TW200907064A - Rapid serotypes K1/K2 test for klebsiella pneumoniae virulent stains - Google Patents

Abstract

The present invention relates to a test kit for detecting Klebsiella pneumoniae virulent strain K1/K2 and use thereof.

Description

200907064 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a rapid test agent for Kjb Klebsiella virulent strain K1/K2. [Prior Art] Krebs pneumonia lake is a Gram-negative rod g, which is a pathogen of an opportunistic infection in the gut of the intestine. In the past two years, a new type of invasive Krebs' lung (the inflammatory infection has gradually increased to a global community-recovery important community infection. Klebsi's pneumonia g caused by suppurative silk disease, madness And a considerable ratio of domain dyeing, in Taiwan, it ranks second in the Gram-negative rod H. Eighty percent of Taiwan's liver lining is caused by Klebsiella pneumoniae's community-based infection, non-like In-hospital infection, rapid development of the disease and may be accompanied by endophthalmitis leading to blindness. Epidemiological analysis most commonly occurs in Taiwan's serotype K1 & K2 type 2 'The second type of strain is the most toxic, most likely to transfer to the eye to cause serious eyes Endophthalmia must be removed from the eyeball to save life. At present, there are more than 7 cases of life-long blindness caused by C. comatus liver pus cancer in Taiwan. The external literature points out serious complications such as endophthalmitis. The occurrence occurs within 48-72 hours after the formation of Klebsiella purulent hepatic sputum cancer. Therefore, if the two virulent strains are determined early in clinical practice, the treatment policy is changed immediately, and the application is more expensive.帛素 can be tested _ inflammation _ occurs. Suppurative liver and sputum cancer caused by Klebsiella is an emerging infectious disease. At present, in addition to prevailing in Asian countries and regions, 'European countries also have _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The method of learning to find miscellaneous disease @子, in the case of the cause of the polymerase chain reaction (polymerasechain coffee, such as pcR), as a rapid diagnosis of 200907064 method, the current research team of Taiwan University found _ gene used to do pcR However, only the monthly b measured ΚΙ ® plant red 2 mother strain can not be lake, spCR method needs to prepare for the fastest time 'with B inch need to have the instrument and skillful technology, and work irritated 'must please Specialized operation. SUMMARY OF THE INVENTION The object of the present invention is to solve the early test speed identification of the K-frequency and K2 type of virulent strain so that the private bed doctor can be provided immediately - an accurate answer for timely treatment of suppurative hepatic carcinoma The problems brought by the new _. The external literature pointed out that serious complications such as endophthalmitis occur within 48 hours after the formation of Klebsiella purulent hepatic abscess, so if In the short day-to-day period, the K. pneumoniae strain kl/k2 rapid test agent can be used to detect whether the bacteria is a virulent strain within 5 minutes, and a major breakthrough will be obtained in clinical diagnosis and treatment. A method for the development of a virulence strain of Klebsiella K1 and K2 by the method of immunochromatogaphic test (ICT). The invention relates to a test reagent for a Klebsiella pneumoniae strain K1/K2. 