Detection method of brucella, kit and applications of kit
A technology of Brucella and detection method, which is applied in the field of Brucella detection, can solve problems such as unrealizable, and achieve the effects of pollution prevention, high sensitivity, effective and accurate quantitative detection
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Embodiment 1
[0049] This embodiment provides a method for obtaining the sample to be tested. The nucleic acid of the sample to be tested can be extracted by a commercial spin column extraction method, a commercial magnetic bead extraction method or a commercial boiling cracking method.
[0050] The invention provides a method for obtaining Brucella DNA from a serum sample by adopting a spin column method to obtain a sample to be tested. The specific method is as follows:
[0051] 1) Before use, add 17 mL of absolute ethanol to buffer GD, add 60 mL of absolute ethanol to rinse solution PW, and shake well.
[0052] 2) Take 1ml of serum sample to be tested, centrifuge at 13,000rpm for 10min, and aspirate the supernatant as much as possible.
[0053] 3) Add 200 μl buffer GA to the cell pellet, shake until the cell is completely suspended.
[0054] 4) Add 20 μl Proteinase K and 5 μl Brucella internal standard quality control solution to the tube, and mix well.
[0055] 5) Add 220 μl buffer G...
Embodiment 2
[0064] Qualitative detection of Brucella
[0065] Present embodiment 2 provides a kind of qualitative detection method of Brucella:
[0066] 1. PCR amplification reaction of Brucella
[0067] 1) Reagents and components used in the PCR amplification reaction of Brucella DNA
[0068] (1) Brucella primer probe mixture:
[0069] The specific components of the Brucella primer-probe mixture are upstream primer Bcsp-F (50 μM) 0.6 μL, downstream primer Bcsp-R (50 μM) 0.6 μL, Taqman fluorescent probe Bcsp-FP (20 μM) 0.5 μL, purified water 3.3 μL.
[0070] The preferred components of the Brucella primer-probe mixture are: upstream primer Bcsp-F (50 μM) 0.6 μL, downstream primer Bcsp-R (50 μM) 0.6 μL, Taqman fluorescent probe Bcsp-FP (20 μM) 0.5 μL, Internal standard upstream primer (20 μM) 0.5 μL, internal standard downstream primer (20 μM) 0.5 μL, internal standard probe (10 μM) 0.375 μL, purified water 1.925 μL.
[0071] The upstream primer Bcsp-F comprises the base sequence show...
Embodiment 3
[0094] Present embodiment 2 provides a kind of quantitative detection method of Brucella:
[0095] 1. PCR amplification reaction of Brucella DNA (sample to be tested)
[0096] 1) Reagents and components used in the PCR amplification reaction of Brucella DNA
[0097] (1) Brucella primer probe mixture:
[0098] The components of the Brucella primer-probe mixture are: upstream primer Bcsp-F (50 μM) 0.6 μL, downstream primer Bcsp-R (50 μM) 0.6 μL, Taqman fluorescent probe Bcsp-FP (20 μM) 0.5 μL, internal Standard upstream primer (20 μM) 0.5 μL, internal standard downstream primer (20 μM) 0.5 μL, internal standard probe (10 μM) 0.375 μL, purified water 1.925 μL.
[0099] The upstream primer Bcsp-F comprises the base sequence shown in SEQ ID NO:1;
[0100] The downstream primer Bcsp-R comprises the base sequence shown in SEQ ID NO:2;
[0101] The Taqman fluorescent probe Bcsp-FP comprises a base sequence as shown in SEQ ID NO: 3, and the Taqman fluorescent probe Bcsp-FP has a fl...
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