Kit used for rapid detection of enterobacter sakazakii in milk, and applications thereof

A technology of Enterobacter sakazakii and a kit, which is applied in the direction of microorganism-based methods, measurement/inspection of microorganisms, microorganisms, etc., can solve the problems of unsuitable on-site detection, errors in naked eye observation, time-consuming and labor-intensive, etc., and achieve suitable on-site detection, The effect of short time consumption and simple equipment requirements

Active Publication Date: 2014-01-01
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional electrophoresis detection method is time-consuming and labor-intensive, and the dye

Method used

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  • Kit used for rapid detection of enterobacter sakazakii in milk, and applications thereof
  • Kit used for rapid detection of enterobacter sakazakii in milk, and applications thereof
  • Kit used for rapid detection of enterobacter sakazakii in milk, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. DNA extraction of tested milk samples: Add 1 mL of DNA extraction solution (0.1 mol / L Tris-HCl, 0.1 mol / L ethylenediaminetetraacetic acid EDTA, 0.1 mol / L to 1 mL of tested milk. L Sodium Phosphate Na 3 PO 4 , 1.5mol / L sodium chloride NaCl, 1% CTAB hexadecyltrimethylammonium bromide, pH8.0), 10 μL proteinase K, 10 μL lysozyme, 10 μL lysozyme at 37°C at 200r / min Incubate at rotational speed for 1.5 hours; add 200 μL of 20% SDS to it and incubate at 65°C for 1 hour; add an equal volume of chloroform and centrifuge at 13,000 r / min for 1 minute and take the supernatant; add 0.6 times the volume to the obtained supernatant to pre-cool Mix the isopropanol upside down; transfer it to the adsorption column AC and centrifuge at 10,000r / min for 30 seconds, then add 700μL of rinsing solution; centrifuge at 10,000r / min for 2 minutes, pour off the liquid, add 500μL of rinsing solution, and centrifuge to remove the rinsing solution as much as possible ; Add 50 μL of elution buffe...

Embodiment 2

[0031] 1. The DNA extraction method of the tested milk sample is the same as in Example 1

[0032] 2. Use the extract as a template for LAMP amplification, and the reaction system is a 50 μL reaction system:

[0033]

[0034]

[0035] LAMP amplification conditions are: react at 95°C for 5 minutes, quickly place at 2°C and add Bst enzyme, react at 63°C for 60 minutes, and passivate at 80°C for 5 minutes.

[0036] 3. Test strip detection of amplified products: Take 2 μL of probe 5'-CGAAGACTCTCGCACTCGCAGCA-3' and hybridize with the product at 64°C for 5 minutes, then take 10 μL of the hybridization product and incubate with 110 μL buffer for 5 minutes, and finally put the test strip Put it in and incubate for 15 minutes, observe whether there is a band on the detection line of the test strip for result interpretation. The result is as figure 1 , 2 As shown, the blank is not amplified and there is no band in the detection line.

[0037] This implementation method has simpl...

example 3

[0038] Detection of Enterobacter sakazakii in example 3 milk

[0039] 1. The DNA extraction method of the tested milk sample is the same as in Example 1

[0040] 2. Use the extract as a template for LAMP amplification, and the reaction system is a 50 μL reaction system:

[0041]

[0042]

[0043] LAMP amplification conditions are: react at 90°C for 10 minutes, quickly place at 4°C and add Bst enzyme, react at 65°C for 40 minutes, and passivate at 90°C for 5 minutes.

[0044] 3. Test strip detection of the amplified product: Take 2 μL of probe 5’-CTAGGTCACACGAAGACTGCAGC-3’ and hybridize with the product at 64°C for 5 minutes, then take 10 μL of the hybridization product and incubate with 110 μL buffer for 5 minutes, and finally put the test strip Put it in and incubate for 15 minutes, observe whether there is a band on the detection line of the test strip for result interpretation. The result is as figure 1 , 2 As shown, the blank is not amplified and there is no band...

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Abstract

The invention discloses a kit used for rapid detection of enterobacter sakazakii in milk, and applications thereof, and belongs to the field of food safety technology. The kit comprises a loop-mediated isothermal amplification (LAMP) reaction system, Bst DNA polymerase, a probe and a colloidal gold test strip. The kit is capable of solving a problem that result interpretation of existing LAMP detection methods is not capable of realizing on-site detection; result interpretation method of the test strip of the kit is simple, no instrument is needed, and interpretation can be realized by the naked eye.

Description

technical field [0001] The invention relates to a kit for rapidly detecting Enterobacter sakazakii in milk and an application thereof, belonging to the technical field of food safety. Background technique [0002] Enterobacter sakazakii is a Gram-negative, non-spore-forming, facultative anaerobic bacterium. It can cause disease in people of any age, but is most at risk in premature, low-birth-weight, or immunocompromised infants, causing neonatal sepsis, meningitis, and necrotizing enterocolitis. Therefore, Enterobacter sakazakii is one of the must-test indicators for microorganisms in food in many countries. However, traditional detection methods are complicated to operate, time-consuming and require complex and expensive instruments. Loop-mediated isothermal amplification technology was established by Notomi et al. in 2000. This technology designs 4 kinds of specific primers for 6 regions of the target gene, uses Bst DNA polymerase with strand displacement activity, and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12R1/01
CPCC12Q1/04C12Q1/6804C12Q2531/119C12Q2563/131C12Q2565/625
Inventor 姜毓君满朝新董鑫悦
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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