TW305937B - An enzyme-linked immunosorbent assay for rapid detection of staphylococcus aureus in processed foods - Google Patents

Abstract

For rapid detection of Staphylococcus aureus in foods, a sandwich enzyme-linked immunosorbent assay (ELISA) was invented. The assay was based on the detection of protein A which is a specific protein secreted by S. aureus. Following a 24-h incubation in astaphylococcal selective broth (SSB) containing mannitol as the carbon source, the culture supernatant was added to the microtiter plate coated with anti-protein A immunoglobulin G (IgG). After incubation, peroxidase-labeled anti-protein A IgG was used to produce the signal of antigen-antibody reaction. The sensitivity of the assay for protein A was 0.1 ng/ml. For 37strains of S. aureus studied, all produced protein A, and the amount (13-1, 100 ng/ml) of protein A secreted by different strains varied to a large degree. Of other 57 strains (including 19 Staphylococcus spp.) of other bacteria tested, two strains (S. capitis subsp. capitis CCRC 12161 and S. lentus CCRC 12926) were found to produce very low amount of protein A (0.6-1 ng/ml) after 24-h incubation. Staphylococcus aureus was detected by ELISA in several processed foods which were naturally contaminated with the bacterium. Twenty two processed foods artificially inoculated with S. aureus at levels of <2 CFU/g and 10-20 CFU/g, respectively, were also found positive by the present immunoassay. As compared to the conventional culture methods which take 5-6 days to complete, the ELISA method can detect low number of S. aureus in processed foods with a total analysis time of only 28 h.

Description

S05937 A6 B6 V. Description of the invention (1) This description part will be divided into: (1) Background description • (2> Process of invention * (3) Results of the invention • (4) Discussing the content of the invention (V) Reference Literature • A total of five points. (1) Background description X. aureus can produce several toxins. • It can cause gastroenteritis (丨 丨, 18). Therefore, the presence of this bacterium in processed food has potential for consumer health. Danger (3) · If the food is improper

I Insulation at temperature will lead to the formation of enterotoxins • In addition • This bacterium is a very important pathogenic bacteria • Often cause people 丨

Class I want to dye (20) · So it is useful if a rapid detection method of the bacteria can be developed · Staphylococcus aureus is easily damaged by heat • It is also sensitive to many disinfectants • So in processed foods or The discovery of this bacterium on the processing equipment indicates that the surgical condition is not good (3) · Because the number of Staphylococcus aureus contained in processed foods is generally not high • Isolation of Staphylococcus aureus usually undergoes an enrichment step ( 2,3. 丨 5) · After proliferation · Selective medium Baird-Parker agar (BPA) is usually used to isolate the bacteria. The coagulase test is usually used to identify whether the colony is Staphylococcus aureus. The entire inspection process takes 5-6 days (two times for growth culture and selective plate culture |

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Day. Colony identification is at least one day> In addition. If the coagulation enzyme test is not very sure (4+ or less) or only part of the coagulation • then the genus will do several auxiliary tests (3,15,23) It is a staphylococcus aureus i »Bacteria · If a specific metabolite of <RTI ID = 0.0> A. Aureus </ RTI> can be found in the propagation culture medium, it can be used as an indicator of the presence of the bacterium It can be greatly reduced. Protein A seems to meet this demand. This protein is a specific product of X. aureus, about 99¾) strains will produce this protein (9). Because of the high sensitivity of enzyme immunoassay Degree. And has been widely used in many laboratories. Therefore, the present invention uses protein A anti-ns and enzyme immunoassay. Developed a rapid detection method of Staphylococcus aureus · (B) the process of invention 1. Yin Yan strain and Peifei __5 _ Heng 4 (210X297 public) ....................................... ................ 3t .............................. # .. ........................ End {please read "Notes on the back" first Then fill out this page)

