Abstract:
The invention provides primers designed on base of the difference of sequences among staphylococcal enterotoxin subtypes, i.e., C 1 , C 2  and C 3 . The invention also provides a PCR method for detecting subtypes of staphylococcal enterotoxin type C by using the above-mentioned primers. The invention relates also to DNA probes useful for the identification of subtypes C 1 , C 2  and C 3  of staphylococcal enterotoxin type C in various food and clinical samples. The primers of the invention comprises following sequences:
       ENTC1 5′-ACAGA GTTAT TAAAT GAAGG-3′;   ENTC2 5′-GTATC AGCAA CTAAA GTTAT-3′;   ENTC3 5′-AAGAG ATTAT TTATT TCACGT-3′;   ENTCR 5′-ATCAT ACCAA AAAGT ATTGC-3′.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to primers designed on base of the difference of sequences among staphylococcal enterotoxin subtypes, and to a PCR method for detecting subtypes (i.e., C 1 , C 2  and C 3 ) of staphylococcal enterotoxin type C by using the above-mentioned primers. The invention relates also to DNA probes useful for the identification of subtypes C 1 , C 2  and C 3  of staphylococcal enterotoxin type C in various food and clinical samples. 
     2. Description of the Prior Art 
     Staphylococcal aureus is one of the important pathogens for food poisoning. Staphylococcal aureus can produces a variety of staphylococcal enterotoxin, i.e. staphylococcal enterotoxin A-B-C-D-E and also, to a lesser extent, G-H-I and J (Genigeorgis, 1989; Betley and Harris, 1994; Su and Wong, 1995; Munson et al., 1998; Zhang. et al., 1998). 
     Staphylococcal enterotoxin C comprises a group of highly conserved proteins, and exhibits a considerable immunological crossover reaction (Marr. et al., 1993). Protein sequence of staphylococcal enterotoxin C present predominantly subtypes of SEC1, C2 and C3 and other minor subtypes (Bohach and Schlievert, 1987; Couch and Betley, 1989; Hovde. et al., 1990; Marr et al., 1993). Since homologies of the amino acid sequences among subtypes C1, C2 and C3 of staphylococcal enterotoxin C are greater than 98% (Bohach and Schlevert, 1987; Hovde. Et al., 1990; Betley. et al., 1992), the identification thereof is not easy. 
     At present, methods for detecting staphylococcal enterotoxin types include, for example, commercially available immuno-detecting kit based on the serological characteristics of staphylococcal enterotoxin, such as, staphylococcal enterotoxin reverse phase latex agglutination (SET-RPLA) kit (Denka. Seiken, Tokyo, Japan); and kits developed based on enterotoxin themselves, such as staphylococcal enterotoxin ELISA (SET-EIA) (Sarasota, Fla., USA) or TECRA (Bioenterprises Pty. Ltd, Roseville, Australia). These kits detect mainly staphylococcal enterotoxin A, B, C, D and E. No publication in literatures reported any DNA assay for the identification of staphylococcal enterotoxin subtypes C1, C2 and C3. As detecting method by means of a gene, though there was a study for detecting staphylococcal enterotoxin types A, B, C, D and E by polymerase chain reaction (PCR) with a specific DNA probe (Johnson. et al., 1991; Tsen and Chen, 1992; Tsen et al., 1994; Schmitz. et al., 1998), no DNA probe or PCR method is available for subtyping staphylococcal enterotoxin C1, C2 and C3, except of the PCR primers for the identification of staphylococcal enterotoxin subtypes C 1 , C 2  and C 3  developed by the invention. Moreover, in case of food poisoning and large-scale food poisoning caused by staphylococcal enterotoxin type C, no method is available for identify further the subtype of the staphylococcal enterotoxin type C. 
     There are a few relating patents such as, for example, ROC patent No. 80100762 titled “Rapid identification of Staphylococcal aureus by the agglutination of micro-latex particles”; ROC patent No. 82106016 titled “Rapid identification of Staphylococcal aureus in processed food by enzymatic immunological analytical method”; EP 1160333A2, “Oligonucleotides and method for detection of mecA gene of methicillin-resistant  Staphylococcus aureus ”; U.S. Pat. No. 6,022,682, “Article and method for detection of enterotoxigenic staphylococci (1996)”; U.S. Pat. No. 563,536 (1995) 7, “Method for detecting staphylococci”; U.S. Pat. No. 5,582,974, “Nucleic acid probes for the detection of  Staphylococcus aureus  (1993)”; U.S. Pat. No. 5,443,963, “Method for detecting staphylococci (1994)”; U.S. Pat. No. 5,132,210 (1989), “Diagnostic test for Staphylococcal mastitis”; U.S. Pat. No. 4,849,341 (1986), “Diagnostic test for Staphylococcal mastitis in cattle”; U.S. Pat. No. 5,770,375 (1997), “Probe for diagnosing  Staphylococcus epidermidis ”; U.S. Pat. No. 5,776,712 (1998), “Methods and materials for the detection of  Staphylococcus aureus ”; U.S. Pat. No. 5,496,706 (1996), “Methods and materials for the detection of  Staphylococcus aureus ”; U.S. Pat. No. 5,437,978 (1993), “Detection for  Staphylococcus  spp.”; and U.S. Pat. No. 5,702,895 (1996), “Method and kit for detecting methicillin-resistant  Staphylococcus aureus”.    
     SUMMARY OF THE INVENTION 
     Accordingly, the main object of the invention is to provide primers useful in the polymerase chain reaction for the identification of subtypes C 1 , C 2  and C 3  in staphylococcal enterotoxin type C, which primers are designed based on parts of gene sequences with greater difference among of staphylococcal enterotoxin subtypes C 1 , C 2  and C 3 , and which are assigned into three pairs of PCR primers with following specificities: 
     SEQUENCE ID NO. 1: ACAGA GTTAT TAAAT GAAGG
         ATCAT ACCAA AAAGT ATTGC       

