TW514640B - Methods for identification and detection of listeria monocytogenes - Google Patents

Abstract

Listeria monocytogenes is a G(+), facultatively anaerobic, non-spore forming rod which can grow over a wide range of temperature. The wide growth temperature range of L.monocytogenes, coupled with its pathogenicity, makes this microorganism a potential hazard in food products. Conventional procedures for the identification of L.monocytogenes are tedious and time-consuming. Immunoassays such as ELISA and latex agglutination for rapid detection and identification of L.monocytogenes by antibodies specifically against L.monocytogenes 68-kDa surface protein were disclosed.

Description

Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 514640 A7 V. Description of the Invention (i The present invention is a method for rapid identification and detection of Listeria monocytogenes (Lα), and more specifically, a method using surface proteins resistant to this bacteria Immunoassay for the identification and detection of Listeria monocytogenes with antibodies or antiserum. Listeria monocytogenes is an important food poisoning bacterium, widely distributed in nature. Unlike most pathogenic bacteria, Listeria monocytogenes Bacteria have the characteristics of low temperature growth, high temperature resistance and high salt concentration (Gahan and Collins, 1991). In recent years, the number of cases of listeriosis in European and American countries has increased year by year, and there have been reports of as high as 23% mortality. (Farber and Peterkm, 1991). Due to its ability to infect people and livestock with food, the contamination of Listeria monocytogenes in food has received extensive attention and research. Traditional methods for the detection of Listeria monocytogenes (FDA and USDA) Suggested method) Pre-proliferation, selective culture, and a series of biochemical, hemolytic, pathogenic, and serum tests are required. It takes less than 7 days (Lovett, α / · 5 1987). In recent years, a radioactive DNA probe (probe) method, enzyme immunoassay method, immunofluorescence microscopy, and DNA have been used. There are quite a few studies on DNA hybridization or polymerase chain reaction for rapid determination of Listeria monocytogenes, and commercial kits have been introduced, such as Gene-Trak (Framingham, MA, USA), Listertest (VICAM, Watertown, MA, USA) and Listeria-Tek (Organon Technika Corp .: Durham, NC, USA), but these methods can only detect bacteria of the genus Listeria (L / wer / a spp.) And cannot Identification of Listeria monocytogenes (B. m⑽oc ioge / ια) (Eley, 1990; Johnson and Lattuada, 1993). Therefore, if a fast, simple and accurate identification and detection method can be developed for food Hygiene and safety regulations will help. The present invention is to provide a species of Listeria monocytogenes with species specificity. The paper size is applicable to the Chinese National Standard (CNS) Λ4 specification (2 丨 0> < 297 male Li), v '< r (Please read the notes on the back before filling this page)

A7 B7 V. Description of the invention (2) ------—— White matter 'has a molecular weight of 68, daitQn, and it is used as an antigen to build a cleaned or purified antibody, and cooperate with immunoassay Method technology, rapid identification and detection of Listeria monocytogenes. In the mouth, M provides a method for rapid identification of Listeria monocytogenes and a method for detecting whether the test sample contains Listeria monocytogenes. The method of the present invention is extremely simple, economical, and does not determine whether the suspicious colony is a Listeria monocytogenes within 4 days, and has sensitivity and specificity (ecifidty). The method of this invention is to perform immunoassay reaction on 68,000 Da Er size cells of Listeria monocytogenes. A species-specific surface protein with a molecular weight of 685qq Dalton, which is species-specific, is used as an antigen to immunize Dou to produce antisera and prepare and purify antibodies, and The antibody is used for immunoassay and can be used in conjunction with the selective medium of Listeria monocytogenes. It can also be combined with simple biochemical tests (such as the activity of hemolysin, cyclohexyl phosphate phosphate lyase, or arylamine knife lyase) that may cause cross-reactivity in L. / foot oc⑽ and B _ from zmer / and Liz Distinguish from Temonas. For the purpose of experimental research, part of the present invention has been published publicly in JO 1996. Of The Food Protection, Vol. 59, No. 11, 1996, pages 1176-1181 (Please read the precautions on the back before (Fill in this page). Binding and printing of the consumer cooperation agreement of the Central Bureau of Commerce of the Ministry of Economic Affairs ¾ A more detailed description of the subject matter of the present invention. The technical content and characteristics are shown in the following figures, descriptions and examples. The present invention provides a species-specific Listeria monocytogenes protein with a molecular weight of 68,000 Daltons, which is used as an antigen to immunize Animals, using a few serum or purified antibodies, combined with the technique of immunoassay methods can be established 3

