A Novel Marker for the Species-Specific Detection and Quantitation of Shigella sonnei by Targeting a Methylase Gene

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1 J. Microbiol. Biotechnol. (2012), 22(8), First published online May 15, 2012 pissn eissn A Novel Marker for the Species-Specific Detection and Quantitation of Shigella sonnei by Targeting a Methylase Gene Cho, Min Seok 1, Tae-Young Ahn 2, Kiseong Joh 3, Oh-Sang Kwon 4, Won-Hwa Jheong 4, and Dong Suk Park 1 * 1 National Academy of Agricultural Science, Rural Development Administration, Suwon , Korea 2 Department of Microbiology, Dankook University, Cheonan , Korea 3 Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin , Korea 4 Water Supply and Sewerage Research Division, Environmental Infrastructure Research Department, National Institute of Environmental Research Environmental Research Complex, Incheon , Korea Received: November 7, 2011 / Revised: April 9, 2012 / Accepted: April 18, 2012 Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qpcr) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qpcr primer set for the accurate diagnosis of Shigella sonnei was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management. Keywords: Detection, diagnosis, quantitation, methylase, Shigella sonnei Shigella sonnei is a nonmotile, nonspore-forming facultative anaerobic Gram-negative bacterium. This pathogen is generally transmitted through uncooked food or contaminated water. In the United States, 70% of shigellosis cases are caused by S. sonnei. Worldwide, it is estimated that million people suffer from shigellosis annually: million in developing countries and 1.5 million in developed countries [4, 9]. It has long been reported that Shigella and Escherichia coli are closely related, but in the 1940s, Shigella strains were separated from E. coli and subgrouped into four species: S. dysenteriae (serogroup A), S. flexneri (serogroup B), S. sonnei (serogroup C), and S. boydii (serogroup D). According to the comparative genome analysis of the two genera, Shigella evolved from many different strains of E. *Corresponding author Phone: ; Fax: ; dspark@rda.go.kr coli. Because the genome of Shigella is highly evolved, it has become a highly specific human pathogen owing to its extensive evolutionary progress involving its repeated gain and/or loss of function compared with E. coli [25]. Therefore, it is important and essential to develop a simple and reliable approach for the sensitive and specific detection of the targeted pathogen. Currently, S. sonnei is isolated from fecal, food, and environmental samples using semi-selective media, and the subsequent identification step consists largely of pathogenicity tests and/or molecular typing techniques [2, 3, 5, 7, 8, 10, 11, 14-16, 18, 19, 22]. Many molecular assays based on ipah, IS1, tuf, uida, and the 16S-ITS-23S gene region are generally used for the detection and identification of Shigella species, but there have been serious defects in the identification and diagnosis of S. sonnei isolates because these assays detect other Shigella and enteroinvasive E. coli (EIEC) [6, 13, 17]. In general, it has been reported that bacteria use methylase to differentiate between foreign genetic material and their own, thus protecting their DNA from their own immune system. To determine their potential annealing specificity, primer sequences based on the methylase gene were evaluated with similarity searches against the sequence database in NCBI ( The BLASTN searches showed similarity to the Eco57I restriction endonuclease sequences (GenBank Accession No. ABO ) from Shewanella loihica PV-4, the Eco57I restriction endonuclease sequences (GenBank Accession No. ABM ) from Shewanella sp. W3-18-1, and the plasmid bglu_4p sequences (GenBank Accession No. CP ) from Burkholderia glumae BGR1. BLAST searches with the predicted protein sequence (BLASTX) revealed the closest similarity to the