     SEQUENCE ID NO. 2: GTATC AGCAA CTAAA GTTAT
         ATCAT ACCAA AAAGT ATTGC       

     SEQUENCE ID NO. 3: AAGAG ATTAT TTATT TCACGT
         ATCAT ACCAA AAAGT ATTGC       

     Another object of the invention is to provide a method for the identification of subtypes of staphylococcal enterotoxin C, in order to identify further the subtype of the staphylococcal enterotoxin type C in case of food poisoning and large-scale food poisoning caused by staphylococcal enterotoxin type C. 
     Yet another object of the invention is to provide DNA probes useful in PCR reaction for the identification of subtypes C 1 , C 2  and C 3  in staphylococcal enterotoxin type C, which probes comprise of the above primers SEQ. ID NO. 1-SEQ. ID NO. 3 based on the specificities of above-mentioned primers with respect to staphylococcal enterotoxin subtypes C 1 , C 2  and C 3  gene. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention, as well as its many advantages, may be further understood by the following detailed description and drawings in which: 
         FIG. 1  shows the PCR detection of SEC  S. aureus  and bacterial strains other than SEC  S. aureus  using experimental conditions as described in Materials and Methods. (A) Lane a, 100-bp ladder; Lanes b through q represent PCR products amplified from SEC 1  S. aureus  (FRI 137), SEC  S. aureus  (CCRC12654), SEA  S. aureus  (CCRC 12657), SEB  S. aureus  (CCRC 12653), SEC2  S. aureus  (FRI 361), SEC3  S. aureus  (FRI 913), SED  S. aureus  (CCRC 12660), SEE  S. aureus  (CCRC 12656), SETSST-1  S. aureus  (CCRC 13831), SEG  S. aureus  (FRI 572), SHE  S. aureus  (FRI 569), SEI  S. aureus  (FRI 445),  S. aureus  (CCRC 011),  S. epidermids  (CCRC 11030),  Streptococcus mutans  (CCRC 10793),  Bacillus cereus  (CCRC 10603),  Clostridium perfringens  (CCRC 10914). (B) Lane a, 100-bp ladder; Lanes b through q represent PCR products amplified from SEC2  S. aureus  (FRI 361), SEA  S. aureus  (CCRC 12657), SEB  S. arueus  (12653), SEC1  S. aureus  (FRI 137), SEC3  S. aureus  (FRI 913), SED  S. aureus  (CCRC 12660), SEE  S. aureus  (CCRC 12656), SETSST-1  S. aureus  (CCRC 13831), SEG  S. aureus  (FRI 572), SHE  S. aureus  (FRI 569), SEI  S. aureus  (FRI 445),  S. aureus  (CCRC 011),  S. epidermids  (CCRC 11030),  Streptococcus mutans  (CCRC 10793),  Bacillus cereus  (CCRC 10603),  Clostridium perfringens  (CCRC 10914). (C) Lane a, 100-bp ladder; Lanes b through q represent PCR products amplified from SEC2  S. aureus  (FRI 361), SEA  S. aureus  (CCRC 12657), SEB  S. aureus  (12653), SEC1  S. aureus  (FRI 137), SEC3  S. aureus  (FRI 913), SED  S. aureus  (CCRC 12660), SEE  S. aureus  (CCRC 12656), SETSST-1  S. aureus  (CCRC 13831), SEG  S. aureus  (FRI 572), SHE  S. aureus  (FRI 569), SEI  S. aureus  (FRI 445),  S. aureus  (CCRC 011),  S. epidermids  (CCRC 11030),  Streptococcus mutans  (CCRC 10793),  Bacillus cereus  (CCRC 10603),  Clostridium perfringens  (CCRC 10914). 
       In  FIG. 1 , lanes that do not show bands are empty as the PCR product was not produced by the non-staphylococcal strains represented by those lanes. Such shows the specificity for the identification of subtypes C 1 , C 2  and C 3  of staphylococcal enterotoxin type C.  
         FIG. 2  shows the sensitivity for PCR detection of SEC3  S. aureus  strain (FRI 913) using ENTC3/ENTCR primers. Experimental conditions were as described in Materials and Methods. Lane a, 100-bp ladder; Lanes b through h represent PCR products amplified from N×10 0  to 10 6  CFU per assay of  S. aureus  cells. N equals to 1˜9. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The invention will be now described below with reference to the drawings. 
     Reagents and Materials: 
     A. Bacterial Strains: 
