The Development of a Loop-Mediated Isothermal Amplification (LAMP) Procedure for Plague Diagnostic


Plague caused by Yersinia pestis is one of the infectious diseases subject to the International Health Regulations (IHR). Permanent monitoring of the focal plague areas is mandatory in order to enable prompt control measures to prevent the spread of the disease. Therefore, the availability of efficient diagnosis tests is of paramount importance. Here, we describe a loop-mediated isothermal amplification (LAMP)-based procedure for rapid Y. pestis detection. We constructed a set of LAMP primers, which were used in assays to establish the reaction conditions that would lead to the quick visualization of the results by evaluating the test tube with the naked eye. The primers were specifically designed to target the caf1 gene located on pFra/Tox (pMT), a prototypical plasmid of Y. pestis. The LAMP procedure was performed at 65°C for 45 min in a water bath and allowed for the detection of at least 10 pg of bacterial DNA. Due to its simplicity, specificity, sensitivity and rapidity, the LAMP technique is an additional tool that may be implemented in routine plague diagnoses, especially in emergencies.

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de Lira Nunes, M. , Mendes-Marques, C. , de Almeida, A. and Leal, N. (2014) The Development of a Loop-Mediated Isothermal Amplification (LAMP) Procedure for Plague Diagnostic. American Journal of Analytical Chemistry, 5, 1069-1077. doi: 10.4236/ajac.2014.516114.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Perry, R.D. and Fetherston, J.D. (1997) Yersinia pestis—Etiologic Agent of Plague. Clinical Microbiology Reviews, 10, 35-66.
[2] WHO (2008) International Health Regulations 2005. 2nd Edition, World Health Organization, Geneva, 74 p.
[3] Schneider, M.C., Najera, P., Aldighieri, S., Galan, D.I., Bertherat, E., Ruiz, A., Dumit, E., Gabastou, J.M. and Espinal, M.A. (2014) Where Does Human Plague Persist in Latin America? PLoS Negeted Tropical Diseases, 8, e2680.
[4] Stenseth, N.C., Atshabar, B.B., Begon, M., Belmain, S.R., Bertherat, E., Carniel, E., Gage, K.L., Leirs, H. and Rahalison, L. (2008) Plague: Past, Present, and Future. PLoS Medicine, 5, e3.
[5] WHO (2010) Human Plague: Review of Regional Morbidity and Mortality, 2004-2009. Weakly Epidemiology Record, 85, 40-48.
[6] Leal, N.C., Abath, F.G.C., Alves, L.C. and Almeida, A.M.P. (1996) A Simple PCR-Based Procedure for Plaque Diagnosis. Revista do Instituto de Medicina Tropical de Sao Paulo, 38, 371-373.
[7] Leal, N.C. and Almeida, A.M.P. (1999) Diagnosis of Plague and Identification of Virulence Markers in Yersinia pestis by Multiplex-PCR. Revista do Instituto de Medicina Tropical de Sao Paulo, 41, 339-342.
[8] Silva, L.C., Vasconcelos, S.S., Mendes-Marques, C.L., Almeida, A.M.P. and Leal, N.C. (2012) Plague Diagnosis STNPCR and MPCR Kits Assembly, Reaction Reproducibility, and Reagent Stability. Advances in Experimental Medicine and Biology, 954, 143-147.
[9] Souza, G., Abath, F., Leal, N.C., Farias, A. and Almeida, A.M.P. (2007) Development and Evaluation of a Single Tube Nested-PCR Based Approach (STNPCR) for the Diagnosis of Plague. Advances in Experimental Medicine and Biology, 603, 351-359.
[10] Parida, M., Sannarangaiah, S., Dash, P.K., Rao, P.V.L. and Morita, K. (2008) Loop Mediated Isothermal Amplification (LAMP): A New Generation of Innovative Gene Amplification Technique; Perspectives in Clinical Diagnosis of Infectious Diseases. Reviews in Medical Virology, 18, 407-421.
[11] Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. (2000) Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Research, 28, e63.
[12] Mori, Y., Nagamine, K., Tomita, N. and 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.
[13] Njiru, Z.K. (2012) Loop-Mediated Isothermal Amplification Technology: Towards Point of Care Diagnostics. PLoS Neglected Tropical Diseases, 6, 1572-1575.
[14] Chaudhary, A.A., Hemant, Mohsin, M. and Ahmad, A. (2011) Application of Loop-Mediated Isothermal Amplification (LAMP)-Based Technology for Authentication of Catharanthus roseus (L.) G. Don. Protoplasma, 249, 417-422.
[15] Galyov, E.E., Smirnov, O.Y., Karlishev, A.V., Volkovoy, K.I., Denesyuk, A.I., Nazimov, I.V., Rubtsov, K.S., Abramov, V.M., Dalvadyanz, S.M. and Zav’yalov, V.P. (1990) Nucleotide Sequence of the Yersinia pestis Gene Encoding F1 Antigen and the Primary Structure of the Protein: Putative T and B Cell Epitopes. FEBS Letters, 277, 230-232.
[16] Prentice, M.B. and Rahalison, L. (2007) Plague. The Lancet, 369, 1196-1207.
[17] Chu, M. (2000) Laboratory Manual of Plague Diagnostic Tests. Centers for Disease Control and Prevention, World Health Organization.
[18] Leal, N.C., Silva, S.C., Cavalcanti, V.O., Figueiroa, A.C.T.A., Nunes, V.V.F., Miralles, I.S. and Hofer, E. (2008) Vibrio parahaemolyticus Serovar O3:K6 Gastroenteritis in Northeast Brazil. Journal of Applied Microbiology, 105, 691-697.
[19] Costa, A.P.R., Vilela, M.A., Mendes-Marques, C.L., Almeida, A.M.P. and Leal, N.C. (2013) Biochemical and Molecular Characteristics of Listeria monocytogenes Isolates from a Prosthetic Mitral Heart Valve-Bearing Patient’s Blood Cultures. Journal of Health & Biological Science, 1, 161-121.
[20] Sanger, F., Nicklen, S. and Coulson, R. (1977) DNA Sequencing with Chain-Terminating Inhibitors. Proceedings of the National Academy of Sciences of the United States of America, 74, 5463-5467.
[21] Tomita, N., Mori, Y., Kea, H. and Notomi, T. (2008) Loop-Mediated Isothermal Amplification (LAMP) of Gene Sequences and Simple Visual Detection of Products. Nature Protocols, 3, 877-882.
[22] Melo, A.C., Almeida, A.M.P. and Leal, N.C. (2003) Retrospective Study of a Plague Outbreak by Multiplex-PCR. Letters in Applied Microbiology, 37, 361-364.

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