2 1114 Cho et al. Fig. 1. Specific PCR amplification of a methylase gene fragment of Shigella sonnei using the SS352F/R primer set. Ethidium bromide-stained agarose gel electrophoresis of PCR products. Lane M, size marker (1 kb DNA plus ladder; Gibco BRL); lanes 1-40 are described in Table 1; lane 41, distilled water. N-6 DNA methylase family protein from E. coli 1357 (GenBank Accession No. EFZ ). However, the region to be amplified with the designed primer pair revealed no significant match with either the BLASTN or BLASTX searches. In this study, a species-specific primer set based on the methylase gene of S. sonnei Ss046 was designed (GenBank Accession No. CP , region: , protein ID AAZ ) with a predicted PCR product of 325 bp (Fig. 1). The primers SS325F (5'-ACGCGTTAA AGATGATGCCTGTT-3') and SS325R (5'-TGCCGCTAA AATCCTTCTGTCCT-3') were synthesized by Bioneer Corporation. The specificity of the SS325F/R primer set in conventional and qpcr assays was demonstrated by testing a total of 40 reference bacterial species (Table 1). All of the bacterial strains were obtained from the American Type Culture Collection in the United States, the Belgian Coordinated Collections of Micro-organisms in Belgium, the Korean Culture Center of Microorganisms and the National Culture Collection for Pathogens in the Republic of Korea, and the National Collection of Type Cultures in the United Kingdom. The culture media and incubation conditions used were in accordance with the Handbook of Microbiological Media [1]. The genomic DNA from all of the microorganisms was prepared using a DNeasy Tissue kit (Qiagen) according to the manufacturer s protocols. All of the amplifications were performed with approximately 50 ng of genomic DNA, the SS325F/R primer set, and GoTaq DNA polymerase (Promega), according to the manufacturer s instructions. The amplifications were performed using a PTC-225 thermocycler (MJ Research) with the following cycling conditions: an initial denaturation of 5 min at 95 o C; 35 cycles of 1 min at 95 o C, 30 s at 56 o C, and 1 min at 72 o C; and a final extension of 7 min at 72 o C. Each amplified PCR product was electrophoresed through a 1.5% agarose gel, stained with ethidium bromide, visualized with a UV transilluminator, and imaged using a gel imaging system. The specificity and sensitivity of the qpcr assay were determined in a 20 µl reaction. All of the amplifications were performed with approximately 5 ng of purified DNA of each sample, the primer set, and the KAPA SYBR Green FAST qpcr Kit (Kapa Biosystems), according to the manufacturer s instructions. The qpcr amplifications were performed using a CFX96 real-time PCR system (Bio-Rad) and the following cycling conditions: an initial denaturation of 3 min at 95 o C, 45 cycles of 10 s at 95 o C and 20 s at 56 o C, and a melting curve at 65 o C to 95 o C, with an increment of 0.5 o C per cycle. The determination of the cycle threshold (Ct) and the data analysis were automatically performed by the CFX Manager Software system (Bio- Rad). The specificity of the SS325F/R primer pair was determined with both the conventional and SYBR Green qpcr analyses (Table 1). As expected, a 325 bp DNA fragment was amplified with conventional PCR (Fig. 1), and the amplification plot and a unique dissociation peak at o C were observed with the qpcr assay (data not shown). To determine the limit of quantification (LOQ) and the limit of detection (LOD), cloned DNA, genomic DNA, and a cell suspension of S. sonnei ATCC were serially diluted 10-fold and tested with SYBR Green qpcr (Table 2). A DNA fragment of 325 bp was ligated into the pgemt easy cloning vector (Promega) according to the manufacturer s instructions. The copy number of the cloned DNA was calculated using the following equation [20]: copies/µl = [ (copy/mol) amount (g)]/ [length (bp) 660 (g/mol/bp)]. All of the samples were analyzed in triplicate. The LOQ assay exhibited a good linear response and a high correlation coefficient (R 2 = 0.996). A standard regression analysis of the linear part of the slope resulted in a coefficient of , which yielded a PCR efficiency of 89.9% (Fig. 2B). The melting curve derived from the amplification plot is shown in Fig. 2C, and the analysis of the melting temperature and melting peaks of S. sonnei