     Standard strains used in the method according to the invention and sources thereof listed in Table 1 are consisted of 10  S. aureus  strains producing enterotoxin of types A, B, C1, C2, C3, D, E, G, H, I and 1 strain producing toxic shock syndrome toxin-1 (TSST-1). Table 2 lists other non- S. aureus , including  Bacillus cereus, Bacillus coagulars, Micrococcus varians, Staphylococcus epidermidis, Streptococcus mutans, Escherichia coli . Table 3 lists 39  S. aureus  strains producing enterotoxin C, which were provided by the Third Branch of Disease Control Agency, Department of Health, Taiwan, ROC (previously the Middle Examination Station of the Preventive Medical Institute) and were isolated from samples collected in food poisoning cases during 1995 to 2000. Table 4 lists 10  S. aureus  strains producing enterotoxin of type C provided by the Food Research Center (FRI) of the University of Wisconsin, Madison, Wis., USA. 
     B. Media: 
     The medium used in the method according to the invention contains, as ingredients, Tryptic Soy Broth (TSB), Brain Heart Infusion (BHI), Baird-Parker agar base (BP), Mueller-Hinton agar, Plate Count Agar (PCA), and Egg Yolk Tullurite (EY) (all available from Detroit, Mich. USA). 
     C. Reagents: 
     Reagents used in the method according to the invention include Sodium dodecyl sulfate (SDS), Ethylenediamine tetra-acetic acid (EDTA), lysozyme, RNase and mineral oil (all available form Sigma Chemical Company, St. Louis, Mo., USA). Tris-(hydroxymethyl)-aminomethane, Triton X-100, proteinase K, dATP, dCTP, dGTP and dTTP (Boegeringer Mannhein GmbH Biochemica, Mannheim, Germany); Lysostaphin (Applied Microbiology, New York, USA); Agarose (Bio-Rad, Hercules, Calif., USA); N-Lauroylsarcosine Na-salt (Heidelberg, N.Y., USA); thermo-stable DNA Polymerase, ProZyme, (PROtech Technology Ent. Co., Ltd., USA); 100 bp ladder (Pharmacia, Uppsala, Sweden); chloroform (Alps, Taiwan), MgCl 2 , NaCl, Sodium Citrate, NaOH, Boric Acid, KCl (Wako Pure Chemical Industry, Ltd., Osaka Japan). All reagents used in the method according to the invention are of reagent grade of molecular biology grade. 
     D. Immunological Analytical Kits: 
     Immunological analytical kits, SET-RPLA (Staphylococcal Enterotoxin A, B, C, D, detection kit by Reversed Passive Latex Agglutination) is available from Denka Seiken, Tokyo, Japan). 
     E. Buffer Solutions: 
     Buffer solutions used in the method according to the invention are as follow: 
     1. 50×TAE Buffer: 
     242 g Tris-HCl, 57.1 ml glacial acetic acid 100 ml 0.5 M EDTA, pH8.0, and water to 1000 ml. 
     2. PIV Buffer: 
     1 M NaCl, 10 mM EDTA, pH 7.6. 
     3. Chloroform-Isoamyl Alcohol Mixture: 
     Chloroform and isoamyl alcohol mixed at volume ratio of 24:1. 
     4. Lysostaphin Buffer (EC Buffer) 
     6 mM Tris-HCl (pH 7.6), 1 M NaCl, 100 mM EDTA, 0.5% Brij 58, 0.2% deoxycholate, and 0.5% sodium lauroyl sacosin. 
     5. 10×PCR Buffer 
     100 mM Tris-HCl, pH 8.3; 500 mM KCl, 60 mM MgCl 2 , 0.1% Gelatin, and 1% Triton X-100. 
     F. PCR Thermocycler: 
     PCR thermocycler used in the PCR is the Perkin Elmer Gene Amp PCR system 9600 (Perkin-Elmer Coporation, Norwalk, Conn., USA). 
     G. Detection of Staphylococcal Enterotoxin 
     Detection of staphylococcal enterotoxin is carried out by RPLA according to the instruction provided by Denka Seiken and comprises following steps: 
     1. Production of Staphylococcal Enterotoxin 
       S. aureus  on a TSA slant was inoculated in 5 ml BHI broth at 37° C., and incubated by shaking at 160 rpm for 18˜24 hours. 
     2. Treatment of Samples 
     One ml aliquot of the above bacterial suspension was centrifuged in a 1.5-ml micro-centrifuge tube at 3000 rpm for 20 minutes. The supernatant thus obtained was used for assay. 
     3. RPLA Assay 
     To each well of a V-shaped 96-well plate, 25 μl each of sensitized latex A, B, C, or D was added, and, after shaking at 60 rpm for 10 minutes, was incubated at room temperature for 18-24 hours and then observed the result. Separately, standard enterotoxin A, B, C, and D was added into sensitized latex A, B, C, or D and used as positive control, while standard enterotoxin A, B, C, and D was added into non-sensitized control latex A, B, C, or D and used as negative control. 
     H. Extraction of Total DNA from  Staphylococcus aureus    
