, R. and Edmondson, P. 2010. The Mastitis Organisms, pp. 33–59. In: Mastitis control in dairy herds, 2nd ed., CAB International, Oxfordshire, U.K. – reference: 3. Fox, L. K. 2012. Mycoplasma mastitis: causes, transmission, and control. Vet. Clin. North Am. Food Anim. Pract. 28: 225–237. – reference: 14. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28: E63. – reference: 16. Unno, H., Inada, M., Nakamura, A., Hashimoto, M., Ito, K., Hashimoto, K., Nikaido, M., Hayashi, T., Hata, E., Katsuda, K., Kiku, Y., Tagawa, Y. and Kawai, K. 2015. Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens. J. Vet. Med. Sci. 77: 1007–1009. – reference: 12. Keefe, G. P. 1997. Streptococcus agalactiae mastitis: a review. Can. Vet. J. 38: 429–437. – reference: 15. Okada, J., Horiuchi, H., Hashimoto, K., Hirosawa, D., Kurosaki, Y., Kawamoto, K., Yasuda, J., Makino, S., Gemma, N. and Nikaido, M. 2012. Mobile Automatic Detection System for Bacillus anthracis using Electrochemical DNA Chip. J Biosens. Bioelectron. 3: 4. – ident: 17 – ident: 3 – ident: 16 doi: 10.1292/jvms.14-0159 – ident: 11 doi: 10.1136/vr.101598 – ident: 13 doi: 10.1021/ac0715468 – ident: 12 – ident: 1 doi: 10.1079/9781845935504.0033 – ident: 10 – ident: 19 – ident: 2 doi: 10.1038/350091a0 – ident: 14 doi: 10.1093/nar/28.12.e63 – ident: 18 doi: 10.3168/jds.S0022-0302(00)75126-5 – ident: 4 doi: 10.1016/j.mimet.2009.02.001 – ident: 9 – ident: 8 – ident: 5 doi: 10.1292/jvms.12-0362 – ident: 7 doi: 10.1051/vetres:2003022 – ident: 6 doi: 10.1292/jvms.16-0049 – ident: 15 doi: 10.4172/2155-6210.1000126 |
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| Snippet | Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection... Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection... |
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| SubjectTerms | Bacteria Bacteriology bovine mastitis Dairy industry Deoxyribonucleic acid DNA DNA chip LAMP Mastitis Microorganisms milk pathogen Pathogens Primers |
| Title | Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip |
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