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Journal of Clinical Microbiology, September 2003, p. 4388-4394, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4388-4394.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Typing of Salmonella enterica Serovar Typhi Isolates from Various Countries in Asia by a Multiplex PCR Assay on Variable-Number Tandem Repeats

Yichun Liu,1 May-Ann Lee,1 Eng-Eong Ooi,2 Yeo Mavis,3 Ai-Ling Tan,3 and Hung-Hiang Quek1*

Biomedical Science Laboratory, Defence Medical Research Institute, Defence Science and Technology Agency,1 Environmental Health Institute, National Environment Agency,2 Department of Pathology, Singapore General Hospital, Singapore3

Received 8 June 2003/ Returned for modification 23 April 2003/ Accepted 9 June 2003

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

* Corresponding author. Mailing address: Biomedical Science Laboratory, Defence Medical Research Institute, 10 Medical Dr. No. 02-14, Singapore 117597, Singapore. Phone: (65) 97380437. Fax: (65) 67791677. E-mail: nmiv16{at}nus.edu.sg.

Journal of Clinical Microbiology, September 2003, p. 4388-4394, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4388-4394.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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