'Characteristics comprising a carrier on which a K. pneumoniae virulence strain K1/K2 200907064 is immobilized. The carrier of the test reagent of the present invention includes, but is not limited to, a nitrocellulose membrane. For the favorable sample flow, the carrier can adhere to the sputum sheet. The label in the assay reagent of the present invention can show the reaction of the antibody with the sample, including but not limited to colloidal gold. The antibody in the test reagent of the present invention is an antibody against any K. cholerae strain K1 and K2 toxic strain, which can be conjugated with colloidal gold to produce a conjugate. In a preferred embodiment, the mouse _a_kpl colloidal gold Sputum (CGC) or mouse-a-kp2 colloidal gold co-host. In the test reagent of the present invention, the colloidal gold and the antibody are combined and coated on the integral diffusible carrier to form a colloidal gasket. The colloidal gold gasket can be adhered to the carrier on one side and adhered to the specimen spacer on the other side. The present invention relates to a method for detecting Krebs is a virulent strain K1/K2 of Klebsiella pneumoniae, comprising (a) providing a sample, and (b) adding the sample to the detection reagent of the present invention, and (c) observing the antibody Whether there is an aggregation reaction with the specimen. In the method of the present invention, the test system Klebsiella pneumoniae strain K1 or K2 forms a solid line at the encounter of the antibody. In the method of the present invention, the test reagent includes a control line to indicate that the reagent is functioning properly. The method of the present invention can be used for any test including, but not limited to, clinical liver and abscess, cerebral ridge 200907064 myeloid or urine test. [Examples] Example 1: The present invention is a rapid test for the development of a virulence strain of Klebsiella K1 and K2 by immunochromatogaphic test (ICT), the steps of which are as follows: (1), Cray Preparation of antiserum against virulence strains of Burdock Κ1 and Κ2: Immunization in mice with low doses of virulence strains of Κ1 and Κ2, and addition of secondary antibody: goat-α-mouse-IgG- HRP (Goat-a-mouse-IgG-HRP) was assayed for its titer by enzyme-linked immunosorbent assay (ELISA), and the resulting antibody was purified by protein A chromatography. (1) Spray film and drying: The purified antiserum (mouse _a_kpi and mouse _a_kp2) was diluted to a nitrocellulose membrane (NC membrane, length 3 〇 cm) after the film spray machine was diluted. After being fixed, it will be placed in a constant temperature and humidity clean room for 24 hours and then subjected to a blocking operation. (B), preparation of colloidal gold: appropriate amount of anti-serum antibody and colloidal gold (25nm) for reaction binding and concentration. The prepared mouse _a_kpi CGC and mouse-a-kp2 CGC were fixed on a glass fiber colloidal gold gasket with a spray volume of 3.〇4/cm by a spray film machine, and TEMP was used in a constant temperature and humidity chamber. =37t:, RH = 5%, dry for 30 minutes. (4) Reagent assembly operation: tear off the protective layer of nitrocellulose membrane (Fig. 1), stick the nitrocellulose membrane (position 1), and press the nitric acid membrane with finger to the abdomen to ensure the tannic acid fiber membrane. Stick firmly. Then peel off the protective layer of the absorbent pad, stick the absorbent pad (position 2), and press the Top pad with your fingertips to ensure that the Top pad is firmly attached. Confirm the top edge of the Top pad and the upper edge of the 200907064 plastic bottom card (4). The body of the body is placed 3) The table is peeled off, and the cut frequency gold gasket is pasted along the upper edge of the protective layer where the sample gasket is nowhere. Pressing the fingertips with your fingertips will press the Lai gold material, and it is true that the gold-plated gasket is not strong. The light-faced golden enamel sheet overlaps with the fiber membrane by 2±1. Finally, the specimen is removed from the protective layer, and the cut specimen is aligned along the lower edge of the bottom card (position 彳), and the fingertips are indeed pressed. The mold sample gasket overlaps with the colloidal gold gasket by 5 ± 2 mm. The above assembly (four) will be carded, and the paper will be cut into the city _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Machine packaging. Example 2: 1. The liver pus and the body should be diluted with physiological food (4) as the specimen of bismuth copper. (If the specimen is brain (four) liquid, urine or other body fluid, it may not need to be diluted. If the specimen is cultivated, it can be cultivated. For bacteria, pick up 2-3 colonies and mix well with physiological saline. 