Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis

[0043] Example 1, Preparation and Purification of Polyclonal Antibody

[0044] 1.1 Preparation of bacteria for immunization

[0045] LB solid plate was streaked to activate Salmonella typhimurium and Escherichia coli O157:H7, pick a single colony and carry out shaking culture in LB liquid at 37℃; Shake culture. Collect bacteria by centrifugation at 4000g for 5 minutes; resuspend and wash the bacteria twice in PBS, and finally resuspend them in PBS and perform ultrasonic crushing; centrifuge at 13000g and 10 minutes to enrich the bacteria fragments, resuspend in PBS, wash twice, and resuspend ; Add formalin solution (37% formaldehyde) at 0.7:100, mix well, place at room temperature, and inactivate the bacteria for 24 hours; centrifuge at 13000g for 10 minutes, take the precipitate, wash it twice with PBS (to remove formaldehyde); collect by centrifugation Precipitate, store at -80°C for future use (you can weigh it first and divide it into equipment).

[0046] 1.2 Preparatio...

[0049] Embodiment 2, the preparation of immunomagnetic beads

[0050] 2.1 Accurately prepare 1mg / mL BSA solution with 0.005M borate buffer.

[0051] 2.2 Take 5 mg of magnetic beads and wash them once with ethanol (to remove the surfactant).

[0052] 2.3 Wash twice with sterile MEST (pH5.5~6.0), remove the supernatant after magnetic separation.

[0053] 2.4 Add EDC / NHSS (each 5 mg) and shake to activate at room temperature for 30 minutes.

[0054] 2.5 Remove the supernatant after magnetic separation, resuspend with sterile MEST (pH5.5~6.0), transfer to a new centrifuge tube, and wash three times.

[0055] 2.6 Take a 1.5mL centrifuge tube, add 100μL 1mg / mL purified polyclonal antibody, make up to 1mL with 0.005M borate buffer, adjust the pH to 8.5~9.0 with NaOH, quickly add to the freshly activated magnetic beads and shake at room temperature Reaction 3h.

[0056] 2.7 After magnetic separation, the supernatant was aspirated, and the magnetic beads coupled to the antibody wer...

[0057] Embodiment 3, sample pretreatment

[0058] Let us take a food sample as an example to illustrate the process of sample processing in the method of the present invention.

[0059] Weigh 1g of food sample, add 9mL of PBS, grind to make a homogenate, centrifuge at 900g for 5min, remove the enlarged food residue, and take the supernatant (this process must be performed aseptically).

[0060] Step 4. Immunomagnetic beads capture target bacteria in food samples

[0061] Take 1 mL of food sample liquid, add 2.0 mg of immunomagnetic beads, incubate with gentle shaking at 37°C for 45 min, separate with a magnetic frame, wash twice with PBST, and resuspend the separated magnetic beads in 50 μL of PBS.