     A platinum ear amount of bacteria was incubated in 3 ml TSB broth at 37° C. for 12 hours. A 0.5 ml aliquot of bacteria suspension was centrifuged in a micro-centrifuge at 5000 rpm for 7 minutes. The supernatant was discarded. After adding 250 μl of lysostaphin buffer and shaking homogeneously, 25 μl lysostaphin (2 mg/ml), 25 μl lysozyme (2 mg/ml), and 20 μl RNase (2 mg/ml) were added and the mixture was reacted at 37° C. for 2-3 hours to a clear solution. 25 μl proteinase K (10 mg/ml) was added and the mixture was reacted again at 65° C. for 3-4 hours to a clear solution. The reaction mixture was then extracted with an equal volume of saturated phenol-chloroform. After centrifuging at 13000 rpm for 10 minutes, the supernatant was transferred to a new micro-centrifuge and extracted again with an equal volume of saturated phenol-chloroform. The procedure was repeated once. Finally, it was extracted once with equal volume of chloroform. To the supernatant thus obtained was added two volume of 95% ethanol and the resulting mixture was allowed to precipitated at −70° C. for 1 hour. Thereafter, the mixture was centrifuged at 13000 rpm for 15 minutes. The supernatant was discarded. The pellet washed once with 70% ethanol, centrifuged, dried, an appropriate amount of sterile distilled water (30-40 μl) was added to dissolve it and then stored at 4° C. till used. 
     I. PCR Primers 
     Since there is a homology of greater than 97% among gene sequences of enterotoxin C1, C2 and C3, the part of sequence having difference among them is used to design primers useful in PCR. For this purpose, sequence data was obtained by searching through Gopher system Internet connected to biological molecular database Gene Bank/EMBL/DDBJ. The sequence data was subjected to multiple sequence format alignment by means of Wisconsin Sequence Analysis Software Package developed by Genetic Computer Group (GCG) to find out the difference among gene sequences, thereby, based on the difference thus obtained, 4 PCR primers having detection specificity to genes of enterotoxin C1, C2 and C3 were designed. These primers were assigned into 3 pairs, SEQ. ID. NO. 1, SEQ. ID NO. 2 and SEQ. ID NO. 3, having specificity genes of enterotoxin C1, C2 and C3, respectively. 
     J. Synthesis of Oligonucleotide Primers 
     Those 4 PCR primers were synthesized with a DNA Synthesizer by Biotechnology Scientific Co., Taipei, Taiwan. 
     K. Polymerase Chain Reaction 
     To a 0.65-ml micro-centrifuge, 1 μl each of 10 mM dATP, 10 mM dCTP, mM dGTP, and 10 mM dTTP, 5 μl of 10×PCR buffer, 1 μl each of primers (50 pmol/μl), suitable amount of target DNA, suitable amount of 1 unit ProZyme, and sterile distilled water to a total amount of 50 μl. Finally, the mixture was covered with mineral oil. Reaction conditions used in each set of PCR was listed in Table 5. 
     L. Test of PCR Specificity and Sensitivity of Primers 
     (1) Specificity: 
     To a 0.5-ml micro-centrifuge was added a formulated PCR reaction solution with a composition of: 200 μM dNTP (N=A-T-G-C), 1×PCR buffer, 25 or 50 pmole primers, a suitable amount of DNA, 0.4 unit Prozyme, and sterile distilled water to a total volume of 50 μl, and the mixture thus prepared was covered with one drop of mineral oil. Thereafter, the micro-centrifuge was placed in a PCR thermocycler and a three-step PCR was carried out under following conditions: 20 seconds at 94° C. to denaturing DNA into single strand; 35 cycles of lowering the temperature to the annealing temperature for each set of primers (Table 5), keeping at this temperature for 20 seconds for annealing the primers and raising the temperature to 72° C. for 30 seconds for polymerase extension; and finally, keeping at 72° C. for 5 minutes. All steps were controlled by the computer program and 10 μl aliquots of PCR products were taken for analysis as described below. Each sample was analyzed by electrophoresis on 2.0% agarose in 1×TAE buffer, stained with ethidium bromide, observed under a UV box and photographed. 
     (2) Sensitivity: 
     The bacteria suspension was serially diluted at 10×. DNA extraction was carried out with phenol/chloroform as described above. DNA thus obtained was dissolved in 10 μl sterile distilled water and the resulting solution was added in a previously prepared 40 μl PCR solution (200 μM dNTP, N=A, T, G, C). Thereafter, the solution was covered with one drop of mineral oil and PCR was performed as described above. 