6 6 AB V. Description of the invention (2) The strains of almond used in this experiment are shown in Table 1. All 94 strains are used to test the production of protein A. Among these strains, 37 are Staphylococcus aureus. 19 strains are from Culture Collection & Research Genter (GCRC) from Hsinchu Food Industry S Exhibition Institute. 18 other strains are isolated from different types of food and identified by traditional methods (3 ) · Other non-Aureus aureus strains for testing are 33 species. A total of 57 strains, also from the Hsinchu City Food Industry Development Institute's Han Species Preservation and Research Center. Except Vibrio enteritidis waAfltf / wfyiimy) and V · vufn v. cui are cultured in trypticase soy agar (TSA), which contains 3 sodium sulfide vapors. Other bacteria are cultured on TSA. • The protein secretion of different strains is measured by enzyme immunoassay (ELISA) When the ability of A is inoculated to the staphylococcal selective broth (SSB) by the strains raised overnight on the TSA • It contains (g / 1): mannitol 20.0 • tryptose 15.0 • sodium pyruvate 10.0 . Glycine 10.0 * Lithium chloride 5.0 · Yeast extract 3.0 · # 气化 纳 (sodium azide) 0.05 and sulfamethazine 0.02 · and cultivated at 37 · 0 24 soil for 2 hours • After 24 hours in SSB culture • Growth status is divided into &quot; ++ &quot; (well growing. &Gt; 108 CFU / m 丨) ··· + · '(poor growth · &lt; 1〇8 CFU / ml) or ···· (not growing) · At the same time. To compare the amount of protein A secretion · Inoculate part of the Staphylococcus aureus strains in tryptic soy broth (TSB>! which contains 10¾ sodium vaporized sodium 1 % Sodium pyruvate • Incubate at 37t for 24 ± 2 hours · |! 2. Anti-protein A IgG (anti-protein A IgG) and immunoglobulin IgG-wasabi peroxidase conjugate (IgG-peroxidase conjugate ) Preparation of protein A for immunization is obtained from the protein S (Pharmacia, Upassalla, Sweden) purified by preparative gel electrophoresis (preparative ge 丨 electrophoresis) * The purification process, immunized animals' Xuanji Fang Zhongqiu The quasi-bureau printed (please read the notes of "Leave the first note before filling in this book)) The harvest of blood treatment (6) ' Bismuthization of immunoglobulin G (IgG) (16). And ISG and horseradishperoxidase (horseradishperoxidase) Hu Hu (conjugation ^ 12) have been published in the literature * 3. Enzyme immunoassay (Enzyme-Linked Immunosorbent Assay , ELISA) T 4 (210X297 膻) _6_ A6 B6 S05S37 5. Invention of Longming (3 &gt; Dissolve anti-protein A immunoglobulin IgG in acid-acid physiological saline (phosphate-buffered saline, PBS, pH 7.2> ; The internal badger degree is 5 μ # πα. Take 0.1 rol and add 96 phase (well) into the eaves hole of the microtiter plate. • Place it at 371C for 1 hour so that IgG is S in the phase hole * Take PBST ( PBS contains 0.02 »of Tween 20> Wash 5 times ♦ Add 0.1 ml of 2 kiss cow blood to each hole

I albumin (bovine serum albumin, BSA) placed at 37t for 1 hour • Wash the microtiter plate with PBST 5 'times • Add 0.1 ml of quasi-protein A solution or the supernatant after incubation (both use 2 »BSA is the diluent) * React at 371C for 1 hour • Wash 5 more times with PBST • And add 0.1 | ml to each well. 2» Antibody and enzyme coupling 10,000 times diluted with BSA》 (anti-protein A-HRP ) · Placed in | 371C for 1 hour • After washing 5 times with PBST • Add 0.1 ml of enzyme matrix per hole (3,3 ·, 5,5 · * I tetramethylbenzidine; kpl, Gaithersburg, MD, USA> · Place in 37_C 5-10 The color of the reaction of the spectroscopy is colored. When the color is colored, add 0.1 ml of 1 M grade phosphoric acid (H3P〇4) to each hole. The reaction solution turns yellow

Color • Absorbance at 450 nm can be measured in a microtiter plate photometer • Negative control test is to replace the sample or standard solution with SSB or bovine blood albumin at 2〇 / 〇 • Perform enzyme in the same way Immunoassay · I

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I 4. Detection of Staphylococcus aureus in food