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ^ 3. 3. 〇, 〇 05. Description of the invention (, method for identification and detection of Listeria monocytogenes. The present invention also provides discernable antibodies. The Kangxian County L7 本 虹 ⑭ #Early cell animals. "The surface protein of Listeria monocytogenes is used as an antigen to immunize the outer membrane of known Gram-negative bacteria, which contains some species-specific _⑽ specifri 、 焉Antigenic protein f (Kabir, i98〇; Kabir, 1986). According to the electrophoresis analysis of the surface protein of Listeria monocytogenes according to the present invention, it was found that Listeria monocytogenes, which is a Gram-positive bacterium, also has this characteristic. Protein, molecular weight is 68, _Dorton. = The properties of this protein are not well understood, but it can be seen that this is the surface protein of A. monocytogenes of Listeria monocytogenes (see lines 1 and 7 in Figure 1 and Figure 2) The surface protein of Listeria monocytogenes can be purified by conventional procedures, such as the method (Koga) described by Koga's and Kawata's. A preferred embodiment of the present invention uses sodium dodecyl sulfate Polyacrylamide Film Electrophoresis (SDS_ PAGE) Separation A protein band with a molecular weight of 68 000 Daurium is directly cut from the SDS-PAGE film. This method is a convenient and feasible method for the purpose of immunization and to obtain high-purity proteins. The present invention The antiserum or antibody used can be prepared and purified by generally known standard procedures. In the present invention, a surface protein is used as an antigen, and a tape containing the surface protein is directly cut from the electrophoresis film and ground into very fine particles. It is then emulsified with Freund's incomplete adjuvant and then immunized animals. The invention also provides a method for quickly identifying Listeria monocytogenes and a method for detecting whether the sample 47 to be tested contains Listeria monocytogenes .It is an immunoassay reaction for 68 Daulton-sized cell surface proteins of Listeria monocytogenes. This antibody is used in immunoassay methods. —___ 8 This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) ) (Please read the precautions on the back before filling out this page)-Must ·, τ Γ

Printed by the Consumer Cooperatives of the Central Sample Bureau of the Ministry of Economic Affairs

514640 A7 _B7____ 5. Description of the invention (4) The method of the present invention can be completed by any technique known in immunoassay methods. For example, a linked enzyme immunoassay (ELISA), a latex agglutination test (latex agglutination test), and the like are not limited thereto. The immunoassay method used in the method of the present invention can be performed according to a method well known in the art. The linked enzyme immunoassay method of the present invention can be applied to any solid phase, such as a well of a microtiter plate, but is not limited thereto. That is, the antibody is first adsorbed and fixed in a slot of a microtiter disk as a capture antibody; and a light-body (IgG-peroxidase conjugate) (Hudson and Hay, 1989) of the antibody and an enzyme is prepared as a test antibody. With these two methods, a sandwich-linked enzyme immunoassay test was performed on the treated colony proliferation solution to be tested. The linked enzyme immunoassay may include the following steps: (1) an antibody coated with the above protein on a solid phase; (2) reacting a cell surface protein of a strain to be identified with the antibody coated on the solid phase, so that the solid phase A complex of antibody-68,000,000 Daurium cell surface proteins is formed on the surface; (3) The antibody (antibody-enzyme conjugate) labeled with an enzyme is added to form an antibody on the solid phase at 68,000 Daltons Cell surface protein-antibody-enzyme complex; (4) A matrix of enzymes is added to observe the reaction results of the linked enzyme immunoassay. The linked enzyme immunoassay may also include the following steps: (1) an antibody coated with the above protein on a solid phase; (2) the cell surface protein of the g strain to be identified and the antibody coated on the solid phase 33.3. 10,000 (Please read the notes on the back before filling out this page). Binding-Order printed by the Central Consumers Bureau of the Ministry of Economic Affairs, Consumer Cooperatives 514640 A7 B7 V. Description of the invention (5) ~ ^. The reaction will form on the solid phase Antibody_68, 00 Dalton cell surface ^ complex; & Tau (3) Add the 68,000 Dalton cell surface protein secondary antibody and the secondary antibody labeled with the enzyme antibody (antibody) The antibody-antibody conjugate of the secondary antibody ') includes an antibody-68,000 dal cell surface protein, a complex of an antibody-antibody secondary antibody antibody complex on the solid phase; human (4) The substrate of the enzyme is added to observe the reaction and analysis of the linked enzyme immunoassay method. The enzyme used can be a peroxidase; the cell surface protein of the strain to be determined can be extracted with a surfactant. , Such as Teep〇l or sulfur Sodium dodecyl sulfate ° The test steps in a specific embodiment of the present invention include the following procedures: first, the antibody is adsorbed and fixed in the slot of the microtiter disk to contain a blocking protein (such as bovine serum protein) Bovine serum albumin (BSA) buffer fills any remaining protein-absorbable sites in the cavity. Add the treated colony proliferation solution to be tested, wash with bovine serum protein-containing buffer solution, add the antibody-enzyme coupling for testing, and then add the enzyme matrix (3,3 ', 5,5'-tetramethylbenzidine kpl, Gaitherburg, MD, USA) for color reaction. After proper coloration, the reaction was stopped by adding 1M arsenic acid (H3P04), and the absorbance at 450 nm was measured in a microplate photometer. In the negative control test, a sterile culture solution containing 2% bovine serum protein was used in place of the bacterial culture solution, and a linked enzyme immunoassay was performed in the same manner. The latex agglomeration method of the present invention can select any latex particles suitable for observing the color of the agglutination reaction. The latex particles are first coated with an antibody, that is, an antibody containing an appropriate concentration of anti- Listeria surface protein is reacted with the latex particles to complete. The concentration of antibodies used _ιο__ ^ Paper wave scale is applicable to China National Standard (CNS) A4 specifications (210X297 male H 83 3. 10,000 (Please read the precautions on the back before filling this page)

