Tubercle bacillus drug tolerance detection reagent kit and tubercle bacillus drug tolerance detection method
A detection kit and technology for Mycobacterium tuberculosis, applied in biochemical equipment and methods, microbial determination/examination, chemical library, etc., can solve the problems of undetectable patients, long turnaround time, accuracy limitations, etc. and sequencing, reducing operational steps, and improving accuracy
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Embodiment 1
[0040] Embodiment 1 A kind of tubercle bacillus drug resistance detection kit
[0041] The present embodiment is a kind of tubercle bacillus drug resistance detection kit, comprises tubercle bacillus nucleic acid detection reagent and tubercle bacillus drug resistance detection reagent:
[0042] (1) Mycobacterium tuberculosis nucleic acid detection reagents include primer pair 1 for IS6110, primer pair 2 for IS6110, probe primers for IS6110, internal quality control template, probe primers for internal quality control template, QuantiNova ProbePCR Master Mix and DEPC water.
[0043] The primer pair 1 for IS6110 includes IS6110-FW1 shown in SEQ ID NO.1 and IS6110-RV1 shown in SEQ ID NO.2; the primer pair 2 for IS6110 includes the sequence shown in SEQ ID NO.3 IS6110-FW2 and IS6110-RV2 shown in SEQ ID NO.4; the probe primer sequence for IS6110 is shown in SEQ ID NO.5, the 5' end of the probe primer is modified with the fluorescent group FAM, and the 3' end Modified with quench...
Embodiment 2
[0059] Example 2 A library construction method for the detection of drug resistance of Mycobacterium tuberculosis
[0060] In this embodiment, a method for constructing a library for detecting drug resistance of Mycobacterium tuberculosis, using the kit described in Example 1 for library construction, includes the following steps:
[0061] (1) Collect sputum samples, extract DNA, and then perform PCR amplification detection on the nucleic acid of Mycobacterium tuberculosis:
[0062]A. Add 100 μL of digestive solution to 1 mL of sputum sample, vortex to mix, and then incubate at 65°C for 10 minutes; B. Centrifuge the mixture obtained in step A at 13200g for 10 minutes, discard the supernatant; C. Add 100 μL Tris-HCl solution to the precipitate obtained in B, vortex to mix, then centrifuge at 13200g for 10 minutes, discard the supernatant; D, add 100 μL lysate to the precipitate obtained in step C, and incubate at 65°C for 45 minutes; E. Add 100 μL Tris-HCl solution to the mixt...
Embodiment 3
[0108] Embodiment 3 A kind of method for detecting drug resistance of Mycobacterium tuberculosis
[0109] This embodiment is a method for detecting drug resistance of Mycobacterium tuberculosis. The library is constructed using the method described in Example 2, and the concentration of the constructed library is measured and then sequenced using a sequencer. The specific steps are as follows:
[0110] 1. Library concentration determination and 2100 determination
[0111] (1) use 3.0 Fluorometer for library concentration determination, use agilent 2100bioanalyzer for length determination;
[0112] (2) Record the concentration data in the test sample information record form;
[0113] (3) The library is stored at -20°C.
[0114] 2. On-board inspection
[0115] (1) Information recording and preparation of reagents on the machine
[0116] ①Record the reagent information used on the machine in the "Molecular Pathology Reagent Use Record Form";
[0117] ②Take out the SBS Reag...
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