I The process of detecting and inoculating Staphylococcus aureus from food is shown in Figure 1 • 31 food samples were purchased in batches from supermarkets • Including high and low moisture content, raw and processed foods • Each The samples were analyzed by aerobic plate counting method (2 丨). Then, according to the traditional method of the American Public Health Association (iHA Public Health Association, APHA) (丨 5) and the enzyme immunoassay method, the sample S was tested for the presence of golden grapes The presence of cocci (qualitative analysis) · APHA method • 225 ml of 0.25 mM phosphoric acid balance solution (5) plus 25 g of food in the bottle "in the 7J bottle of the product • smashed at high speed for 2 minutes and then blunt • Take out 50 ml of all foodstuffs and buttercup liquid. • Add to TSB-H containing 50 ml of double gelatinity.% Grade sodium -1¾ sodium pyruvate triangle flask. • Take 37 stems and 48 hours for 2 hours. Take the shoveling liquid of the rafting liquid on Baird-Parker agar (BPA> at 37.C for 48 ± 2 hours. It can be closed on the BPA. Then use the Coagulase test and other auxiliary tests. Confirmation (3, 15> · A 4 (210X297 Koji) .......................................... ... ............ 5t .............................. .................. Line {Please first read "Notes on the back and then fill out this page" Printed by the Central Ministry of Economic Affairs of the Ministry of Economic Affairs Printed by the Hyunjibu Department of Central Committee of the Ministry of Economic Affairs «. 305 ^ 37 A6 B6 V. Description of the invention (4) When using enzyme immunoassay to detect lysates • Take 50 m of the homogenized solution of food S and add it to a 50 ml double-concentration SSB corner bottle • Incubate at 37t for 24 ± 2 畤• Take 0.5 ml of culture medium and centrifuge at 3000 X g for 5 minutes. • Take the supernatant into a microcentrifuge tube containing 0.5 ml of 2Ϊ bovine blood protein. * Determine protein A in the supernatant by enzyme immunoassay. If traditional methods are used to detect the presence of Staphylococcus aureus in food samples, then the most probable number method is used to detect the failure of Staphylococcus aureus in each gram of sample. 5. Food inoculation test i Foods that have not been contaminated with golden yellow grape vines analyzed by traditional methods are used for the inoculation test. • The gold 丨 yellow bud grape Ufl «rei« aFM100 · This strain is the strain that produces the least protein A. Table 1) Incubate overnight on TSA • Take a single colony to float in 10ml 0.25mM phosphoric acid In the slow street solution • Confirm the bacterium failure by the plate counting method (approximately 107 CFU / ml>, use the same phosphoric acid slow street solution as the floating solution of the bacteria to make 10; dilute ito column • take 50 ml of food to homogenize The liquid is added to TSB-10¾ sodium vapor-l &lt; Jb prophyl acid sodium containing 50 ml of double stress. And 50 ml of food homogenous liquid is added to a triangular bottle containing 50 ml of double SSB. 1 ml of appropriately diluted bacterial sleeping float is inoculated into a triangular flask containing food homogenization liquid and culture solution. This results in a low inoculation volume (&lt; 2 CFU / ml) and a high inoculation volume (10 ~ 20 CFU / ml), after cultivating 24 soil for 2 hours at 37t, the SSB culture solution was used to measure the production of protein A by ELISA. • The sample was cultured in \ \ TSB-HMb sodium chloride-1Ϊ sodium pyruvate sample. 2 hours later • The presence of Staphylococcus aureus is detected by traditional methods. The inoculation process is also shown in circle 1. There are two other food-like S-paths of Staphylococcus aureus that are heat-injured. Its The inoculation method is similar. Only the bacteria-eating fluid is heated in a 50t water bath for 5 minutes. Then do the dilution and inoculation! Test · f 4 (210X297 2 · «) ................................................... ......................................................... .... &lt; r…: ..................... &amp; {Please read the precautions on the back before writing this page) _8 A6 B6 V. Description of the invention (5) (3) The results of the invention 1. The S sensitivity of protein A was measured by enzyme immunoassay. The S sensitivity of protein A analyzed by sandwich type EUSA was 0.1 ng / ml (ie 10-12 μ Calculated by | the molecular weight of protein A is 67,000) · This is approximately equal to the bacterial concentration of 10 · 107 CFU / ml (different from different bacterial strains) · Figure 2 is the relationship between the concentration of protein A and the absorbance at 450 nm Curve • When the content of protein A i is between 1-100 ng / ml • The linear relationship between absorbance and protein A content is the best • 2. The ability of different strains to secrete protein A Table 1 is 37 strains of Staphylococcus aureus And 57 other strains in SSB cultured at 37 deaths for 24 hours • Growth status and protein A secretion • Most staphylococci (staphylococci) grow well in the SSB culture medium • Generally, the iodine of the body can be used. 〇8-l〇9CFU / mi * Most Gram-negative bacteria have an iodine level of 107 CFU / ml or less • And some strains cannot grow • At the same time, it can be seen from Table 1 that all S. aureus strains produce protein A. However, Secretion of protein A in SSB • There is a large degree of difference between different S. aureus strains * For example, the amount of S. aureus aFM 003 secreted (1,100 ng / ml) is higher than aFM 100 (13 ng / ml> Some strains that are 85 times higher than β are also cultivated in SSB for 48 hours. The enzyme production of lysine protein A is also detected by enzyme immunoassay. But 48 hours of culture II and 24 hours are not significantly different. 12 strains of Staphylococcus aureus &amp; TSB-101b Tsukihina-1¾ protein A secretion in sodium pyruvate is also determined in the present invention and its secretion Q is generally only 5 to a few tenths in SSB One (Table 1) · There are two strains of rugosa (S. 〇 // «along subsp. CCRC 12161 and 孓 CCRC 12926) after training in SSB for 24 hours • can produce trace amounts of protein A (respectively 0.6 And 1.0 ng / ml) · As shown by the above results · SSB seems to be a good selection for S. aureus Sexual rafting fluid. And does not inhibit the production or secretion of protein A. 9 A 4 (210X297 * 1: hair) ............................... ................................................................. ........... Order ...: ........................ Line {Please first read the notes on the back Fill in this page} 305S37 A6 B6 V. Description of the invention (5) Table 1. Growth status of different bacterial strains in Staphylococcal selective broth (SSB) after 24 hours of cultivation Growth status in SSB a Protein A secretion b nq / ml_