514640 A7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (6 degrees depends on the price of the antibody and the needs of the manufacturer. Those skilled in the art can easily determine the appropriate concentration of this solution. In the specific embodiment, the antibody wave length of 70 tons / shank is used. The antibody coating step includes the following procedures: first wash the latex particles (such as salt-filling buffer) with a buffer solution, and then the antibody and milk dissolved in the buffer solution The fine particles are mixed to make the antibodies adsorb on the latex particles, and then the excess antibodies are removed by centrifugation, and washed with human buffered protein (such as bovine serum albumin) buffer solution. According to this, the coated latex particles are prepared: It can be used as a reagent for the agglutination reaction test. The method of the present invention can also be completed on a glass slide. For example, two circles are drawn on the slide with a marker pen, and one or two colonies are picked out by a sterile toothpick and moved to the circle on the broken piece. Add one drop (25 μΐ) of the control latex microparticle reagent to one of the circles, and add one drop of antibody-coated latex microparticle reagent to the other circle, and evenly apply η β λ ^ + with a toothpick % j ”soil open and slightly shake back and forth for 4 minutes' to observe whether a coacervation reaction has occurred and record it. If a coacervation reaction is found in the circle of the latex particle reagent with the coated antibody, the control group still maintains the milky white spoon-like shape as a positive reaction. ; If there is no agglutination reaction in the two circles on the slide, it is recorded as a negative reaction. Occasionally, if the agglutination reaction occurs in both circles, it shows that it has auto-aggludnation. At this time, the test Invalid, it should be identified by traditional methods. The present invention can also be used with the selective culture of Listeria monocytogenes, or use selective media to screen out suspected colonies in the sample to be determined for identification. Samples detected by the method of the present invention 'Suspicious colonies can be isolated first in a selective soil lipid culture medium. According to conventional techniques (Lovett d (//., 1987), the sample can usually be cultured in (001 // protein water, peptone water). The solution is proliferated and then inoculated onto a selective soil lipid medium (such as Oxford medium or lithium chonde-phenylethanoUmO Xalactam). This paper is applicable to paper National Standards (CNS) A4 size (210X 297 mm) 10,000 83. 3. (Please read the Notes on the back to fill out Page) - installed ·

1T »line 514640

V. Description of the invention (Cultivation and screening of suspicious colonies. The invention can also be combined with simple biochemical tests (such as the activity of lysin, cyclohexanol phosphate-lipolytic enzyme, or arylamine-decomposing enzyme activity) may cause crossover The reaction CDB and "we / Aimen · and Listeria monocytogenes are distinguished. Cyclohexyl phosphate phospholipase (ρι PLC) activity analysis method (Notermans, etal, 1991) can be performed as follows: The to-be-determined strain was inoculated on a τγ plate (1% twone, 0.05% yeast extract, 丨% NaC1, i ° / 〇agar) 'incubate at 37 ° C for 24-48 hours; add 4 to the plate Ml, 55 ° C, agar gelatin containing LW-phosphatidylmositol (10mg / mL) ④ 7% at 20% of heart rate, pH 7.0.) 'Cultivation, observation; opaque circles around colonies are: (+) Strains (ie, have PI-PLC activity). Hemolysin analysis method (chest_ca, ^, ΐ99ΐ) can be performed as follows: The colonies from TSYAE plates are punctured and inoculated to 5% bl00d. = h_ blood agar), cultured under muscles for 48 hours; observed under bright light for colonies Blood spots. The method for analyzing the activity of arylamine-decomposing enzymes (professional, ^ •, calendar) can be carried out in the following manner: using a commercially available inspection agent milk ❿ Franee) (of which DIMtest). Central Bureau of Standards, Ministry of Economic Affairs Printed by employee consumer cooperatives-ruler-sheet; paper literary standard I home country I country I used in centimeters 97 (Please read the precautions on the back before filling this page)