     The invention will be described more detailed by means of the following non-limiting examples. 
     EXAMPLE 1 
     Results of the detection and sensitivity test performed on  S. aureus  stains producing standard staphylococcal enterotoxin C1, C2 and C3 and non-C type strains were listed in Table 1 and 2 as well as shown in  FIGS. 1 and 2 , wherein the PCR conditions were the same as described above. It is found that primers designed according to the invention produced specific PCR products only in their targeted enterotoxin-producing strains. These specific PCR products exhibited a size in consistent with the product size as predicted by the original design, i.e., 402 bp, 501 bp, and 672 bp, respectively. Among them, PCR using the primer set of ENTC1/ENTCR could detect standard enterotoxin-producing strain of type C, CCRC 12654 and FRI 137. The standard enterotoxin-producing strain of type C, CCRC 12654, was derived from strain ATCC 19095 that was isolated in the Food Research Institute of the Wisconsin University, USA, and assigned as FRI 137. Other non-staphylococcal bacterial strains could not produce PCR product. These results suggested that those three sets of primers, ENTC1/ENTCR, ENTC2/ENTCR, and ENTC3/ENTCR, exhibited extremely high degree of specificity with respect to genes of enterotoxin-producing  S. aureus  strains of subtypes C1, C2 and C3. Therefore, they can be used to detect whether genes of enterotoxin C1, C2 and C3 is present in a  S. aureus  strains strain. The result of sensitivity test revealed that, since PCR product could be produced using primer set ENTC1/ENTCR, ENTC2/ENTCR and ENTC3/ENTCR at a concentration of 10 0 , the sensitivity could be as high as 10 0  CFU/ml. 
     EXAMPLE 2 
     PCR detections with primer sets ENTC1/ENTCR-ENTC2/ENTCR-ENTC3/ENTCR designed according to the invention were carried out on 39 enterotoxin-producing  S. aureus  strains type C that had been isolated in 20 cases of food poisoning by the Third Branch of the Disease Control Agency, the Health Administration, Executive Yuan, ROC during 1995-2000. PCR and analytical conditions were same as described above. The result is shown in Table 3. 
     It can be seen from Table 3 that identification of subtype C1, C2 and C3 performed on the above-mentioned 39 enterotoxin-producing  S. aureus  type C by PCR using primer sets according to the invention revealed one staphylococcal enterotoxin subtype C1, 12 strains of subtype C2, and 13 strains of subtype C3, as well as 13 strains of other type C. Therefore, among those pathogenic  S. aureus , staphylococcal enterotoxin subtypes C2 and C3 are the prominent ones with a proportion of about 64%, and only 1 subtype C1 strain, while with about 33% of other subtype Cs. 
     EXAMPLE 3 
     PCR detections with primer sets ENTC1/ENTCR-ENTC2/ENTCR-ENTC3/ENTCR designed according to the invention were carried out on 10 enterotoxin-producing  S. aureus  strains type C (FRI strains No 202, 248, 293b, 406, 412, 414, 418, 423, 429, 623) that had been provided by the Food Research Institute (FRI) of the University of Wisconsin, Madison, Wis., USA. PCR and analytical conditions were same as described above. The result is shown in Table 3. 
     It can be seen from Table 3 that identification of subtype C1, C2 and C3 performed on the above-mentioned 39 enterotoxin-producing  S. aureus  type C by PCR using primer sets according to the invention revealed 8 staphylococcal enterotoxin strains of subtype C2, 1 strains of subtype C3, and 1 strains of other type C, while no subtype C1. Therefore, this result is similar to that of Example 2, i.e., among those pathogenic  S. aureus  type C, staphylococcal enterotoxin subtypes C2 and C3 are the prominent ones. 
     Many changes and modifications in the above-described embodiments of the invention can, or course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is intended to be limited only by the scope of the appended claims. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Specificity of the PCR primers for SEC1, SEC2, SEC3 and SEB 
               