Staphylococcus aureus CCRC 10451 S. aureus CCRCl (m9 5. aureus CCRC 10780 5. aureus CCRC 10781 5. aureus CCRC 10782 5. aur ^ uj CCRC 10786 5. aureus CCRC 10823 5. a «reii5 CCRC 10908 5. CCRC 11551 5. aurc «5 CCRC 11276 S. aureus CCRC 11863 S. aureus CCRC 12154 CCRC 12651 5. auret / 5 CCRC 12652 5. aureus CCRC 12653 5. aureus CCRC 12654 5. aureus CCRC 12655 5. aureus CCRC 12656 5. aure« 5 CCRC 12657 5. aureus aFM 003 S. aureus aFM 004 S. aureus aFM 005 5. aureus aFM 007 5. aureus aFM 010 5. aureus aFM 012 5 i / wmu aFM 023 S. aureus aFM 024 S. aureus aFM 026 S. aureus aFM 047 S. aureus aFM 055 S. aureus aFM 061 S. aureus aFM 071 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 260 (15) 840 450 (175 ) 170 (5) 180 (5) 140 240 (22) 26⑶ 167 (14) 580 140⑹ 100 (5) 250 (6) 150 (5) 72⑷ 233 210 100 152 1100 9⑻ 220 480 330 300 27 870 195 390 310 580 770 Please read the precautions of Μ before you fill in 1Γ binding line (continued on the next page) 10 A 4 (210X297 public) 5. Description of invention (Strain B6 is in the SSB Protein A secretion b Long condition a nq / ml Printed by La Jiye Central Standards Bureau S. aureus aFM 082 S. aureus aFM 100 S. aureus aFM 1Γ6 S. aureus aFM 120 S. aureus aFM 132 Bacillus subtilis CCRC 10029 Bacillus subtilis CCRC 10258 Escherichia coli CCRC 10314 Escherichia coli CCRC 10324 Escherichia coli CCRC 10362 Listeria monoicytogens CCRC 14845 Micrococcus Iteus CCRC 10449 Micrococcus Iteus CCRC 15275 Micrococcus Iteus CCRC 15276 Pseudomonas aeruginosa CCRC 10261 Salmon CCRC 10 10 Salmon CCRC Salmonella typhimurium CCRC 10309 Shigella flexneri CCRC 10265 S. auricularis CCRC 15261 S. auricularis CCRC 15262 S. caapitis subsp. Capitis CCRC 12161 S. caapitis subsp. Capitis CCRC 15231 S. caapitis subsp. Ureolyticus CCRC 15265 S. caapitis subsp. Ure S. camosus CCRC 12922 S. caseolyticus CCRC 15268 5. caseolyticus CCRC 15269 S. cohnii subsp. Cohnii CCRC 12155 S. cohnii subsp. Cohnii C CRC 15224 S. delphini CCRC 15270 S. delphini CCRC 15271 5. epitlermidis CCRC 10783_ ++ + + + + + ++ + ++ + ++ + + ++ 28 13 770 690 405 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00.6 0 0 0 0 0 0 0 0 0 0 0 Please "Precautions on the back of tt before filling in this 1Γ) (Continued from next page) 11 f 4 (210X 297-i: *) V. Description of invention U) Strain S. epidermidis CCRC 15245 S. haemolyticus CCRC 12923 S. haemolyticus CCRC 15238 S. hominis CX: RC 12156 S. hominis CCRC 15228 S. hominis CCRC 15229 S. hyicus subsp. Chromogens CCRC 12924 S. intermedius CCRC 12156 S. intermedius CCRC 15236 S. lentus CCRC 12926 S. lentus CCRC 15259 5. lugdunensis CCRC 1S260 S. saprophyticus CCRC 13977 S. saprophyticus CCRC 15283 S. sciuri subsp. Sciuri CCRC 12927 S. simulans CCRC 10778 S. women CCRC 12929 5. wameri CCRC 13976 S. xylosus CCRC 12930 S. xylosus CCRC 15250 S. xylosus CCRC 15252 Streptococcus mutans CCRC 15256 Streptococcus mutans CCRC 15257 Streptococcus mutans CCRC 15272 Streptococcus mutans CCRC 15273 Vibrio parahaemolyticus CCRC 12 965 Vibrio vulnificus CCRC 10905_ A6 B6 Biomass A secretion in SSB b Long condition a nq / rol + + + + + + + + + 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (Please read the notes on the back of the stick before writing this page)

