Loop-Mediated Isothermal Amplification Method for Rapid Detection of Shigella dysenteriae

Abstract:

The study was aimed to develop a loop-mediated isothermal amplification (LAMP) method which amplifies DNA with high specificity and rapidity for the detection of Shigella dysenteriae. A set of four primers was designed for recognizing six distinct sequences on the target ipaH of S. dysenteriae. By the method, the target DNA was amplified within 1h under isothermal condition at 65 °C. The sensitivities of the LAMP for detecting pure culture and genomic DNA were 1.04 CFU/ml and 1.06 fg/μl, while the sensitivities of PCR method were 1.04×10² CFU/ml and 1.06 pg/μl. Furthermore, the LAMP assay was examined for its ability to detect S. dysenteriae in artificially contaminated lettuce sample, the detection limits of this LAMP assay and the PCR method were 4.60 CFU/g and 4.60×10² CFU/g, respectively.

• Periodical: Applied Mechanics and Materials (Volume 140)
• Pages: 369-373
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.140.369
• Online since: November 2011
• Authors: Qing Ping Zhong, Li Wang, Bin Wang, Hong Yuan Chen
• Keywords: IpaH Gene, Loop-Mediated Isothermal Amplification, Rapid Detection, Shigella dysenteriae

References

[1] V.D. Thiem, O. Sethabutr, L. von Seidlein, Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam, J. Clin. Microbiol. 42 (2004) 2031-(2035).

DOI: 10.1128/jcm.42.5.2031-2035.2004

[2] F.E. Kocka, F.D. Dorigan, Q.T. Abbasi, Clinical-evaluations of the wellcolex color Shigella and Salmonella tests, Diagn. Micr. Infec. Dis. 15 (1992) 1-4.

DOI: 10.1016/0732-8893(92)90050-4

[3] K. Sonjai, R. Soisangwan, Y. Sakolvarre, Validation of salmonellosis and shigellosis diagnostic test kits at a Provincial hospital in Thailand, Asian Pac. J. Allergy. 19 (2001) 115-127.

[4] K.E. Sapsford, A. Rasooly, C.R. Taitt, Detection of Campylobacter and Shigella species in food samples using an array biosensor, Anal. Chem. 76 (2004) 433-440.

DOI: 10.1021/ac035122z

[5] H. Houng, O. Sethabutr, P. Echeverria, A simple polymerase chain reaction technique to detect and differentiate Shigella and enteroinvasive Escherichia coli in human feces, Diagn. Micr. Infec. Dis. 28 (1997) 19-25.

DOI: 10.1016/s0732-8893(97)89154-7

[6] A. Vantarakis, G. Komninou, D. Venieri, Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels, Lett. Appl. Microbiol. 31 (2000) 105-109.

DOI: 10.1046/j.1365-2672.2000.00797.x

[7] T. Notomi, H. Okayama, H. Masubuchi, Loop-mediated isothermal amplification of DNA, Nucleic Acids Res. (2000) 28.

DOI: 10.1093/nar/28.12.e63

[8] F. Maruyama, T. Kenzaka, N. Yamaguchi, Detection of bacteria carrying the stx(2) gene by in situ loop-mediated isothermal amplification, Appl. Environ. Microb. 69 (2003) 5023-5028.

DOI: 10.1128/aem.69.8.5023-5028.2003

[9] Y. Hara-Kudo, N. Konishi, K. Ohtsuka, Detection of Verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAMP method: A collaborative study, Int. J. Food Microbiol. 122 (2008) 156-161.

DOI: 10.1016/j.ijfoodmicro.2007.11.078

[10] T.Y. Song, C. Toma, N. Nakasone, Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method, Fems. Microbiol. Lett. 243 (2005) 259-263.

DOI: 10.1016/j.femsle.2004.12.014

[11] R. Savan, A. Igarashi, S. Matsuoka, Sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method, Appl. Environ. Microb. 70 (2004) 621-624.

DOI: 10.1128/aem.70.1.621-624.2004

[12] P.A. Gaudio, O. Sethabutr, P. Echeverria, Utility of a polymerase chain reaction diagnostic system in a study of the epidemiology of shigellosis among dysentery patients, family contacts, and well controls living in a shigellosis-endemic area, J. Infect Dis. 176 (1997).

DOI: 10.1086/516531