3 A NOVEL MARKER FOR SPECIES-SPECIFIC DETECTION AND QUANTITATION 1115 Table 1. Bacterial strains used in the PCR specificity test. No. Bacterial strains Source This study a 1 Shigella sonnei T KCCM b 2 Shigella sonnei KCCM Shigella sonnei KCCM Shigella sonnei NCTC Shigella flexneri (serovar serotype 2a) T KCCM Shigella flexneri (serovar serotype 2b) KCCM Shigella flexneri (serovar serotype 2b) LMG Shigella boydii (serovar serotype 2) T KCCM Shigella dysenteriae (serovar serotype 2) NCTC Shigella dysenteriae (serovar serotype 3) NCTC Shigella dysenteriae (serovar serotype 4) NCTC Shigella dysenteriae (serovar serotype 6) NCTC Shigella dysenteriae (serovar serotype 7) NCTC Shigella dysenteriae (serovar serotype 8) NCTC Shigella dysenteriae (serovar serotype 9) NCTC Escherichia coli (O1:K1:H7) T LMG Escherichia coli (K-12 met- str-r F-) LMG Escherichia coli LMG Escherichia coli (O157:H42) NCCP Escherichia coli (O157:H7) LMG Escherichia coli (O157:H7) NCTC Escherichia coli (O157:H-) NCTC Salmonella enterica subsp. enterica serovar Typhimurium T ATCC Salmonella enterica subsp. enterica serovar Typhimurium ATCC Salmonella enterica subsp. enterica serovar Vichow ATCC Salmonella enterica subsp. enterica serovar Paratyphi A ATCC Salmonella enterica subsp. enterica serovar Paratyphi B ATCC Salmonella enterica subsp. enterica serovar Typhi ATCC Salmonella enterica subsp. enterica serovar Choleraesuis ATCC Salmonella enterica subsp. enterica serovar Enteritidis ATCC Salmonella enterica subsp. arizonae ATCC Salmonella enterica subsp. diarizonae ATCC Salmonella enterica subsp. houtenae ATCC Salmonella enterica subsp. indica ATCC Salmonella bongori T ATCC Campylobacter jejuni subsp. jejuni T LMG Campylobacter jejuni subsp. jejuni LMG Campylobacter coli T LMG Legionella pneumophila subsp. pneumophila T KCCM Helicobacter pylori T LMG ATCC, American Type Culture Collection, United States; KCCM, Korean Culture Center of Microorganisms, Republic of Korea; LMG, The Belgian Coordinated Collections of Microorganisms (BCCM), Belgium; NCCP, National Culture Collection for Pathogens, Republic of Korea; NCTC, National Collection of Type Cultures, United Kingdom. T Type strain. a Experiments to assess the specificity of real-time PCR assays. b +, detected; -, not detected. with SYBR Green qpcr revealed a reproducible melting temperature of o C and specific peaks (Fig. 2D). The LOD of genomic DNA and the cell suspension determined with the SYBR Green qpcr assay was 5 fg/µl (fg per µl ) and CFU/ml (CFU per ml ) of S. sonnei, respectively. The assay showed excellent quantification characteristics and accurate detection. In conclusion, in this study, the specificity and the sensitivity of a primer set based on the methylase gene of the pathogen were demonstrated with the S. sonnei type strain and a number of isolates, other Shigella species,

4 1116 Cho et al. Table 2. Mean Ct end-point fluorescence of 10-fold serial dilutions of Shigella sonnei cloned DNA, genomic DNA, and a cell suspension determined with a real-time PCR assay. Cloned DNA a Genomic DNA Cell suspension Plasmid copies/µl Weight/µl CFU/ml ± ± ± ± ± ± ± ng 500 pg 50 pg 5 pg 500 fg 50 fg 5 fg ± ± ± ± ± ± ± a ± ± ± ± ± ± , not tested. Fig. 2. Specificity, melting peak, and standard curve of the SS352F/R primer set with SYBR Green qpcr. (A) Fluorescence intensity as a function of the concentration of the template. For each assay, a series of 10-fold dilutions of cloned DNA (range, to copies/µl) was used as the template for PCR (1-7, sample dilutions; 8, no-template control). (B) Standard curve derived from the amplification plot. (C) Melting curve analysis (1-7, sample dilutions; 8, no-template control). (D) Melting peak analysis (1-7, sample dilutions; 8, no-template control). The negative first derivative of the relative fluorescence units ( d(rfu)/dt) is plotted as a function of the temperature. The amplified product, 81.50oC. The high peak indicates the amplified product; the low peak is the no-template control. E. coli, and closely related bacterial isolates. Thus, the quantitative PCR-based method can be used for the rapid detection of S. sonnei and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen

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