a ++, good growth; seven poor growth;-, no growth b The numbers in parentheses are golden yellow unloaded grapes grown in TSB-1 () ¾ sodium sulfide trans 1⁄4 pyruvate sodium 24 small rice * secreted protein A's S% h P i A4 (210X297 ^: No) 12 ^ Qi Bad Central Central Huahua Seal «. A6 B6 V. · Description of the invention &lt; 9 &gt; 3. Detection of Staphylococcus aureus in food Of the 31 food samples • 9 of the traditional analysis methods contained Staphylococcus aureus (bacterial failure is between 4 * 240 CFU / g). • Among the 9 suitable samples, 6 were detected by enzyme immunoassay. Presence of protein A • The 6 samples are all heat-processed products • The 3 samples with false negatives (beef, pork and male) are fresh foods and contain high levels of bacteria (l〇5 -l〇6cFU / ml) · Other 22 processed foods that do not contain A. aureus were used for inoculation test. The strain used for inoculation was aFM 100 a. aureus. · The inoculation amount was divided into low amounts (&lt; 2 CFU / g) and high setting (10 * 20 CFU / g) · After inoculation, analysis by Futong method and enzyme immunoassay • The results are shown in Table 2 • Even at low inoculation In all 22 food samples, the presence of Aureus aureus can be detected by these two methods. When the amount of bacteria is high, the bacteria can also be completely detected (the result of high inoculation of Dong, not listed in Table 2) · Staphylococcus aureus if inoculated with heating injury (50t, 15 points setting) · Whether at high or low inoculation doses can also be measured by two analytical methods (Table 2) · (4) Contents of the invention In the present invention, an enzyme immunoassay method is proposed to rapidly detect Staphylococcus aureus in processed foods. The antibody used is protein A antibody. The enzyme immunoassay method detects protein A Very high sensitivity (0.1 ng / ml or 10-12 M) · This may be due to the use of anti-protein A IgG instead of normal serum IgG (protein serum IgG) and protein A will interact with many animal immunoglobulins The Fc fragment of G (ISG>) has a specific binding ability (丨 〇) · The advantage of using anti-protein A IgG is in addition to the Fc fragment · The Fab fragment of IgG can also bind protein A with strong and specific binding • If the normal rabbit IgG is used in the immunization method The anti-S5 of non-protein A. The S sensitivity is 丨 ng / ml. This sensitivity is one-tenth of the anti-hermetic protein. Protein A exists on the cell wall of the golden staphylococcus aureus (丨 7) and cell wall The covalent bond of pepiidoglycan is about 99 T. The Staphylococcus aureus strain contains 13 T4 (210X 297 ^ 'Α') on the cell wall ... ....................................... ^ ......... .....................hit…:.......................... / *. Please first "« Precautions on the back and then fill in this? Γ) A6 B6 V. Explanation of 奁 明 (i〇) Table 2. Results of detection of golden staphylococcus aureus in food by enzyme immunoassay and traditional methods鮏: -3-¾-Central Approved Print ^ The number of bacteria in food (CFU / g) The number of original Staphylococcus aureus (MPN / g) The number of inoculated Staphylococcus aureus (CFU / ιϊ) Test method Enzyme free traditional Epidemic analysis analysis method beef (raw) 6.7 X 106 15 • + chicken (raw) 2.4 X 1〇5 23-+ pork (raw) 2.5 X 105 4-+ beef 2.0 X 1〇5 240 + + beef 6.3 X 105 64 + + Fried chicken legs 7.0 X 101 4 + Imitation crab meat 1.6 X 102 9 + Gong Jiu 3.1 X 102 23 + oil noodles 1.5 X 105 43 + cheese 1.9 X 1〇3 NDb 0.8 + + milk 61 8.4 X 1〇4 ND 0.8 + + cheese 1.0 X 102 ND 0.9C + + corn Slices 2.5 X 101 ND 0.9 + + Cornflakes 5 ND 0.9 + + Xianli 2.6 X 101 ND 1.0 + + Yujiu 1.1 X 102 ND 1.1 + + Fried male pieces 1.5 X 102 ND 1.0 + + Fried chicken pieces 7.0 X 101 ND , 0.9C + Hamburger 7.0 X 101 ND 1.2 + + Yugui &lt; 10 ND 1.1 + + salad dressing 5 ND 1.0 + + salad dressing &lt; 10 ND 1.0 + + milk powder 6.3 X 102 ND 0.8 + + milk powder 3.5 X 102 ND ll + + milk powder 1.2 X 102 ND 0.9C + + peanut mule 2.0 X 101 ND 1.6 + + peanut inspection &lt; 10 ND 1.6 + + sticky meat 1.8 X 105 ND 1.6 + + station meat 1.7 X 103 ND 1.6 + + tribute Maru 6.5x 101 ND 1.1 + + Rice 5.0x 101 ND 1.2 + + (Continued on next page) U A 4 (210X25) 7 (public) ........................ ......................... St .................. ............ ^ ...: .................. {Please first read the notes on the back and then fill out this page) A6 B6 «£ 邙 中 中 掃 擃 變局 印 装 V. Invention Shuangming (11) a Unless otherwise specified • No Various test foods are processed. B. The presence of Staphylococcus aureus is not detected. C. Inoculation of heat-treated Staphylococcus aureus 15 T 4 (210X297) ............. .............................. X ............. .................. &lt; Γ…: .............. xk {Please first "« Back Note I need to fill out this I) A6 B6 5. Description of the invention (12) Protein KA (9) · However, the content of protein A in different strains is significantly different (6,8.) · Although protein A is the One of the main components of the bacterial cell wall · But about 8.30 »protein A will be secreted in the log phase (19, 22) · and only these secreted protein A can be used for enzyme immunoassay Measured • Although protein A is a special product of Staphylococcus aureus · but this characteristic has never been applied to enzyme immunoassay (ELISA) before the present invention • As an important rapid detection of microorganisms use·