514640

V. Description of the invention (Z-1) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs The sandwich-linked enzyme immunoassay method of the present invention can obtain a sensitivity of 98%, and the false negative rate is very low (1.96%). Although in a specific embodiment of the present invention, 6 strains of L · / train ocwa and 3 strains of L · we / Mzmer / produced false positives, this cross-reaction can be solved by simple biochemical tests, such as the production of lysin A, phosphate ring Is hexanoic acid-specific phospholipase C (Plplc) active? Is there any arylamidase activity?丨 Cross-reacted B ⑽ and L.㈣ / Mitten r / · and Listeria monocytogenes were distinguished. Notermans et al. (Notennans α /., 1991) tested 468 Listeria bacteria and found that only Listeria monocytogenes and ζναηονζ ... have cyclohexyl phosphate phosphatase activity 'this enzyme Acting on the matrix L-oc-phosphatidylinositol, it produces insoluble diacylglycerol and opaque rings around the cells (Leimeister-WSchtei * with α / ·, 1991). In addition, 99.4% L. z> 2⑽c⑽ can produce arylamine-decomposing enzymes, while 97.7% of Listeria monocytogenes does not have this enzyme activity (BiUe with α /., 1992), and the arylamine knife carrageenase is hydrolyzed. The enzyme matrix amin〇-mouth mouth susceptible to ylamide, the generated naphthylarmne can interact with the coloring agent Fast Violet β β, and orange-red reaction occurs, which can distinguish between Listeria monocytogenes and M Mα. Therefore, the test of cyclohexyl hexanoic acid lipolytic enzyme or arylamine degrading enzyme enzyme activity can make Listeria monocytogenes (Polyhexahexanophosphate phosphate lipolytic enzyme positive reaction, arylamine degrading enzyme enzyme negative). Differential from other Listeria bacteria that produce false positives. Compared with the traditional biochemical reaction, which requires more than $ days to monitor Listeria monocytogenes, the method of the present invention uses the enzyme immunoassay method and the national standard of the national standard .....- v; 83. 3. l 〇, 〇〇〇 (Please read the precautions on the back before filling out this page). 1T- 514640 A7 Printed by the staff of the Central Bureau of Standards, Ministry of Economic Affairs, Consumer Cooperation. Du V. Description of the invention (8) Cooperated with hexanoic acid Phospholipase or arylamine degrading enzyme activity test can complete the identification of suspicious Listeria monocytogenes within 24 hours. It can be seen that the method of the present invention is an effective and reliable method for identifying Listeria monocytogenes. The following examples further illustrate the features of the present invention, but by no means limit the scope of the present invention, which is limited only by the scope of the attached patent application. Examples Example 1: A total of 93 strains of experimental strains and a test strain were cultured, including 51 Listeria monocytogenes, 25 Listeria monocytogenes bacteria, and 17 non-Listeria monocytogenes strains (Table 丨). Among the 51 strains of Listeria monocytogenes tested, 28 strains are known, including 12 serovars from serogroups to 4; the remaining 23 strains are unknown. These Listeria monocytogenes are from the Collection Collection and Research Center (CCRC), American Type Culture Collection (ATCC, Rockville, MD, USA), Collection of the Institiute Pasteur (CIP5 Paris France) and National Collection of Type Cultures (NCTC, London, UK). Except for the similarity, all non-Listeria bacteria are from the species preservation and research center of the Institute of Food Development σσ. Q Listeria strains are stored in Tenghua egg white with 0.6% yeast extract (YE) Soy agar medium (trypticase soy agar, TSA; Difco Laboratories, Detroit, MI, USA). Non-Listeria bacteria are stored on tryptic soy agar medium. Strains to be analyzed by linked enzyme immunoassay (ELISA) are first activated on tryptic soy broth (TSB) containing Tenghua Egg S with 0.6% yeast pomegranate extract for 20-24 hours. 13 This paper size applies Chinese National Standard (CNS) M specifications (210X 297mm) 83. 3. 10,000 Please read the notes on the back before %% Gutter on this page 514640 A7 B7 V. Description of the invention (9 Table 1: Experiments The total number of strains and bacteria sourced from the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs

Listeria monocytogenes L. grayi L. innocua L. ivanovii L. murrayi L. seeligeri L. welshimeri Bacillus cereus Bacillus megaterium Bacillus subtillis Brevibacterium acetylicum Citrobactor freundii Enterobacter cloacae Escherichia hermcinnic 15i delicense 15a , 15386-15388, 15397-15403, CIP 55143, 7832, 7835, 7836, 8147, NCTC 10887, 10890, 11994, ATCC 19115 2 CCRC 14842, 15356 6 CCRC 14843, 15357-15358, 15384-15385, 15404 3 CCRC 14844 , 15383, NCTC 11007 4 CCRC 14849, 15359-15360, 15394 2 CCRC 15361, 15395 8 CCRC 14851, 15362-15364, 15396, ATCC 43550, 43551, 49591 1 CCRC 10250 1 CCRC 10252 1 CCRC 10029 1 CCRC 10378 1 CCRC 12291 1 CCRC 12313 1 CCRC 13892 1 CCRC 10032 1 ATCC 14870 1 CCRC 10692 14 This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) 83. 3. 10,000 (Please read the precautions on the back before filling in this Page) "Binding · Binding · 514640 A7 B7 V. Description of the invention (10)

Micrococcus 1 CCRC 11273 candidus Micrococcus leteus 1 CCRC 11271 Micrococcus varians 1 CCRC 11272 Proteus vulgaris 1 CCRC 10486 Pseudomonas 1 CCRC 10485 mendocina Salmonella 1 CCRC 10241 typhimurium Staphylococcus 1 CCRC 10783 epidermidis (please read the notes on the back page first)

