Method for rapidly detecting salmonellae

A rapid technology for Salmonella, applied in biochemical equipment and methods, microbiological determination/inspection, and resistance to vector-borne diseases. Wide range of application promotion, simple and fast operation, and short time required

Pending Publication Date: 2017-05-24
BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] The routine method of Salmonella detection is currently used by most domestic testing institutions, the main steps are pre-enrichment (8-18h), enrichment (18-24h), plate culture separation (18-24h), biochemical test And serological identification, due to its shortcomings such as long detection cycle, complicated procedures, and various reagents required, it is far from meeting the requirements of modern detection.

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  • Method for rapidly detecting salmonellae
  • Method for rapidly detecting salmonellae
  • Method for rapidly detecting salmonellae

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0036] In Example 1 of this preparation, Antibody 1 was used to prepare the immunomagnetic beads used in the method of the present invention.

[0037] Surface-modified superparamagnetic Fe with carboxyl groups activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) 3 o 4 Polystyrene composite microspheres (purchased from Shanghai Aorun Micro-Nano New Material Technology Co., Ltd., PM3-020 polymer magnetic balls, the concentration is 10mg / mL, the particle size is 180nm, and the surface is modified with carboxyl functional groups) are used as magnetic balls , take 2 mg of magnetic spheres and disperse them in the phosphate buffer solution, the concentration of the magnetic spheres in the dispersion is 2 mg / mL. Mix 0.2mg of specific antibody (dissolved in 1mL of phosphate buffer) with 2mg of magnetic balls, keep in a shaker (200r / min) for 3h at room temperature, and then wash by magnetic separation to remove uncoupled magnetic beads For ...

Embodiment 1

[0039] This example is used to illustrate the sensitivity, specificity and accuracy of the method of the present invention.

[0040] Different bacterial strains (see Table 1) were mixed with 2mL of PBS to obtain bacterial suspensions of different concentrations (i.e. samples to be tested), and immunomagnetic bead capture, bacterial culture, PCR amplification and electrophoresis detection were carried out in the following manner, wherein, The samples to be tested in the dead bacteria group were obtained by inactivating the bacteria suspension under the conditions of 121°C and 0.103MPa for 30min.

[0041] Table 1

[0042]

[0043] Note:" Picture 1-1 "express figure 1 1 lane in , and so on.

[0044] (1) Immunomagnetic bead capture

[0045] Add the bacterial suspension to the immunomagnetic bead centrifuge tube obtained in Preparation Example 1, oscillate gently on a vortex mixer, place in a mixer or a shaker, and rotate (or oscillate) at room temperature (25°C) ) for 30 ...

Embodiment 2

[0063] Carry out the detection of Salmonella according to the method of embodiment 1, difference is, the sample used is as shown in table 4, and the sequence of the specific primer (the FimY gene that can amplify 526bp) that PCR amplification uses is as follows:

[0064] Forward primer (FimYF): 5'-GCCGGTAAACTACACGATGA-3' (SEQ ID NO: 3)

[0065] Reverse primer (FimYR): 5'-GAGTTACTGAACCAACAGCT-3' (SEQ ID NO: 4)

[0066] Table 4

[0067]

[0068] Note:" Figure 3-1 "express image 3 1 lane in , and so on.

[0069] Electrophoresis results such as image 3 Shown: the experimental group 1-2 and the positive control group 3-4 present an obvious 526bp band, while the negative control group 5 and 6 have no obvious band. From image 3 It can also be seen that the method of the present invention can be used to specifically and accurately detect Salmonella.

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Abstract

The invention belongs to the field of detection of pathogenic bacteria and discloses a method for rapidly detecting salmonellae. The method comprises steps as follows: (1) immunomagnetic beads and a to-be-detected sample in a form of liquid are mixed to capture possible salmonellae in the to-be-detected sample, and the immunomagnetic beads are obtained through coupling of antibodies and magnetic balls; (2) the immunomagnetic beads possibly capturing the salmonellae are mixed with a liquid culture medium, and the obtained mixture is cultured under the conditions under which salmonellae can be proliferated; (3) thalli obtained through culture in step (2) are subjected to PCR (polymerase chain reaction) amplification by use of specific primers of the salmonellae, and existence of the salmonellae in the to-be-detected sample is judged according to a PCR amplification product. The method is simple and rapid to operate, takes a short time, has higher sensitivity and accuracy and high applicability, is particularly favorable for application and popularization in a wide range and has great significance for safety monitoring and supervision of food.

Description

[0001] This application is a divisional application of a Chinese invention patent application with an application date of August 14, 2014, an application number of 201410400047.7, and a title of “A Method for Rapid Detection of Salmonella”. technical field [0002] The invention belongs to the field of detection of pathogenic bacteria, and in particular relates to a method for rapid detection of Salmonella. Background technique [0003] Pathogenic bacteria (Pathogenic bacteria) Microorganisms that can cause disease are called pathogenic microorganisms or pathogenic bacteria. Pathogenic microorganisms include bacteria, viruses, spirochetes, rickettsia, chlamydia, mycoplasma, fungi, and actinomycetes. Generally speaking, pathogenic bacteria refer to bacteria in pathogenic microorganisms. The pathogenicity of bacteria is related to its virulence, invasion quantity and invasion portal. While the vast majority of bacteria are harmless or even beneficial, a significant number ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10
CPCC12Q1/6804C12Q2531/113Y02A50/30
Inventor 杜美红孙永军许迪莘杨寅陈婷
Owner BEIJING CENT FOR PHYSICAL & CHEM ANALYSIS
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