Patent Application: US-26146702-A

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:
the invention will be now described below with reference to the drawings . 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 . 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 ). 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 . 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 ). buffer solutions used in the method according to the invention are as follow : 242 g tris - hcl , 57 . 1 ml glacial acetic acid 100 ml 0 . 5 m edta , ph8 . 0 , and water to 1000 ml . 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 . 100 mm tris - hcl , ph 8 . 3 ; 500 mm kcl , 60 mm mgcl 2 , 0 . 1 % gelatin , and 1 % triton x - 100 . pcr thermocycler used in the pcr is the perkin elmer gene amp pcr system 9600 ( perkin - elmer coporation , norwalk , conn ., usa ). detection of staphylococcal enterotoxin is carried out by rpla according to the instruction provided by denka seiken and comprises following steps : 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 . 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 . 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 . 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 . 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 . those 4 pcr primers were synthesized with a dna synthesizer by biotechnology scientific co ., taipei , taiwan . 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 . 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 . 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 . 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 fig1 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 . 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 . 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 . 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 . 3 . becker , k ., r . roth , and g . peter . 1998 . rapid and specific detection of toxigenic staphylococcus aureus : use two multiplex pcr enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes , exfoliative toxin gene , and toxic shock syndrome toxin 1 gene . j . clin . microbiol . 36 : 2548 - 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