-UF Pack. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, CCRC, Culture Collection and Research Center, ATCC, American Type Culture Collection, Rockville, MD, USA Example 2: Surface Purification of the protein The purification of the surface protein was performed according to the method described by Koga and Kawata (Koga and Kawata, 1983) and modified. First, Listeria monocytogenes (Lberza m⑽oe is supplied by CCRC 14845) was cultured in 500 ml TSB containing 0.6% yeast extract for 20-24 hours (35-37 ° C), and then centrifuged (3,000 X g, 15 The cells were collected, washed with 0.1 M Tns-HCi buffer (pH 7.8), centrifuged again, and the cells were suspended in 10 ml of a 1% (w / v) sodium dodecyl sulfate (SDS) solution. Heat in a 70 ° C hot water bath for 2 hours, and centrifuge at 1,300 X g for 20 minutes to take the supernatant. This is the Listeria cell surface protein. It was dialyzed against 70 mM phosphate buffer (pH 6.3) and stored frozen until use. ___ 15___ The paper size is in accordance with the Chinese National Standard (CNS) A4 specification (210X: Z97 mm) " " 83 3. 10,000 ~ A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (U). Analysis of Listeria monocytogenes ATCC19115, CCRC 14842-14849, 14851 The surface protein obtained by the method of Example 2 was analyzed with sodium dodecyl sulfate polypropylene amidamine film (SDS-PAGE). The film thickness is 075 nmi and the concentration is 12.5%. After electrophoresis, the cells were stained with Commassie brilliant blue. As shown in Figure 1, except for the 5th C. monocytogenes Ccrc 14848 (ATCC 19114), the surface protein profiles of the other 6 Listeria monocytogenes are very close, all of which have a molecular weight of 68,000 Dalton. It can be seen from Figure 2 that the surface proteins of other Listeria bacteria (lines 2-6) have different electrophoretic patterns. Example 4: Preparation of Antigen To perform preparative SDS-PAGE, 2.8 mg of total surface protein was applied to each 3 mm-thick film. After electrophoretic separation (80 V, 18 hours), the film was immersed in 4 In sodium M solution (Higgins and Dahmus, 1979), shake at room temperature for 10-15 minutes. The 俟 protein band is transparent, and the protein band with a molecular weight of 68,000 Daer is directly cut from the film for the purpose of immunizing white rabbits. Example 5: Preparation of antiserum The film containing the protein with a molecular weight of 68,000 Daulton obtained in Example 4 was crushed into fine pieces and then 'emulsified with Freund's incomplete adjuvant of the same volume, with multiple points subcutaneously Injection into white rabbits, each with 2 ml of emulsified antigen. Additional injections were performed in the same manner every 3 weeks for a total of 4 additional injections. Blood was collected from rabbit ears 10 days after the last injection, and the blood was allowed to stand at 37 ° C for 1 hour, and the serum was centrifuged at 4 ° C every other day. The serum was heated in a 56 ° C water bath for 30 minutes to destroy complements, and the antibody was purified by diethylamine ethyl (DEAE) ion exchange analysis. The method can refer to Limm et al. 16 (Please read the note on the back first) Please fill in this page for more details)