In this "Mingzhong Liang I, I tried 37 strains of A. aureus. All produced protein A (Table 1). Therefore, the false-negative reaction was 0¾. • However, its yield (B-1,100ng / tnl &gt.; There are great differences between different strains. Two strains of the genus Xanthococcus (brother αι / Λίί subsp. CCRC 12161 and // iftuCCRC 12926) after 24 hours of cultivation in SSB • A very small amount of protein A ( &lt; 1 ng / inl) secreted • Therefore • the current enzyme immunoassay may still have a false positive reaction • however • these two strains of bacteria should be reached to about l〇7-l〇8CFU / ml or more. Immunoassay detects the presence of protein A. In the present invention, E is adjusted to an appropriate culture solution. It can selectively grow X. aureus. It does not inhibit protein A secretion. It is one of the successful inventions. Important factors • Because X. aureus has a high tolerance to toilets, it is common to use a medium containing high toilets (such as 10¾ sodium chloride>) to do selective proliferation of the bacteria. • However, under high a Inhibits the secretion of protein a (Table 1) · makes the enzyme immune The analysis method is more difficult to detect • There are more false negative reactions • The components of the SSB in the present invention are: mannitol, 'gas chemistry (lithium chloride), glycine, glycine, sodium propionate (Sodium pyruvate) 'fi azide and sulfamethazine> mannitol is a carbon source. Sodium propionate can help the recovery of injured golden chrysanthemum (14)愎 fi sodium azide (sodium azide), hindering agent (sulfamethazine) and Tsukiwa Si (lithium chloride) shellfish U is used to inhibit the growth of Gram-negative nucleus and other plants (丨 5) The strains used in the experiment * (S. aFM 100> is the strain with the lowest protein A secretion (Table 1> · Even so • Only about 1 gold per gram in various processed foods (Table 2) Staphylococcus aureus bacterium * after 24 hours of cultivating · Enzyme immunoassay can also effectively detect its 16 f 4 (210X297 g) ......................... ................................ St .................... ............. ίτ…: .................... margin {Please first run #Notes on the back then fill in } 305937 A6 B6 V. Description of the invention (13) {U first "read the precautions on the back and then fill out this page] Existence · Several kinds of foods that are contaminated with Staphylococcus aureus can also be analyzed, but Enzyme immunoassay method for detecting raw foods (such as raw beef, raw pork and meat) is not as sensitive as traditional methods. Sometimes this bacteria cannot be detected. This may be due to the complex microbial phase (raw Bacteria count l〇5-l〇6CFU / g) · There is a strong competition between bacteria. Therefore, the enzyme immunoassay method of the present invention can be used in processed foods, and samples with low adult bacteria count · Use traditional methods to detect low-bacterial Staphylococcus aureus in foods • It takes at least 5-6 days (proliferation culture 2 days • selective plate culture 2 days and 1-2 days of coagulation enzyme test) Enzyme immunoassay requires only 28 hours (24 hours growth in SSB • Enzyme immunoassay requires only 4 hours> Although there have been reports recently (7) latex microparticle agglutination reaction method can be used to quickly identify food source of S. aureus • However, this type of test requires Only isolated colonies or pure bacteria can be cultured. • Cannot be detected directly from the proliferation culture medium. • Generally speaking, the sensitivity and specificity of the enzyme immunoassay method described in this book are quite good (Qihe and Table A) In addition, the immunoassay may also be used clinically. The inventor once inoculated a urine sample of about 1 CFU / m 丨 aureus aureus aFM 100 in a urine sample. Incubated in SSB at 37′C for 24 Hours • The presence of Staphylococcus aureus can be detected by the analysis method in the present invention · Although the enzyme immunoassay has been used to detect Salmonella (1, 13) and other food poisoning bacteria (4) · The present invention is The first application of this analysis method for the rapid detection of Staphylococcus aureus · A4 (210X297 public release) 赀 浒 邙 Central fe quasi-bureau printed A6 _ B6__ Five, invented "Ming (W) (五-) Reference 1. Anderson, JM, and PA Hartman. 1985. Direct immunoassay for detection of |