               
                 genes of  Staphylococcus aureus  strains 
               
             
          
           
               
                 Source 
                 PCR for SEC subtype 
               
             
          
           
               
                 and 
                 Enterotoxin 
                   
                   
                 ENTC1/ 
                 ENTC2/ 
                 ENTC3/ 
               
               
                 Strain # 
                 type a   
                 RPLA 
                 PCR 
                 ENTCR 
                 ENTCR 
                 ENTCR 
               
               
                   
               
               
                 CCRC 
                 A 
                 A 
                 A 
                 − 
                 − 
                 − 
               
               
                 12657 
               
               
                 CCRC 
                 B 
                 B 
                 B 
                 − 
                 − 
                 − 
               
               
                 12653 
               
               
                 CCRC 
                 C 
                 C 
                 C 
                 + 
                 − 
                 − 
               
               
                 12654 
               
               
                 FRI 137 
                 C1 
                 C 
                 C 
                 + 
                 − 
                 − 
               
               
                 FRI 361 
                 C2 
                 C 
                 C 
                 − 
                 + 
                 − 
               
               
                 FRI 913 
                 C3 
                 C 
                 C 
                 − 
                 − 
                 + 
               
               
                 CCRC 
                 D 
                 D 
                 D 
                 − 
                 − 
                 − 
               
               
                 12660 
               
               
                 CCRC 
                 E 
                 ND b   
                 E 
                 − 
                 − 
                 − 
               
               
                 12656 
               
               
                 CCRC 
                 TSST-1 
                 TSST 
                 TSST-1 
                 − 
                 − 
                 − 
               
               
                 13831 
               
               
                 FRI 572 
                 G 
                 ND 
                 ND 
                 − 
                 − 
                 − 
               
               
                 FRI 569 
                 H 
                 ND 
                 ND 
                 − 
                 − 
                 − 
               
               
                 FRI 445 
                 I 
                 ND 
                 ND 
                 − 
                 − 
                 − 
               
               
                   
               
               
                   a Enterotoxin types are according to the information from strain source. 
               
               
                   b  ND: Not determined. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 PCR assay for bacteria strains other than SEC  S. aureus  using 
               
               
                 ENTC1/ENTCR, ENTC2/ENTCR and ENTC3/ENTCR primer pairs. 
               
             
          
           
               
                   
                 PCR for SEC subtype 
               
             
          
           
               
                   
                 Source and 
                 ENTC1/ 
                 ENTC2/ 
                 ENTC3/ 
               
               
                 Species 
                 strain # 
                 ENTCR 
                 ENTCR 
                 ENTCR 
               
               
                   
               
               
                 
                   Staphylococcus aureus 
                 
                   
                   
                   
                   
               
               
                   S. aureus  SEC1 
                 FRI 137 
                 + 
                 − 
                 − 
               
               
                   S. aureus  SEC2 
                 FRI 361 
                 − 
                 + 
                 − 
               
               
                   S. aureus  SEC3 
                 FRI 913 
                 − 
                 − 
                 + 
               
               
                 
                   S. aureus 
                 
                 CCRC 011 
                 − 
                 − 
                 − 
               
               
                 (non-enterotoxigenic) 
                 CCRC 029 
                 − 
                 − 
                 − 
               
               
                   
                 CCRC 033 
                 − 
                 − 
                 − 
               
               
                 Other bacteria strains 
               
               
                 
                   Staphylococcus epidermids 
                 
                 CCRC 11030 
                 − 
                 − 
                 − 
               
               
                 
                   Staphylococcus xylosus 
                 
                 CCRC 12930 
                 − 
                 − 
                 − 
               
               
                 
                   Streptococcus mutans 
                 
                 CCRC 10793 
                 − 
                 − 
                 − 
               
               
                 
                   Alcaligenes faecalis 
                 
                 CCRC 10828 
                 − 
                 − 
                 − 
               
               
                 
                   Bacillus cereus 
                 
                 CCRC 10603 
                 − 
                 − 
                 − 
               
               
                 
                   Bacillus psychrophilus 
                 
                 CCRC 11738 
                 − 
                 − 
                 − 
               
               
                 
                   Bacillus subtilis 
                 
                 CCRC 10258 
                 − 
                 − 
                 − 
               
               
                 
                   Bacillus thuringiensis 
                 
                 CCRC 14683 
                 − 
                 − 
                 − 
               
               
                 