Line 1T · This paper size is in accordance with China National Standard (CNS) A4 specification (2 丨 〇 × 297mm) 33. 3. 10,000 514640 A7 _ _B7_. V. Document of the invention description (12) (Linn ei α / .5 1973). The antibody titer was determined by a linked enzyme immunoassay. The total surface protein of 5 pig / ml was adsorbed on the microtiter plate. After adding antisera of different dilution times, protein A-horseradish peroxidase was used as a secondary antibody for enzyme immunoassay. The antibodies were tested. The power price is about 1 X 107. Example 6: Affinity purification of antibodies. 8 ml of EAH-Sepharose 4B colloid (Code No. 17-0569-01, Pharmacia, Uppasalla, Sweden) was printed in a consumer cooperative of the Central Standards Bureau of the Ministry of Economy. Saturate and wash with 0.5 M NaCl, 80 mg. Listeria monocytogenes or L./cisoc ⑽ total surface protein (10 mg / ml) and 80 mg 2N-ethyl-N obtained by the method of Example 2, (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Code No. E-6383, Sigma, St. Louis, USA) is dissolved in deionized water (pH is adjusted to about 4 in advance), and then mixed with colloid evenly, at room temperature Rotate the reaction overnight, and the pH value of the solution needs to be adjusted between 4.5-6 (adjusted by 0.IN HC1) during the reaction. After the reaction, the colloid was washed with 0.1 M acetate buffer (pH 4.0), packed into a column of 0.7 x 18 cm, and then 10 mM Tris-HCl buffer (pH 7.5), 0.1 to 0 (: 111, 1 ^ 1 buffer solution (? 112.5) and 1011 ^ 1, 1 ^-^ «: 1 buffer solution (?" [8.8) parental interwashing "and finally, phosphate-buffered saline (phosphate-buffered saline) (PH 7_2; PBS) after equilibration. The antibody purified by DEAE in Example 5 was put into an affinity column containing Listeria monocytogenes surface protein, and the unbound protein was washed out with PBS, and then The 0.1 M glycine-HCl buffer solution (pH 3.0) was used to wash out the antibody against the surface protein of molecular weight 68,000 Dalton and dialyzed against PBS. The purified antibody was concentrated with polyethylene glycol 10,000 and passed into L. / 湖 本⑽ In the affinity column of the total surface protein, and flow wash with PBS, flow wash to 280 nm _____17 83. 3. 10,000 (Please read the precautions on the back before filling this page) This paper size applies Chinese national standards ( CNS) A4 size (210X297 mm) 514640 Printed by the cooperative A7 B7 V. Description of the invention (l3) The absorbance is zero, and the antibody is stored at -20 ° C. Example 7: Preparation of antibody and wasabi perhydrogenase conjugate Preparation 4 mg wasabi peroxidase was dissolved in 1 Add 200 μ1 of freshly prepared sodium periodate in de-ionized water. After 20 minutes at room temperature, dialyze with 1 mM sodium acetate buffer (pH 4.4) at 4 ° C overnight. Add wasabi peroxidase after dialysis 8 mg of the antibody purified in DEAE column (dialyzed in 0.1 M sodium acetate buffer, pH 9.5) in Example 5 and reacted at room temperature for 2 hours. Finally, 100 μΐ of freshly prepared sodium borohydride (4 mg / ml), react at 4 ° C for 2 hours, dialyze with 0.1 M borate buffer (PH 7.4), add an equal volume of 0.1 M borate buffer (pH 7.4) containing 0.4% bovine serum protein, 60% glycerol, and place in Store at -20oC. Example 8: Pre-treatment of the bacterial liquid for determination of the strains. The strains listed in Table 1 were propagated overnight in TSB-YE, and 1 ml of the proliferating bacteria was taken at 10 ° / 〇 in Teepol (primary alkyl (C9-C13) sodium sulfates, Sigma, St. Louis, USA ) To a final concentration of 0.1%. After boiling for 3 minutes, centrifuge and add an equal volume of 2% bovine serum albumin to the supernatant and mix well. Example 9: Enzyme-linked immimosorbent assay (ELISA) The antibody purified from the DEAE column in Example 5 was dissolved in PBS to a concentration of 10 pg / ml, and 0.1 ml was added to the% wells. In the slot of the microtiter plate, react at 37 ° C for 1 hour to coat the antibody in the slot. Wash 5 times with PBST (PBS containing 0.05% of Tween 20), and add 0.1 ml of 1% BS A to each well, and place the spacer at 4 ° C. _ 18_____ This paper scale applies Chinese national standards ( CNS) A4 size (210X297 mm) 81110 00 (Please read the precautions on the back before filling out this page) -Packing · -Line · 514640 A7 B7 V. Description of the invention (14) After washing the microtiter plate 5 times with PBST Add 0.1 ml of the treated proliferation bacteria solution, and react at 37 ° C for 1 hour, then rinse with PBST. The conjugate of antibody and wasabi peroxidase was diluted 5,000 times in PBS containing 0.1% Tween 20 and 1% BSA. 0.1 ml was added to each well, reacted at 37QC for 1 hour, rinsed with PBST, and then oi ml enzyme was added. Matrix 3,3 ', 5,5'-tetramethylbenzidine (TMB): H2〇2 (v: v = 1: 1, Kirkegaard & Perry Laboratories, Gaithersbyrg, MD, USA) reacted for several minutes to perform a color reaction (blue color). After proper coloration, add 0.1 ml of 1 M phosphoric acid to stop the reaction. At this time, the reaction solution is yellow. The microtiter plate photometer (Bio-Tek, Model EL 309, Winooski, Vermont, USA) can be used to measure the absorbance at 450 nm. . < The various Listeria bacteria and other bacterial strains are shown in Table 2 by using this method. Table 2: Results of various enzyme-linked enzyme immunoassay tests (please read the precautions on the back before filling out this page) L. monocytogenes (Listeria 51 50 1 Monascus) L. grayi 2 0 2 L. innocua 6 6 0 L. ivanovii 3 0 3 L. seeligen 2 0 2 L. welshimeri 8 3 5 Bacillus cereus l 0 1 Bacillus megaterium l 0 1 Bacillus sub til Its 1 0! Brcvibacterium acetv11cum 1 0 1 19 This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 cm) Line 83. 3. [〇, 〇〇〇514640 A7 B7 V. Invention Instructions (15) Citrobactor freundii Enterobacter cloacae Escherichia hermannii Escherichia intermedia Jonesia denitrificans Klebsiella pneumoniae Micrococcus candidus Micrococcus leteus Micrococcus varians Proteus 0ul 0 0 first read 0 first 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (Fill in this page again) The .ir is very similar to the method described by Mcliure (McClure, 1990), specificity, false positive and false negative ratios. Stem sensitivity is calculated by dividing the positive (true positive) response in Listeria monocytogenes by the number of all Listeria monocytogenes tested. Obligate-specificity is obtained by dividing the number of non-Listerial gg strains in the non-Listerial strains (true negatives) by dividing the total number of non-Listerial gg strains. The false positive ratio is the percentage of non-Listerial strains divided by the number of all positive strains (false positives plus the number of true positives). Residual ratio is the percentage of the number of negative reaction strains in P. pastoris divided by the number of anti-counties with M sex (the number of false negative reaction responses). I 夂 The results are summarized in Table 3. The number of strains in New Zealand is applicable to the specificity of printing sensitivity of the Consumer Cooperative of the Central Samples Bureau of the Ministry of Economic Affairs of China. 33.3.1 (3,000 98514640 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. 5. Description of the invention (16) 51 bacteria Listeria spp. 21 bacteria other bacteria 17 50 9 0 12 17 57.1 100.0 " Tested ―51 i strain was positive, only 1 strain was negative, sensitivity was 98%, false negative The response rate is 96% (1/51). For other bacteria of the Listeria genus, the false positive rate is 15 25% (9/59). For other non-Listeria species, § 'sensitivity and The specificity is 1%. Painting _ Example ο: Milk star gel i ^^ (latexagglUftmTltest) A. Covering the latex particles Take 1.09 μηι latex particles (JapanSyntheticRubber, Tokyo, Japan) 1 liter with PBS ( (pH 7.2) Wash twice and resuspend in 10 ml of pBS. Add the same volume of antibody (70 μ§ / ίη1) purified by affinity column, react at room temperature for 2 hours, wash twice with PBS, In suspension in 10 ml containing 5% bovine serum albumin and 0.05% sodium azide (Na%) In PBS, the final concentration of latex particles was 0.05% (w / v). The control latex was purified from normal rabbit serum to obtain immunoglobulin ^ (IgG), which was coated on the latex particles in the same way. B. Latex aggregation reaction Method (latex agglutination test) Use a pen to draw two circles with a diameter of 15 cm on a slide, and use a sterile toothpick to pick out one or two colonies and move them to a circle on the slide. One of the circles is added with one drop (25 μΙ ) Control latex microparticle reagent, add a drop of antibody-covered milky month microparticle reagent 'coated with a toothpick and shake it back and forth slightly for 4 minutes, observe whether a coacervation reaction occurs and record. If you add antibody-coated latex Condensation found in the circle of the microparticle reagent -___ 21 (Please read the precautions on the back before filling this page). 燊 'Γ