2. Open the wrapping paper of the reagent on the table. Draw the specimen to be tested with a micropipette. The specimen to be tested is dropped into the specimen test area (S-Weu). 3, and then "sucking Na Na touch __ secret pure as an oil (four) 3 drops to be tested specimen specimen area (S_well), 3_5 health _ can be read. 4, the criteria for interpretation are as follows: (1). If only - the red line appears in the c handsome ye. Coffee uine), indicating that the test is normal. However, the specimen is not a Krebs, and even K. pertussis is not the most toxic K1 or K2 strain. 200907064 (2). If there are two «red yokes, _ (4) in c post (four) and another - appear in area i (service e) or zone 2 (4), you can determine that the strain is a strain of Ki or K2. Example 3: Picking strains of three different serotypes (packaged, K2 and coffee 2) test results: the reagent on the left side in the second figure is shown as a strain of Κ1/Κ2 type, and the reagent on the middle side is shown as a phantom strain. On the right side, the test surface is K2 type g strain. The serotyping method of this (10) system was used as a control test, and the results did not find cross-reaction with other serotypes of different serotypes, indicating that the specificity of the reagent was extremely high. [Simple description of the diagram] Figure 1 shows the sequence of reagent assembly. S - page shows the results of strain tests for different serotypes (including magic, ^ & n〇n_K1/K2). [Main component symbol description] 1 nitrocellulose membrane 2 absorbent gasket 3 colloidal gold gasket 4 specimen gasket

Claims (1)

200907064 X. Application for Patent Park: 1. A test reagent for the strain K. pneumoniae virulence strain Κ1/Κ2, which is characterized in that a carrier is immobilized on the anti-K. pneumoniae strain Κ1/Κ2 antibody. 2. The test reagent of claim 1, wherein the carrier is a nitrocellulose medium. з. As requested! The test reagent of the item, wherein the antibody is a small colloidal gold co-light (CGC). 4. The test reagent of claim 1, wherein the antibody is a mouse _ ton 2 colloidal gold co-host. 5. The test reagent of claim 1, which further comprises colloidal gold as a marker. 6♦ If the test reagent of claim 5 is applied, wherein the knee gold and the antibody have been combined and coated in an integral diffusible carrier to form a colloidal gasket. 7. The test reagent of claim 6, wherein the colloidal gasket is adhered to the carrier. 8. If applying for the test reagent of item 7, it also includes a sample gasket, which is adhered to the sturdy body. For example, the test reagent of Item 8 is further included, and the water absorbing pad is attached to the carrier. Said - a method for detecting Krebs is a K. pneumoniae strain K1/K2, comprising (8) providing a sample, and (9) adding the sample to a test reagent such as the application for a special fiber, and (c) observing Whether the antibody and the sample have an aggregation reaction. и. - A method for detecting Krebs is a strain of K. pneumoniae strain K1/K2, comprising (4) providing a sample, and (9) adding the sample to a detection reagent as in claim 5, and (6) observing the antibody and detecting Whether the body has an aggregation reaction. For example, the method of claim π patent|& surrounding the 11th item, wherein the test system Klebsiella pneumoniae virulence strain Κ1 ' will form a solid line at the encounter of the antibody. For example, the method of claiming the patent item i!, wherein the test system Klebsiella pneumoniae strain 11 200907064 Κ2, will form a solid line at the encounter of the antibody. 