                   Brevibacterium linens 
                 
                 CCRC 10041 
                 − 
                 − 
                 − 
               
               
                 
                   Clostridium perfringens 
                 
                 CCRC 10914 
                 − 
                 − 
                 − 
               
               
                 
                   Enterobacter aerogenes 
                 
                 CCRC 10370 
                 − 
                 − 
                 − 
               
               
                 
                   Erwinia carotovora 
                 
                 CCRC 11298 
                 − 
                 − 
                 − 
               
               
                 
                   Escherichia coli 
                 
                 ATCC 35401 
                 − 
                 − 
                 − 
               
               
                 
                   Hafnia alyei 
                 
                 CCRC 10906 
                 − 
                 − 
                 − 
               
               
                 
                   Klebsiella marcescens 
                 
                 CCRC 10629 
                 − 
                 − 
                 − 
               
               
                 
                   Kluyvera ascorbata 
                 
                 CCRC 11645 
                 − 
                 − 
                 − 
               
               
                 
                   Micrococcus roseus 
                 
                 CCRC 11577 
                 − 
                 − 
                 − 
               
               
                 
                   Micrococcus varians 
                 
                 CCRC 11272 
                 − 
                 − 
                 − 
               
               
                 
                   Morganella morganii 
                 
                 CCRC 10706 
                 − 
                 − 
                 − 
               
               
                 
                   Proteus vulgaris 
                 
                 CCRC 10728 
                 − 
                 − 
                 − 
               
               
                 
                   Salmonella enteritdis 
                 
                 ATCC 13076 
                 − 
                 − 
                 − 
               
               
                 
                   Salmonella typhimurium 
                 
                 ATCC 14028 
                 − 
                 − 
                 − 
               
               
                 
                   Serratia marcescens 
                 
                 CCRC 13880 
                 − 
                 − 
                 − 
               
               
                 
                   Proteus vulgaris 
                 
                 CCRC 10728 
                 − 
                 − 
                 − 
               
               
                 
                   Yersinia entreocolitica 
                 
                 CCRC 10807 
                 − 
                 − 
                 − 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 PCR identification of the SEC1, C2 and C3 subtypes for SEC  S. aureus  strains 
               
               
                 obtained from randomly selected diarrhea patients engaged with food-borne outbreaks that 
               
               
                 occurred in central Taiwan between 1995 and 2000. 
               
             
          
           
               
                 Out- 
                 Ratio of a   
                   
                 SEC strains 
                 Food 
                 No of strains for each 
               
               
                 breaks 
                 diseased 
                   
                 identified b  by 
                 Samples 
                 SEC subtype c   
               
             
          
           
               
                 no 
                 person 
                 Date 
                 Location 
                 SET-RPLA 
                 associated 
                 C 1   
                 C 2   
                 C 3   
                 Other C 
               
               
                   
               
             
          
           
               
                 1 
                 121/402 
                 March 1995 
                 Yunlin 
                 1 
                 School lunch 
                   
                   
                 1 
                   
               
               
                 2 
                 12/51 
                 March 1995 
                 Nantou 
                 4 
                 Meal boxes 
                   
                 4 
               
               
                 3 
                  66/405 
                 March 1995 
                 Taichung 
                 2 
                 Meal boxes 
                   
                 2 
               
               
                 4 
                 21/38 
                 October 1995 
                 Miaoli 
                 1 
                 Meal boxes 
                   
                   
                 1 
               
               
                 5 
                  22/201 
                 January 1996 
                 Taichung 
                 1 
                 Meal boxes 
                   
                   
                 1 
               
               
                 6 
                  34/393 
                 January 1996 
                 Taichung 
                 2 
                 Meal boxes 
                   
                   
                 2 
               
               
                 7 
                  15/1935 
                 March 1996 
                 Nantou 
                 1 
                 School lunch 
                   
                   
                 1 
               
               
                 8 
                  20/120 
                 April 1996 
                 Taichung 
                 1 
                 Banquet 
                   
                   
                 1 
               
               
                 9 
                  20/279 
                 January 1997 
                 Taichung 
                 1 
                 Meal boxes 
                   
                   
                 1 
               
               
                 10 
                  44/862 
                 March 1997 
                 Taichung 
                 3 
                 Meal boxes 
                   
                 1 
                 1 
                 1 
               
               
                 11 
                 1036/1938 
                 March 1997 
                 Taichung 
                 9 
                 Meal boxes 
                 1 
                 3 
                 3 
                 2 
               
               
                 12 
                  6/25 
                 October 1997 
                 Taichung 
                 3 
                 Meal boxes 
                   