I This paper size applies to China National Standards (CNS) A4 specification (210X297 mm) S3. 3. 10,00 (514640 A7 ______ B7 — V. Description of the invention (17) Polymerization reaction ”but the control group still maintains milky uniformity , Recorded as a positive reaction; if no agglutination reaction occurred in the two circles on the slide, it was recorded as a negative reaction. Occasionally, it was found that both agglutination reactions occurred in both circles, which is auto-agglutination The results are unexplainable. The test results of different Listeria bacteria using this method are shown in Table 4. The printing soil printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs: various Listeria bacteria were tested by latex agglutination reaction. Results Total number of bacterial strains Positive reaction Negative reaction L. ~~ 45 40 5 monocytogenes L. welshimeri 3 3 0 L · innocua 5 0 5 L. ivanovii 4 2 2 L. seeligeri 2 1 1 L. murrayi 4 1 3 L. grayi 1 0 1 References Bille, J., B. Catimel, E. Bannernan, C. Jacquet, M. N. Yersin, I. Caniaus, D. Monget, and J. Rocourt. 1992. API Listeria ^ a new and ( please (Please read the notes on the back and fill in this page). Binding-booking-promising one-day system to identify Listeria isolates. Appl. Environ. Microbiol. 58: 1857-1860. Eley, A. 1990. New rapid detection methods for Salmonella and Listeria. British Food 92: 28-3 1. Farber, JM and P. I, Petekin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55: 476-5 11. —_.,.,- 22- This paper size applies to Chinese National Standard (CNS) A4 specification (2 丨 0X297mm) 8 3. 3. 丨 0 · 0 () () 514640 A7 B7 V. Description of the invention (18)

Gahan, C. G. M. and J. K. Collins. 1991. Listeriosis: biology and implications for the food industry. Trends in Food Science & Technology. 2: 89-93.

Higgins, R. C. and Μ. E. Dahmus. 1979. Rapid visualization of protein bands in preparative SDS-polyacrylamide gels. Anal. Biochem. 93: 257-260.

Hudson, L. and F. C. Hay. 1989. Affinity techniques for molecules and cells, p. 322-329. In Practical immunology. 3rd ed. Blackwell Scientific Publications, London.

Johson, J. L. and C. P. Lattuada. 1993. Comparison of nucleic acid hybridization assays and biochemical characterization tests for the confirmation of L. monocytogenes. J. Food Prot. 56: 834-840.

Kabir, S. 1980. Composition and immunological properties of outer membrane proteins of Vibrio cholerae. J. Bacteriol. 144: 382-389.

Kabir, S. 1986. Composition and immunological properties of the cell surface proteins of Vibrio cholerae. J. Gen. Microbiol. 132: 2235-2242.

Koga, T. and T. Kawata. 1983. Isolation and characterization of the outer membrane proteins from Vibrio parahaemolyticus. J. Gen. Microbiol. 129: 3185-3196. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs

Leimeister-Wachter, M., E. Domann, and T. Chakraborty. 1991. Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is co-ordinately expressed with listeriolysm in Listeria monocytogenes. Mol. Microbiol. 5: 361-366 .

Linn, TG, AL Greenleaf, RG Shorenstein, and R. Losick. 1973 Loss of the sigma acitivity of RNA polymerase of Bacillus suhtilis during sporulation. Proc. Natl. Acad. Sci. USA. 70: 1865-1869. 23 83. 3 . 10,000 (Please read the notes on the back before filling this page) This paper size is applicable to China National Standard (CNS) A4 size (210X 297 mm) 514640 Employees' cooperation in cooperation with the Central Standards Bureau of the Ministry of Economic Affairs, printed A7 _B7 _ ^ _ 五(I9) Lovett, J.5 DW Francis, and JM Hunt. 1987. Listeria monocytogenes in raw milk: detection, incidence and pathogenicity. J. Food Prot. 50: 188-192. McClure, FD 1990. Design and analysis of quantitative collaborative studies: minimum collaborative program. J. Assoc. Off. Anal. Chem. 73: 953-960. Notermans, SHW, J. Dufrenne, M. Leimeister-WSchter, E. Domann, and T. Chakraborty. 1991 . Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and non-pathogenic Listeria species. Appl. Environ. Microbi ol. 57: 2666-2670. Schematic illustration Figure 1: Electrophoretic map of surface proteins of different strains of Listeria monocytogenes Surface proteins extracted from eight Listeria monocytogenes, sodium dodecyl sulfate Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) spectrum. One of the unnumbered ones is a protein of known molecular weight. The first and second lines are Listeria monocytogenes ccRC 14845 (ATCC19111), the third and fourth lines are Listeria monocytogenes ccrc 14847 (ATCC 19113), and the fifth line is Listeria monocytogenes. Monoclonal CCRC 14848 (ATCC 19114), 6th line of Listeria monocytogenes ATCC 19115, 7th and 8th lines of Listeria monocytogenes ccrc 14846 (ATCC 19112). The numbers in the left column are molecular weights (kDa), and the arrows represent common proteins. Figure 2: Electrophoresis maps of surface proteins of different species of Listeria, surface protein shells extracted from two Listeria and five other Listeria bacteria, and sodium dodecyl sulfate polypropylene Phenamine film electrophoresis map. One of the unnumbered ones is the peptone of known 77 sub-1, and lines 1 and 7 are Listeria monocytogenes ATCC specifications (27 ^ 29:)-^ 1-! () 〇ό〇 '(please first Read the notes on the back and fill in this page) • Install ·, 1T line 514640 A7 B7 V. Description of the invention (20) 19111 and ATCC 19115; the second line L · CCRC 14842; the third line L. and the CCRC 14843; the fourth line Act / · να / 7〇ν " · CCRC 14844; 5th Act L. wwrray / CCRC 14849; 6th Act L · we / M / mer / CCRC 14851. The numbers in the left column are molecular weights (kDa), and the arrows represent common proteins. (Please read the precautions on the back before filling out this page) -Installation-11 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 83. 3. 10,000 This paper size applies to China National Standard (CNS) A4 (210X297 mm)