14. The method of claim 11, wherein the test reagent comprises a control line to indicate that the reagent is functioning properly. 15. For the method of patent application No. 10 or 11, it can be used for clinical liver abscess, cerebrospinal fluid or urine testing. 12 200907064 VII. Designated representative map: (1) The representative representative of the case is: (1). (2) The symbol of the symbol of this representative figure is simple: 1 nitrocellulose membrane 2 absorbent pad 3 colloidal gold gasket 4 sample gasket 8. If there is a chemical formula in this case, please reveal the chemical formula that best shows the characteristics of the invention: 200907064 (This, Mingyue j month patent month book - ※ Application number: * Body. Do not change any month, please do not fill in the ※ part of the mark) ※ Application period: 亀丨〇%IPC eight charm. Jade-' gas ^ ”: XL classification. Li2Ql^r, ^0 6,01) I~Bamboo·, r文/央文) I called!/ Kleberite® Toxicity _K1 Fine Rapid Test Reagent and its inspection, applicant :(Total 1A) Invention Name: (Chinese / English) C/2/^ /X 〇+ — ... ~ 〇〇06,(6) Name or Name: (Chinese / English) National Yang Ming University / NATIONAL YANG-MING UNIVERSITY Representative: (Chinese / English) Wu Yuhua / LEE Yang, YAN-HWA Residence or Business Office Address: (Chinese / English) 11221 Taipei City North 155, No. 155, Section 2, Nong Nong Street, Civic District 155, SEC. 2, UN〇NG ST., BEIT0U DISTRCT, TAIPEI CITY 11221, TAIWAN (ROC) Nationality: (Chinese/English) Republic of China/ROC II. Invention Person: (5 in total) Name. (Chinese/English) 1·; Ma Changfeng/Fung, Chang-Phone 2_ Xiao Liangji/Siao, Liang-Ji 3. Zhang Beeyi/Chang, Feng-Yee 4. Lin Yongchong/Lin , Jung-Chung 5·Chen Deli/Chen, Te-Li Nationality: (Chinese/English) Bu 5 Republic of China/Roc 200907064 (This specification format, order and bold type, invention patent specification order and bold type please Do not change anything, please do not fill in the ※ mark) Category: ※Application number: ※Application period: 1. Invention name: (Chinese/English) Arm (4) Rapid detection agent for Klebsiella pneumoniae strain K1/K2 1. Applicant: (1 person in total) Name or Name: (Chinese / English) National Yang Ming University / NATIONAL YANG-MING UNIVERSITY Representative: (Chinese / English) Wu Yuhua / LEE WU, YAN-HWA Residence or business address: (Chinese / English) U221 Taipei Beitou District 155, Section 2, Lian Nong Street / N〇. 155, SEC. 2, LIN〇NG ST., BEITOU DISTRCT, TAIPEI CITY 11221, TAIWAN (ROC) Nationality: (Chinese/English) Republic of China/ROC II. Inventor: (5 in total) Name: (Chinese/English) 1. Feng Changfeng/Fung, Chang-Phone ID: F104203272 2. Xiao Liangji/Siao, Liang-Ji ID: F131085806 3. Zhang Fengyi/Chang, Feng-Yee ID: W100154228 4· Lin Yongchong/Lin, Jung-Chung ID: R120879220 5. Chen Deli/Chen, Te-Li ID: 1100442529 Nationality: (Chinese/English) Bu 5 Republic of China/r.〇.c. 200907064 Invention Patent Specification (This manual format, ; ^ sequence and thick and wide words, please do not change any more, ※ Do not fill in the number of the mark] ※ Application number: 7 / '/, /- I ※ Application period: ※ IPC classification: Cl 2 ίκ4ί 〇.〇°^ου I. Name of the invention: (Chinese/English) mn1/ C Uh/S^i (2006 Ci Klebsiella pneumoniae strain K1/K2 rapid test agent / RAPID SEROTYPES K1/K2 TEST FOR KLEBSIELLA PNEUMONIAE VIRULENT STAINS II. Applicant: (1 in total) Name or Name Chinese/English) National Yangming University / NATIONAL YANG-MING UNIVERSITY Representative: (Chinese / English) Wu Yuhua / LEE WU, YAN-HWA Residence or establishment Address: (Chinese/English) 11221 No.155, Section 2, No. 2, Nongguan, Beitou District, Taipei, SEC. 2, LIN0NG ST., BEIT0U DISTRCT, TAIPEI CITY 11221, TAIWAN (ROC) Nationality: (Chinese/English) ) Republic of China / ROC III. Inventor: (5 in total) Name: (Chinese / English) 1 · Feng Changfeng / Fung, Chang-Phone 2 · Xiao Liangji / Siao, Liang-Ji 3. Zhang Fengyi / Chang, Feng-Yee 4 , Lin Yongchong / li n, Jung-Chung 5_ Chen Deli / Chen, Te-Li Nationality :(Chinese / English) 1-5 Republic of China / R 0 c.