                 1 
                   
                 2 
               
               
                 13 
                  23/300 
                 December 1997 
                 Miaoli 
                 1 
                 Banquet 
                   
                   
                 1 
               
               
                 14 
                 3/3 
                 December 1997 
                 Taichung 
                 1 
                 Lunch 
                   
                 1 
               
               
                 15 
                  78/350 
                 September 1998 
                 Nantou 
                 1 
                 Banquet 
                   
                   
                   
                 1 
               
               
                 16 
                  174/1070 
                 October 1998 
                 Taichung 
                 1 
                 Lunch 
                   
                   
                   
                 1 
               
               
                 17 
                  17/299 
                 November 1998 
                 Nantou 
                 2 
                 Lunch 
                   
                   
                   
                 2 
               
               
                 18 
                 121/840 
                 December 1998 
                 Miaoli 
                 2 
                 Other 
                   
                   
                   
                 2 
               
               
                 19 
                  17/571 
                 November 1998 
                 Changhua 
                 1 
                 Meal boxes 
                   
                   
                   
                 1 
               
               
                 20 
                  17/500 
                 June 1999 
                 Miaoli 
                 1 
                 Lunch 
                   
                   
                   
                 1 
               
             
          
           
               
                 Total number of strains identified 
                 39 
                   
                 1 
                 12 
                 13 
                 13 
               
               
                 Total no. of outbreaks associated with 
                   
                   
                 1 
                 6 
                 10 
                 9 
               
               
                 each SEC subtype 
               
               
                   
               
               
                   a The number of diseased persons over the number of total attendants. 
               
               
                   b Fecal samples from selected diseased persons were collected and subjected to SET-RPLA assay bythe Third Branch of National Center for Disease Control, Taichung, Taiwan. Randomly selected SECstrains were used in this study. 
               
               
                   c SEC subtypes were determined by PCR. SEC strains not grouped in SEC1, C2 and C3 subtypes were termed as other SEC subtype. 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 PCR detection of the SEC subtypes for randomly selected SEC strains 
               
               
                 obtained from FRI a . 
               
             
          
           
               
                   
                 PCR for C type 
               
             
          
           
               
                 Lab. 
                 strain 
                 Enterotoxin 
                 ENTC1/ 
                 ENTC2/ 
                 ENTC3/ 
               
               
                 no. 
                 Source 
                 type 
                 ENTCR 
                 ENTCR 
                 ENTCR 
               
               
                   
               
               
                 6013 
                 FRI-202 
                 C 
                 − 
                 + 
                 − 
               
               
                 6069 
                 FRI-248 
                 C 
                 − 
                 + 
                 − 
               
               
                 6070 
                 FRI-293 b   
                 C 
                 − 
                 − 
                 − 
               
               
                 6071 
                 FRI-406 
                 C 
                 − 
                 + 
                 − 
               
               
                 6072 
                 FRI-412 
                 C 
                 − 
                 + 
                 − 
               
               
                 6073 
                 FRI-414 
                 C 
                 − 
                 + 
                 − 
               
               
                 6074 
                 FRI-418 
                 C 
                 − 
                 + 
                 − 
               
               
                 6075 
                 FRI-423 
                 C 
                 − 
                 + 
                 − 
               
               
                 6076 
                 FRI-429 
                 C 
                 − 
                 − 
                 + 
               
               
                 6077 
                 FRI-623 
                 C 
                 − 
                 + 
                 − 
               
               
                 Total 
                   
                 10 
                 0 
                 8 
                 1 
               
               
                   
               
               
                   a FRI: Food Research Institute, Univ. of Wisconsin, Madison, WI, USA. 
               
               
                   b Strain of FRI-293 may belong to SEC subtypes other than SEC1, C2 and C3. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 The PCR conditions used in this study 
               
             
          
           
               
                   
                   
                   
                 Size of PCR 
               
               
                 Target gene 
                 Primers 
                 PCR condition a   
                 product (bp) 
               
               
                   
               
               
                 entC1 
                 ENTC1/ENTCR 
                 94° C./30 sec 
                 402 
               
               
                   
                   
                 55° C./30 sec 
               
               
                   
                   
                 72° C./20 sec 
               
               
                 entC2 
                 ENTC2/ENTCR 
                 94° C./30 sec 
                 501 
               
               
                   
                   
                 59° C./20 sec 
               
               
                   
                   
                 72° C./20 sec 
               
               
                 entC3 
                 ENTC3/ENTCR 
                 94° C./30 sec 
                 672 
               
               
                   
                   
                 59° C./20 sec 
               
               
                   
                   
                 72° C./20 sec 
               
               
                   
               
               
                   a Total cycles are 35, the first denature time is 3 min at 94° C., and the final extension time is 3 min at 72° C. 
               
             
          
         
       
     
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