Claims (1)

86105625 Patent Application for "Determining and Detecting Methods for Listeria monocytogenes" (Amended in April, 91) Species of K-body, "Small and Fine-tuned Swallows", Cell Surface The antigen is prepared by the following methods: ⑴ extraction of cell surface proteins of Listeria monocytogenes S; (2) electrophoresis analysis of the cell surface crusts; (3) taking 68k Dalton protein. 2-A method of determining Listeria monocytogenes g, including the following steps: 培养 culturing the g strain to be selected in a selective medium of Listeria monocytogenes g; Listeria monocytogenes (Z mb mowqytogms · ) The white matter on the cell surface is an antibody prepared by the anti-Gu antibody S for immunoassay reaction. The antigen is prepared by the following methods: a. Extraction of the filament φ protein of Lissinolysis S; b. The cell surface The protein was analyzed by electrophoresis; c. A protein with a size of 68k Dalton was taken; (3) The biochemical test method was used to test the strain to be identified. The biochemical test method is known to be used in Listeria monocytogenes and Z. __ and ㈣. Yang ················ the results of the undefeated test to distinguish Listeria monocytogenes from ζ ζ · _㈣ and Yi Mi Shi &, ⑷ If the strain to be identified is presented on the selective medium of Listeria monocytogenes The typical colony characteristics of Listeria monocytogenes, and the result of the immunoassay reaction showed a positive response, and the results of the biochemical test method showed the same results as those of known Listeria monocytogenes, the strain to be identified is Listeria Monascus. 3. If you apply for the method of item 2 of the supplement, the selective culture medium of E. monocytogenes is Oxford medium or brain ium chloride_phenyleth lion ^ < soil 514640 A8 B8 C8 D8 6. Application scope of patent 4. If you apply for a patent The method of the second item, wherein the immunoassay reaction is a linked enzyme immunoassay or a latex agglutination reaction method. 5. For example, the method of claim 4 of the patent fell, wherein the binding enzyme immunoassay method includes the following steps: (1) coating the antibody on a solid phase; (2) combining the cell surface protein of the strain to be identified with the solid phase The antibody reaction coated on the phase 'makes the antibody-68k Daur cell surface protein complex on the solid phase; (3) Add the antibody (antibody-enzyme conjugate) labeled with enzyme to make the solid phase An antibody-68k Dalton cell-protein-antibody-enzyme complex was formed on the phase; (4) A matrix of human and protein was added to observe the reaction result of the enzyme immunoassay method. 6. The method according to item 4 of the scope of patent application, wherein the binding enzyme immunoassay method includes the following steps: (1) coating the antibody on a solid phase; (2) combining the cell surface protein of the strain to be identified with the solid The antibody reaction coated on the phase, so that a complex of antibody-68k Dalton cell surface protein is formed on the solid phase; (3) a secondary antibody added to the 68k Dalton cell surface protein and an enzyme labeled The primary antibody antibody (anti-secondary antibody-antibody-enzyme conjugate) forms a complex of antibody-68k Doyle cell surface protein-secondary antibody_anti-secondary antibody-antibody-enzyme conjugate on the solid phase. (4) Add the substrate of the enzyme to observe the reaction result of the linked enzyme immunoassay. 7. The method of claim 4 or 5, wherein the enzyme is a peroxidase. 8. The method according to item 4 or 5 of the scope of patent application, wherein the surface protein of the scale-sensitive cells is extracted with a surfactant. 9. The method according to item 8 of the scope of patent application, wherein the surfactant is Teep〇1 or sulfuric acid ten paper standard / T "National Standard (CNS) A4 Specification (21〇 > < 297 mm) ) (Please read the notes on the back before filling this page) , Yan-Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (Please read the notes on the back before filling this page) 514640 •, A8 B8 C8 __ _ 08 6. Scope of patent application Sodium dodecyl sulfate. I9. The method of claim 11 in the scope of the patent application, wherein the biochemical test method is to test the activity of cyclohexanone diacetate (PI pore C), arylamine decomposition enzyme activity or dissolution and the known Listeria Monascus should show results, amine-decomposing enzyme activity, and hemolysin production. Miscellaneous and non-aryl, please read the note on the back first I Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs