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A detection method of Lactobacillus rhamnosus immunomagnetic bead electrochemical sensor

A technology of Lactobacillus rhamnosus and immunomagnetic beads, which is applied in biochemical equipment and methods, electrochemical variables of materials, measurement/inspection of microorganisms, etc., can solve problems such as complicated operation and inability to meet rapid and large-scale detection

Active Publication Date: 2021-05-04
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods are easily affected by factors such as culture conditions, and the detection cycle reaches 5-6 days; although molecular biology methods based on PCR technology have high sensitivity, they are complicated to operate and require expensive instruments, which cannot meet the needs of rapid production and circulation in actual production. , large-scale testing requirements

Method used

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  • A detection method of Lactobacillus rhamnosus immunomagnetic bead electrochemical sensor
  • A detection method of Lactobacillus rhamnosus immunomagnetic bead electrochemical sensor
  • A detection method of Lactobacillus rhamnosus immunomagnetic bead electrochemical sensor

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Experimental program
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Embodiment 1

[0027] Cloning, expression and purification of embodiment 1 Lactobacillus rhamnosus pili protein subunit SpaA gene

[0028] According to the coding sequence of Lactobacillus rhamnosus LGG pili subunit SpaA registered in GenBank, a pair of primers containing NcoI and XhoI restriction enzyme sites were designed and synthesized by Shanghai Sangon Bioengineering Co., Ltd. The genomic DNA of Lactobacillus rhamnosus was extracted by the improved CTAB method, and the SpaA gene was amplified by PCR. The PCR amplification program was: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 50 seconds, 59°C annealing for 1 minute, 72°C extension for 2 minutes, 30 cycles ; 72°C extension for 10 min. The size of the amplified product is 1005bp. Use restriction endonucleases Nco I and Xho I to double-digest plasmid pET28-a and PCR amplification product, recover the target fragment with the kit, and connect the recovered vector and SpaA target gene with T4DNA ligase, 16 Ligated overnig...

Embodiment 2

[0030] Example 2 Preparation of recombinant SpaA pili protein polyclonal antibody

[0031] Healthy New Zealand white rabbits were pre-raised in the new environment for one week, and blood was collected from the ear vein, and the serum was separated as a negative control for the detection of antiserum titer. The purified recombinant SpaA was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and the New Zealand white rabbits were subcutaneously injected into the abdomen at multiple points. Booster immunization (1 / 2 of the immunization dose); 5 days after the last booster immunization, blood was collected from the ear vein. The collected blood was placed at 4°C for 2h, centrifuged at 3000r / min for 10min, the antiserum was collected, and the multiple antiserum after immunization was affinity purified with Protein A, the antiserum titer was 1:160000, and stored at -20°C.

Embodiment 3

[0032] Example 3 Preparation of SpaA Antibody Immunomagnetic Beads and Observation with Transmission Electron Microscope

[0033] Immunomagnetic bead preparation

[0034] Take 2mg of carboxyl-modified magnetic beads PM3-020 in a 1.5mL centrifuge tube, wash twice with 500μL MEST, and discard the supernatant by magnetic separation. Add newly prepared 200 μL 5mg / mL EDC and 200 μL 5mg / mL NHS, mix well, activate at 37°C for 30 min, and discard the supernatant by magnetic separation. Add 500 μL MEST and mix well, transfer the magnetic beads to a new centrifuge tube, and wash twice with 500 μL MEST to obtain activated magnetic beads. Add the SpaA polyclonal antibody to the activated magnetic beads, adjust the total volume to 500 μL with PBST (0.01mol / L, pH 7.4) solution, mix gently, couple at 37°C for 3 hours, discard the supernatant by magnetic separation, and add 1 mL of PBST (pH 7.4 , containing 1% BSA), resuspended magnetic beads, blocked at 37°C for 45min, washed 3 times with ...

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Abstract

The invention relates to the technical field of microbes, and relates to a detection method of a lactobacillus rhamnosus immunomagnetic bead electrochemical sensor. The method of the invention is to quantitatively detect the Lactobacillus rhamnosus in the probiotic product by using the immunomagnetic beads with the specific pili subunit SpaA antibody of the Lactobacillus rhamnosus combined with an electrochemical sensor. The method of the invention is easy to operate, does not require cultivation, and has a detection period of 2-3 hours, is convenient for popularization and use, and is suitable for rapid quantitative detection and quality evaluation of Lactobacillus rhamnosus in probiotic products and fermented products.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to an immunomagnetic bead capable of specifically recognizing Lactobacillus rhamnosus, and an electrochemical sensor detection method based on magnetic beads, which can detect Lactobacillus rhamnosus in a complex system. Enrichment and rapid quantitative detection, the detection process does not require proliferation and culture, the operation is simple, and the detection limit reaches 1.53×10 3 CFU / mL is superior to traditional detection methods, and the detection cycle is shortened from traditional 5-6 days to 2-3 hours. This method can be used for rapid isolation and quantitative detection of active Lactobacillus rhamnosus in probiotic products and environmental samples. Background technique [0002] Lactobacillus rhamnosus is currently recognized as a probiotic lactobacillus, which has physiological functions such as maintaining the intestinal microecological balance, in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/06G01N27/48
CPCC12Q1/06G01N27/48
Inventor 杨振泉薛宇蒋栋磊高璐饶胜其
Owner YANGZHOU UNIV
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