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Colloidal gold immunoassay paper for detecting bacillus in bee larvae and its preparation and application

A technology for bacillus and bee larvae, applied in the direction of measuring devices, instruments, and biological material analysis, can solve the problems of high operational technical requirements, long detection methods, unfavorable rapid detection, etc., and achieve good detection repeatability and easy promotion and use , low cost effect

Inactive Publication Date: 2020-02-18
COMPREHENSIVE TECH SERVICE CENT YILI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems in the prior art that the detection method of honeybee larval bacillus takes a long time, requires high operating technology, and is not conducive to rapid detection on the spot, the present invention aims to provide a colloidal gold immune test paper for detecting honeybee larval bacillus and Its preparation and application establish an accurate, sensitive, and on-site method for rapid detection of bacillus in bee larvae

Method used

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  • Colloidal gold immunoassay paper for detecting bacillus in bee larvae and its preparation and application
  • Colloidal gold immunoassay paper for detecting bacillus in bee larvae and its preparation and application
  • Colloidal gold immunoassay paper for detecting bacillus in bee larvae and its preparation and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Purification of PLMP polyclonal antibody

[0037] 1. Serum pretreatment

[0038] Draw 6 mL of rabbit serum with a clean syringe and filter it with a 0.45 μm filter membrane.

[0039] 2. Caprylic acid precipitates non-IgG proteins

[0040] Take a 50 mL autoclaved centrifuge tube, add 24 mL of acetic acid buffer and 6 mL of rabbit serum filtered through a 0.45 μm filter membrane, shake and mix well, and adjust the pH to 4.5 with 1 mol / L NaOH. Add 2.25 mL of n-octanoic acid, stir at room temperature for 30 min, centrifuge at 10,000 rpm for 20 min, and discard the precipitate. Draw up the remaining liquid with a syringe, filter it with a 0.45 μm filter membrane, add 1 / 10 volume of 0.01mol / L PBS solution (PH=7.4), then adjust the pH to 7.4 with 1mol / L NaOH, and put it in an ice bath follow-up experiment.

[0041] 3. Ammonium sulfate precipitation of IgG

[0042] In an ice-bath environment, an equal volume of saturated ammonium sulfate was added to the above sol...

Embodiment 2

[0043] Embodiment two: the preparation of gold label pad

[0044] 1. Determination of the optimum pH value of colloidal gold

[0045] In this test, 0.2mol / L K was added 2 CO 3 The amount is standard. Take 10 1.5mL centrifuge tubes and mark the numbers, and add 1mL of colloidal gold solution to each centrifuge tube. Add a certain amount of 0.2mol / L K to each centrifuge tube 2 CO 3 Mix thoroughly (as shown in Table 3-2), then add 30 μg of purified polyclonal antibody to each tube, mix again, and let stand at room temperature for 30 minutes. Slowly add 20 μL of 10% NaCl solution to each centrifuge tube, mix well and let stand at room temperature for 2 hours. Observe the color change of the 10 centrifuge tubes and whether there are black particles of sedimentation and aggregation. Select the K for the tube that starts with no aggregation 2 CO 3 The addition amount of solution, then is the optimum 0.2mol / L K of colloidal gold in this test. 2 CO 3 The amount of addition, ...

Embodiment 3

[0059] Example 3: Preparation of detection line and quality control line

[0060] 1. Determination of the detection line coating antibody concentration

[0061] On the NC membrane of the detection line, add dropwise the concentration of 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, 1.0mg / mL, 1.2mg / mL, 1.4mg / mL, 1.6 mg / mL diluted with PBS, respectively. mL, 1.8 mg / mL 1 μL PLMP polyclonal antibody, on the NC membrane of the quality control line, drop 1.0 μL of goat anti-rabbit IgG antibody with a concentration of 1.0 mg / mL diluted with PBS. Use 10 μg / mL purified PLMP protein for detection, observe the color development of the detection line, and determine the optimal concentration of the coating antibody.

[0062] When the C line is coated with 1.5 μL of goat anti-rabbit IgG antibody with a concentration of 1 mg / mL, set 8 concentration gradients of the PLMP protein polyclonal antibody, and use PLMP protein for detection. It can be seen that when the concentration is diluted from 1.0 mg / mL to ...

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Abstract

The invention discloses colloidal gold immunological test paper for detecting the paeniBacillus larvae of bee, preparation and applications thereof. The colloidal gold immunological test paper specifically comprises a sample pad, a gold standard pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the gold standard pad contains colloidal gold-labeled PLMP protein polyclonal antibody, and the nitrocellulose membrane has a PLMP protein polyclonal antibody pre-coated detection line and a goat anti-rabbit IgG solution pre-coated quality control line. According to thepresent invention, the prepared colloidal gold immunological test paper has advantages of strong specificity, good sensitivity, good detection repeatability and stable performance, and provides the negative results for negative bacteria liquids, wherein the specificity can be 100%, the minimum detection concentrations of the bacterial liquid and the positive sample of the paeniBacillus larvae respectively are 10<5> CFU / mL and 10<6> CFU / mL, and the preservation period of the colloidal gold immunological test paper is 3 months.

Description

technical field [0001] The present invention mainly relates to the technical field of molecular biology, specifically, the present invention relates to the technical field of polyclonal antibody preparation. Background technique [0002] American foulbrood (AFB) is an acute and severe infectious disease of bee larvae and bee pupae caused by Paenibacilus larvae (P. larvae). The main pathogenic factors of Bacillus larvae are some extracellular secreted proteases. Studies have shown that these proteases are zinc-containing metalloproteases (Paenibacilus larvae metaloproteases, PLMP) and are involved in larval degeneration. The disease is serious, destructive and only infects bee larvae. In the early stage of infection, the body color of the larvae changes from normal pearly white to brownish-yellow, brown or even dark brown. The final corpse can pull out 2cm-3cm filaments, giving off a fishy smell, and finally clings to the nest wall with dark brown scales. P.larvae spores ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/558G01N33/532
CPCG01N33/532G01N33/558G01N33/56911G01N2333/32
Inventor 王振宝季新成员丽娟何晓杰雷程红葛婷叶尔保勒叶尔兰·阿不都买金阿斯喀·夏热甫汉哈森
Owner COMPREHENSIVE TECH SERVICE CENT YILI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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