Mycobacterium tuberculosis PCR-LFB detection kit
A technology for Mycobacterium tuberculosis and a detection kit, which is applied in the field of pathogen detection, can solve the problems of unfavorable promotion, expensive detection equipment for Mycobacterium tuberculosis, etc., and achieves the effects of easy promotion, short time period and high accuracy.
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[0032] Example 1 Design and synthesis of specific nucleic acid sequence for Mycobacterium tuberculosis identification
[0033] Specific oligonucleotide primers were designed based on the IS6110 gene of the inserted sequence shared by the Mycobacterium tuberculosis complex. The primer sequence is as follows:
[0034] Upstream primer: 5-CGATCGTGGTCCTGC-3’,
[0035] Downstream primer: 5'-AACTACGGTGTTTACGGTGC-3'.
[0036] The modified oligonucleotide probes used to hybridize with IS6110 amplified fragments are:
[0037] P1: 5’-GCGGCAAAGC-FAM-3’;
[0038] P2: 5-Biotin-GAGGGCATCGA-3’.
[0039] The specificity of the above primers and probes was analyzed by the Blast tool software on the NCBI website, which proved that the sequence did not cross-react with other sequences in the nucleic acid database, and it was preliminarily proved that it was specific to Mycobacterium tuberculosis. Oligonucleotide primers and probes were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. The...
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[0040] Example 2 Preparation of lateral flow biosensor
[0041] (1) Preparation of 30nm colloidal gold: there is a maximum absorption peak at 525nm (ultraviolet scanning spectrum), such as figure 2 As shown, the OD value is 0.882, and chloroauric acid: trisodium citrate 1:1.25.
[0042] (2) Colloidal gold and streptavidin (SA) labeling: Take the labeling of 1mL colloidal gold as an example, add 14μL 0.2M K 2 CO 3 Mix well to make the colloidal gold pH near the SA isoelectric point; add 1μLSA (10μg / μl), shake and mix for 10min; add 20μL 10% BSA for 10min, then add 20μL 10% PEG20000 for 10min, 4°C, 7000rpm, centrifuge 15min, remove the supernatant, reconstitute the original volume with the reconstituted solution (10mM PBS+0.5% Triton X-100+0.3% BSA, pH 7.4), cut the gold label binding pad into 0.9cm×30cm size, and use a pipette to The reconstituted markers are spread on the gold-labeled binding pad and dried at 37°C for 3-4 hours before use. The processing capacity of each membrane...
Example Embodiment
[0048] Example 3 Detection method of Mycobacterium tuberculosis
[0049] (1) Treatment of patients' sputum specimens and extraction of Mycobacterium tuberculosis DNA: add 2 times the volume of sputum 4% sodium hydroxide solution, shake and mix, and place at room temperature for 30 minutes, during which time shake and mix to make it fully liquefied; Centrifuge at g for 10 min, discard the supernatant; add 1.5 mL of 0.01mol / L PBS (pH 7.8), shake well and mix. Centrifuge at 6000×g for 10 min, and discard the supernatant. Collect the precipitate, add 100µL of DNA extraction solution (10 mmol Tris, 150mmol NaCL, 0.01% Triton X-100, 0.05% Tween 20, 200µg / µL proteinase K) and shake and mix thoroughly, and then water bath at 60°C for 15 min; heat at 95°C for 5 min , Centrifuge briefly; add an equal volume of chloroform, shake and mix, centrifuge at 12,000 × g for 5 min; take 5 μL of the supernatant and use it directly for PCR amplification.
[0050] (2) PCR amplification reaction procedu...
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