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Method for ELISA rapid detection of thermoduric bacteria

A detection method and heat-resistant bacteria technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of high requirements for equipment and experimental conditions, cumbersome steps, and difficulties in popularization and application

Inactive Publication Date: 2013-07-10
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the detection method of heat-resistant bacteria based on PCR is fast and specific, it requires high equipment and experimental conditions, and the steps are cumbersome, making it difficult to popularize and apply.

Method used

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Examples

Experimental program
Comparison scheme
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Embodiment Construction

[0010] The present invention carries out according to the following specific steps:

[0011] a. Capture of thermostable bacteria

[0012] a.1 Take 10-20g of concentrated apple juice to be tested and dilute it appropriately with sterile water,

[0013] a.2 Use a solvent filter to filter and capture heat-resistant bacteria, the filter membrane pore size is 0.45 μm,

[0014] a.3 Heat-resistant bacteria are retained on the surface of the filter membrane;

[0015] b. Pre-enrichment of thermostable bacteria

[0016] b.1 Use 402 medium to pre-increase bacteria for 14-15 hours, so that the concentration of heat-resistant bacteria in the culture medium reaches 5×10 4 more than one / mL,

[0017] b.2 Centrifuge the enriched culture solution at 10000-12000rpm / min for 10-15min;

[0018] c. Thermostable bacteria coated microtiter plate

[0019] c.1 Use 0.05mol / L, pH9.6 carbonic acid buffer to prepare bacteria suspension as coating solution,

[0020] c.2 Add the coating solution evenly...

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PUM

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Abstract

The invention relates to a method for ELISA rapid detection of thermoduric bacteria. The prior bacteria platiculture number notation has a long detection cycle, and the steps of PCR and QC-PCR detection methods are cumbersome. The method overcomes the defects of the prior method for detecting the thermoduric bacteria comprises the following steps: catching thermoduric bacteria, pre-culturing bacteria, coating an enzyme labeled plate, sealing the enzyme labeled plate, adding an polyclonal antibody of the thermoduric bacteria, adding an enzyme labeled antibody, adding a substrate working solution, and measuring with an enzyme labeling instrument. Because each step comprises a flushing step, if a sample does not contain the thermoduric bacteria, the polyclonal antibody of the thermoduric bacteria and the enzyme labeled antibody are washed away; and if the sample contains the thermoduric bacteria, the required detection concentration is reached by pre-culturing the bacteria, and finally the color of the substrate is developed and the substrate is in positive reaction. The method is simple, convenient and practicable, the detectability reaches the international standard, the detection standard that the thermoduric bacteria in apple condensed juice are not more than 1 in 10 milliliters can be satisfied, and the operation process can be finished in 24 hours to achieve the effect of rapid detection.

Description

technical field [0001] The invention relates to a detection method for heat-resistant bacteria, in particular to an ELISA rapid detection method for heat-resistant bacteria, and belongs to the field of food microorganism detection. Background technique [0002] ELISA (Enzyme-linked Immunosorbent Assay) is the abbreviation of enzyme-linked immunoassay technology. This method is based on immunological reactions and combines the specific reactions of antigens and antibodies with the efficient catalytic effect of enzymes on substrates. highly sophisticated analytical techniques. The basic principles are: ① Make the antigen or antibody bind to the surface of a certain solid phase carrier and maintain its immune activity. ②The antigen or antibody is connected with an enzyme to form an enzyme-labeled antigen or antibody, which not only retains its immune activity, but also retains the activity of the enzyme. During the determination, the tested sample (containing the tested antib...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/531
Inventor 李建科王峰余朝舟
Owner SHAANXI NORMAL UNIV