Diagnostic method for rifampicin resistant mycobacterium tuberculosis
A technology of Mycobacterium tuberculosis and a diagnostic method, applied in the field of warm nucleic acid amplification diagnosis, can solve the problems of complicated operation and expensive equipment, and achieve the effects of high reference value, high specificity and high sensitivity
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[0027] (1) Preparation of template DNA: extract the DNA of the sample to be tested, which is a sample identified as containing Mycobacterium tuberculosis or isolated Mycobacterium tuberculosis;
[0028] (2) Loop-mediated isothermal nucleic acid amplification reaction: Prepare the reaction system in a 200 μl PCR tube: 1 μl of primer mixture, 12.5 μl of reaction solution, 0.5-1 μl of DNA polymerase, 2-5 μl of template DNA, make up with sterilized deionized water to 25μl, then add 20μl of sealing solution, and react the above reaction tube at 63~65℃ for 60~90min;
[0029] The primer mixture contains the above-mentioned 4 specific primers (SEQ ID No.1~4), wherein, the concentration of inner primer 1 is 4~8 pmol / μl, the concentration of inner primer 2 is 4~8 pmol / μl, The concentration of outer primer 1 is 1 pmol / μl, and the concentration of outer primer 2 is 1 pmol / μl;
[0030] The reaction solution contains 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 , 5mM Betaine, the...
Embodiment 1
[0036] Preparation of the loop-mediated isothermal nucleic acid amplification reaction system:
[0037] (1) Reaction solution: 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 , 5mM Betaine, the volume ratio of the four is 8:5:3:10;
[0038] (2) Primer solution: including 4 pmol / μl internal primer 1, 4 pmol / μl internal primer 2, 1 pmol / μl external primer 1, and 1 pmol / μl external primer 2. The sequences of the four primers are:
[0039] Internal primer 1: 5'-GTGCACCCGTCGCACTACGGGCCCGACCTCCAGGCGCTG-3' (SEQ ID No.1);
[0040] Internal primer 2: 5'-GGGTCAACCCGTTCGGGTTCTGCGGTACGGCGTTTCGAT-3' (SEQ ID No.2);
[0041] Outer primer 1: 5'-GCCCGGCGGTCTGTCACGTG-3' (SEQ ID No.3);
[0042] Outer primer 2: 5'-GTCGGCGGTCAGGTACACGA-3' (SEQ ID No.4);
[0043] (3) DNA polymerase: Bst DNA polymerase, the concentration is 8U / μl;
[0044] (4) Chromogen: Fluorescent dye 1×SYBR Green I.
[0045]Use the above reaction system to diagnose rifampicin-sensitive Mycobacterium tuberculosis and r...
Embodiment 2
[0060] Preparation of the loop-mediated isothermal nucleic acid amplification reaction system:
[0061] (1) Reaction solution: 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 , 5mM Betaine, the volume ratio of the four is 9:5:4:11;
[0062] (2) Primer solution: including 8 pmol / μl internal primer 1, 8 pmol / μl internal primer 2, 1 pmol / μl external primer 1, and 1 pmol / μl external primer 2. The sequences of the four primers are the same as in Example 1;
[0063] (3) DNA polymerase: Bst DNA polymerase, the concentration is 8U / μl;
[0064] (4) Chromogen: Fluorescent dye 1×SYBR Green I.
[0065] Use the above reaction system to diagnose rifampicin-sensitive Mycobacterium tuberculosis and rifampicin-resistant Mycobacterium tuberculosis in the following manner. The sample to be tested in this embodiment is the bronchial lavage fluid of a patient identified as Mycobacterium tuberculosis.
[0066] 1. Template DNA extraction:
[0067] 1) Take 2ml of the sample to be tested an...
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