Further Evaluation of Multiplex PCR for Rapid Detection of Salmonella Typhi

Deog-Yong Lee; Jung-Eun Min; Esther Lee; Sung Hun Kim; Hee-Bok Oh; Mi-Sun Park

doi:10.3947/ic.2010.42.4.237

Journal List > Infect Chemother > v.42(4) > 1035017

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Lee, Min, Lee, Kim, Oh, and Park: Further Evaluation of Multiplex PCR for Rapid Detection of Salmonella Typhi

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Infection & Chemotherapy

Infection & Chemotherapy 2010; 42(4): 237-240.

Published online: 30 August 2010

DOI: https://doi.org/10.3947/ic.2010.42.4.237

Further Evaluation of Multiplex PCR for Rapid Detection of Salmonella Typhi

Deog-Yong Lee, Jung-Eun Min, Esther Lee, Sung Hun Kim, Hee-Bok Oh, Mi-Sun Park

Division of Enteric Bacterial Infections, Center for Infectious Diseases, Korea Centers for Disease Control and Prevention, Seoul, Korea.

Correspondence to Mi-Sun Park. Division of Enteric Bacterial Infections, Center for Infectious Diseases, KCDC, 194 Tongil-ro, Eunpyoung-gu, Seoul 122-701, Korea. Tel: +82-2-380-2981, Fax: +82-2-352-4767, pmsun63@korea.kr

Received 15 April 2010 Revised 21 June 2010 Accepted 27 July 2010

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Typhoid fever is a class I legally designated communicable disease in Korea; and if remains as an important public health problem in many developing countries. It takes at least 3-5 days to detect and identify Salmonella Typhi (S. Typhi) by classical diagnostic method. For this reason, multiplex PCR (mPCR) was evaluated in detecting and identifying S. Typhi. In this study, forty-three bacterial strains, which consisted of 42 Salmonella enterica serovars and one Citrobacter freundii. were used to evaluate the promptness of mPCR in detecting and identifying S. Typhi. mPCR was performed with four genes which were known for representing Salmonella spp and/or S. Typhi: invA, fliC-d, viaB and prt. invA and prt gene was amplified in all strains and viaB gene was in only S. Typhi. fliC-d gene was amplified in three serovars: S. Typhi, S. Schwarzengrund and S. Livingstone. After specificity test, mPCR was modified as triplex PCR with three genes (invA, fliC-d, and viaB) and the sensitivity test was performed against S. Typhi-inoculated stool samples. mPCR was able to detect S. Typhi cell suspension of 1×10⁵ cfu/mL. We found that modified multiplex PCR was useful to detect S. Typhi from stool samples within 24h whereas it takes 3-5days to detect by classic diagnosis method.

Keywords: Salmonella Typhi, Multiplex PCR, invA, fliC-d, viaB

Figures and Tables

Figure 1

The specificity and sensitivity of modified multiplex PCR to detect S. Typhi. (A) Specificity of multiplex PCR. invA gene was amplified all Salmonella serovars but viaB and fliC-d was only S. Typhi by modified multiplex PCR. All Salmonella serovar was inoculated into human feces, concentration of 10⁸ cfu/mL. M. 100 bp DNA ladder; Lane 1. S. Enteritidis; Lane 2. S. Typhimurium; Lane 3. S. Typhi; Lane 4, S. Braenderup; Lane 5, S. London; Lane 6. S. ParatyphiA; Lane 7, S. Infantis; Lane 8, S. Montevideo; Lane 9, Negative control (distiled water). (B) Sensitivity of multiplex PCR. Modified multiplex PCR was able to detect 10⁵ cfu/mL of S. Typhi, which contained human feces. M, 100bp DNA ladder; Lane 1, 10⁷ cfu/mL; Lane 2, 10⁶ cfu/mL; Lane 3, 10⁵ cfu/mL; Lane 4, 10⁴ cfu/mL; Lane 5, 10³ cfu/mL; Lane 6, 10² cfu/mL; Lane 7, 10¹ cfu/mL; Lane 8, 10⁰ cfu/mL; Lane 9, 0 cfu/mL; Lane 10, Positive control; Lane 11, Negative control (distiled water).

Table 1

Oligonucleotide Sequence of Target Primers Used in This Study

Table 2

Amplification of target genes by multiplex PCR used in this study

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