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salmonellae in foods and feeds. Appl. Environ. Microbiol. 49: 1124-1127. I 2. Association Official Analytical Chemists. 1990. Staphylococcus aureus in foods,

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Secs. 980.37 and 987.09. In K. Helrich (ed.), Official methods of analysis. 15th ed „AOAC, Arlington, VA. 3. Bennett, RW, and GA Lancette. 1992. Staphylococcus aureus, p. 161-166. In Bacteriological analytical manual. 7th ed., AO AC International, Arlington, VA. 4. Bhunia, A., and MG Johnson. 1992. Monoclonal antibody speciflc for Listeria .monocytogenes associated with a 66-kilodalton cell surface antigen. Appl. Environ . Microbiol. 58: 1924-1929. 5. Butterfield's phosphate-buffered dilution water, p. 511. In Bacteriological analytical manual. 7th ed .. AOAC International, Arlington, VA. 6. Chang, TC, and SHC Ding. 1992. Rapid detection of Staphylococcus aureus in food by flow cytometry. J. Rapid Methods Automation Microbiol. 1: 133-147. 7. Chang, TC, and SH Huang. 1993. Evaluation of a latex agglutination test for rapid identiflcation of Staphylococcus aureus. J . Food Prot. (In press) 8. Cohen, JO 1972. Normally occurring antibodies for S. aureus, p. 357 * 363. In JO Cohen (ed.) F The staphylococci. John Wiley &amp; Sons, New York. 9. Forsgren, A. 1970. Significance of protein A production by staphylococci. Infect. Immun. 2: 672-673. 10. Goding, JW 1978. Use of staphylococcal protein A as an immunological reagent. J. Immunol. Methods. 20: 241-253. 18 f 4 (21〇Χ297 ^ Αβ) ............... ................................. X ............... ................hit…:! ..............: A {Please M «Precautions on the back and then« this page> Printed by the Ministry of Finance and Economics 4i A6 B6 V. Description of the invention (15) 11. Halpin-Dohnalek, M. 1., and EH Marth. 1989. Staphylococcus aureus: production of extracellular compounds and behavior in foods-a review. J. Food Pr〇L 52: 267-282. 12. Hudson, L. , and FC Hay. 1989. Affinity techniques for molecules and cells, p. | 322-329. In Practical immunology. 3rd ed. Blackwell Scientific Publications, London. 13. Kerr, S., HJ Ball, DP Mackie, DA Pollock, and DA Finlay. 1992. Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of salmonella. J. Appl. Microbiol. 72: 302-308. 14. Lancette, GA, and J. Lanier. 1987. Most probable number method for isolation and enumeration of Staphylococcus aureus in foods: collaborative study. J. Assoc. Off. Anal. Chem. 70: 35-38. 15. Lancette, GA, and SR Tatini. 1990. Staphylococcus aureus, p. 533-550. In C. Vanderzant and DF Splittsto esser (eds.). Compendium of methods for the microbiological examination of foods. 3rd ed. American Public Health Association, Washington, DC. 16. Linn, TGt AL Greenleaf, RG Shorenstein, and R. Losick. 1973. Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation. Proc. Natl. Acad. Sci. USA. 70: 1865-1869. 17. Lofkvisl, T., and J. Sjoquist. 1962. Chemical and serological analysis of antigen preparations from Staphylococcus aureus A comparison between the products obtained by Vcrwey's and Jensen's techniques. Acta Pathol. Microbiol. Scand. 56: 295-3 () 4. IX. Minor, TE, and EH Marth. 