Method of stabilizing reagent for amplifying or detecting nucleic acid and storage method
A reagent and nucleic acid technology, applied in biochemical equipment and methods, biological testing, material inspection products, etc., can solve the problem of destroying the cleavability of DNA fragments
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Embodiment 1
[0212] Mycobacterium tuberculosis was used as the experimental material to verify the detection method of the present invention. Based on the nucleotide sequence of the Mycobacterium tuberculosis genome registered in GenBank, numbered AL123456, primers K-F-1033-2 (SEQ ID NO: 7) and K-F for amplifying the region with relatively low GC content in the Mycobacterium tuberculosis genome were synthesized -1133-2 (SEQ ID NO: 8). The length of the region connected to the primer pair and including the primer part is 105 bp. Genomic DNA of Mycobacterium tuberculosis as a template was extracted from dried BCG vaccine (Nippon BCG Seizo) according to a conventional method. Prepare serial dilutions containing 100 gf to 10 pg of genomic DNA in 1 μl of sterile water. The reaction was carried out as follows. Briefly, a reaction mixture was prepared in a final volume of 25 μl containing the following final concentrations: 32 mM HEPES-potassium hydroxide buffer (pH 7.8); 100 mM potassium acet...
Embodiment 2
[0214] (1) Validation of the method for detecting target nucleic acids using RNA probes. Mycobacterium tuberculosis was chosen as the experimental material. 1 ng or 100 pg of the BCG genomic DNA prepared in Example 1 and used as a template was added to 50 μl of the reaction mixture. Using a DNA synthesizer to synthesize primers MTIS2F (SEQ ID NO: 162) and MTIS2R (SEQ ID NO: 163), and probes for detecting MTIS (SEQ ID NO: 11) and MTIS-2 (SEQ ID NO: 12) . Each probe has fluorescent labels 6-FAM (GlenResearch) and TAMRA (Glen Research) at the 5' and 3' ends, respectively. The reaction was performed as described in the Examples except that 4 U of BcaBEST DNA polymerase and 18.75 U of Pfu RNase HII were used. 5 pmol of RNA probe was added to each reaction mixture (final volume 50 μl). From 50 μl of each reaction mixture, 25 μl was used for the ICAN reaction at 58 °C. Smart Cycler (Takara Shuzo) was used for ICAN reaction and detection of amplification products. The result is ...
Embodiment 3
[0220] The storage stability of the reagents used in the method of the present invention was examined as follows.
[0221] (1) Prepare the premix solution for ICAN reaction containing the following substances, and store at 4°C or 30°C for about one month: 1.6mM various dNTPs, 101mM HEPES-potassium hydroxide buffer (pH7.8), 317mM potassium acetate, 12.7 mM magnesium acetate, 0.03% bovine serum albumin, 3.2% dimethyl sulfoxide, 0.56 U / μl of Afu RNase HII and 0.35 U / μl of BcaBEST DNA polymerase.
[0222] Add 16.125 μl of an aqueous solution containing two 50 pmol primers for detecting Mycobacterium tuberculosis, namely K-F-1033(68) (SEQ ID NO: 21) and K-R-1133(68) (SEQ ID NO: 22) to 7.875 μl of ICAN In the premix used for the reaction. In addition, 1 µl of the aqueous solution containing BCG genomic DNA at a concentration of 100 pg / µl or 10 pg / µl prepared in Example 1 was added to the mixed solution. The corresponding mixture was subjected to ICAN reaction at 64°C for 1 hour. ...
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