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Yun, Chae, Na, Chung, Yoo, and Lee: Modified Method of Multilocus Sequence Typing (MLST) for Serotyping in Salmonella Species
Original Article

J Bacteriol Virol 2015;45(4):314-318.

Published online: 9 February 2015

DOI: https://doi.org/10.4167/jbv.2015.45.4.314

Modified Method of Multilocus Sequence Typing (MLST) for Serotyping in Salmonella Species

Young-Sun Yun, Su-Jin Chae, Hye-Young Na, Gyung Tae Chung, Cheon-Kwon Yoo∗, Deog-Yong Lee

Division of Enteric Diseases, Center for Infectious Diseases, Korea National Institute of Health, Osong, Korea

∗Corresponding author: Deog-Yong Lee. Division of Enteric Diseases, Center for Infectious Diseases, Korea National Institute of Health, 187 Osongsaengmyoung 2(i)-ro, Osong-eup, Heungduk-gu, Cheongju-si, Chungcheongbuk-do, 28159, Korea. Phone: +82-43-719-8113, Fax: +82-43-719-8149, e-mail: leedy0610@korea.kr

∗∗ This study was supported by the acute diarrheal laboratory surveillance system (K-EnterNet) (4851-304-201).

Received 11 November 201525 November 2015       Accepted 30 November 2015

Copyright © 2015 Journal of Bacteriology and Virology

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

Multilocus sequence typing (MLST) of Salmonella is useful method for replacing serotyping using antisera but is limited by difficulties associated with in polymerase chain reaction (PCR). We optimized the PCR reaction, especially annealing temperature and extension time (94°C for 2 min; 40 cycles at 94°C for 30 sec, 56.8°C for 1 min, 72°C for 2 min; and 72°C for 10 min). The degradation of PCR product by thermostable nucleases was inhibited by using template DNAs treated proteinase K or purified by a commercialized preparation kit. The resulting modified MLST was used as accurate and fast typing method.

Keywords: Multilocus sequence typing (MLST), Salmonella spp., PCR conditions, Serotyping

REFERENCES

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Figure 1.
Comparison of the amplified PCR products using two commercialized PCR premix kits. Aliquots of 5 μl of PCR products were analyzed on a 1.5% agarose gel at 100 volts for 30 min. The PCR products with the optimized condition showed thicker and cleaner bands than those obtained by the previous condition with two different kits. (A) iNtRON Co., Ltd. PCR premix, Korea (B) SNC Co., Ltd PCR premix, Korea. M: 100 bp of DNA marker; Lanes 1~2, aroC, amplified with the previous and optimized conditions; Lanes 3~4: dnaN, amplified with the previous and optimized conditions; Lanes 5~6, hemD, amplified with the previous and optimized conditions; Lanes 7~8, hisD, amplified with the previous and optimized conditions; Lanes 9~10, thrA, amplified with the previous and optimized conditions; Lanes 11~12, sucA, amplified with the previous and optimized conditions; Lanes 13~14, purE, amplified with the previous and optimized conditions.
jbv-45-314f1.tif
Table 1.
Oligonucleotide sequences of primers for multilocus sequence type
Primers Oligonucleotide sequences (5′ → 3′) Expected Tm. (℃) Product size (bp)
aroC F CCTGGCACCTCGCGCTATAC 64.6 826
R CCACACACGGATCGTGGCG 63.8 826
dnaN F ATGAAATTTACCGTTGAACGTGA 57.5 833
R AATTTCTCATTCGAGAGGATTGC 59.3 833
hemD F ATGAGTATTCTGATCACCCG 56.4  
R ATCAGCGACCTTAATATCTTGCCA 61.8 666
hisD F GAAACGTTCCATTCCGCGCAGAC 66.4 894
R CTGAACGGTCATCCGTTTCTG 61.3 894
thrA F GTCACGGTGATCGATCCGGT 62.5 852
R CACGATATTGATATTAGCCCG 57.4 852
sucA F AGCACCGAAGAGAAACGCTG 60.5  
R GGTTGTTGATAACGATACGTAC 58.4 643
purE F ATGTCTTCCCGCAATAATCC 56.4  
R TCATAGCGTCCCCCGCGGATC 67.2 510
Table 2.
Alleles numbers and sequence types of 20 strains of Salmonella serovars
No. Serovar aroC dnaN hemD hisD purE sucA thrA Sequence types
1 Enteritidis 5 2 3 7 6 6 11 11
2 Typhimurium 10 7 12 9 5 9 2 19
3 I 4,[5]12:i:- 10 19 12 9 5 9 2 34
4 Montevideo 43 41 16 42 35 13 4 81
5 Virchow 2 7 10 10 8 10 14 38
6 Infantis 17 18 22 17 5 21 19 32
7 Typhi 1 1 2 1 1 1 5 2
8 Bareilly 81 5 101 12 124 130 17 362
9 Thompson 14 13 18 12 14 18 1 26
10 Newport 2 2 15 14 15 20 12 31
11 Saintpaul 5 14 18 9 6 12 17 27
12 Stanley 16 16 26 18 8 12 18 51
13 ParatyphiA 45 4 8 44 27 9 8 85
14 Agona 3 3 7 4 3 3 7 13
15 ParatyphiB 2 14 24 14 37 19 8 86
16 Braenderup 12 2 15 14 11 14 16 22
17 Rissen 92 107 79 156 64 151 87 469
18 Mbandaka 15 70 93 78 113 6 68 413
19 Panama 22 11 25 21 10 23 23 48
20 Anatum 10 14 15 31 25 20 33 64
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