1976. Staphyl (x: ucci and their significance in ftwuJs, p. 99-125. Elsevier Scientific Publishing, Inc., Amsterdam. Gan 4 (210X297 public release) ..................................... ...................... ¾ .................................... .... ir ............................... Λ {Please first read the notes on the back Qi Qiwen π) 19 _ A6 B6 i, ft Ming Shuangming (16> 19. Movitz, J. 1976. Fo rmation of extracellular protein A by Staphylococcus aureus. Eur. J. Biochem. 68: 291-299. 20. Nelles, MJ, CA Niswander, WW Karakawa, WF Vann, and RD Arbeit. 1985. Reactivity of type-specific monoclonal antibodies with Staphylococcus aureus clinical isolates and puriHed capsular polysaccharide. Infect Immun. 49: 14-18. 21. Peeler, JT, and LJ Maturin. 1992. Aerobic plate count, p. 17-26. In Bactenological analytical manual. 7th ed., AOAC International, Arlington, VA. 22. Sjoquist, J., J. Movitz, IB. Johansson, and H. Hjelm. 1972. Localization of protein A in the bacteria. Eur. J. Biochem. 30: 190-194. 23. Sperber, WH, and SR Tatani. 1975. Interpretation of the tube coagulase test for identification of Staphylococcus aureus. Appl. Microbiol. 29: 502-505. (6) Brief description of the diagrams Figure 1. Traditional methods or enzyme immunoassays Flow chart for analysis of Staphylococcus aureus in foods Figure 2. Standard curve for quantification of protein A by enzyme immunoassay (please note the * first Xiang Zai wrote this page &gt; .R. • Da · 54 Hu Zhong Zhong Xing Youyang Bureau Printed ο 2 «Ρ4 (210Χ297 male dragon)

Claims (1)

ΤΓ A8 B8 C8 D8 ", Patent application No. 8210016" "Method for rapid detection of Staphylococcus aureus in processed foods by enzyme immunoassay method j Patent case (S5 η month amendment) Enzyme immunoassay method (Enzyme- Linked Immunosorbent Assay) method for detecting Staphylococcus aureus in food. The detection object is protein A. The steps are as follows:-(1). Inoculate food samples in Staphylococcus aureus selective culture solution (staphylococcal selective broth, SSB), which causes S. aureus in food to multiply in the culture broth and produce protein A; wherein the staphylococcal selective broth uses mannitol as the main carbon source and contains sodium azide (Sodium azide) or chloride chloride (lithium chloride) or amine amplifying agent (sulfamethazine); (2). Enzyme immunoassay to detect the step ⑴ the presence of protein A in the culture, the steps are: (i) . An antibody coated with protein A or a substance capable of binding protein A on a solid phase (ii). Add the sample solution after incubation in SSB In step (i), protein A binds to the antibody (or substance that can bind to protein A); (iii). Add antibody (or substance that can bind to protein A) and the enzyme complex of enzyme to form the antibody (or Substance capable of binding to Protein A) —Protein A ”antibody (or a complex of a substance capable of binding to Protein A and a lotus complex; (iv). Add the enzyme matrix in step (iii) to produce a color reaction; ( 3). According to the detection result of step (2), the presence of protein A can be used to infer the presence of Staphylococcus aureus in the food sample. 1 According to the method of item 1 of the patent application, the antibody used may be compatible with protein A The combined substance is immunoglobulin white G (IgG), or Fc fragment of IgG (Fc fragment) 21 This paper size is applicable to China National Standard (CNS) A4 specification (210X297mm) --------- -1 ------ lsτ ------ 0 (Please read the notes on the back before filling out this page) Printed by the Employee Consumer Cooperative of the Central Standard Rating Bureau of the Ministry of Economic Affairs