| 主权项 |
1. A method for amplifying a target nucleotide sequence comprising:
(a) obtaining, from an animal, plant, or food, a sample comprising a target nucleic acid, the target nucleic acid comprising the target nucleotide sequence, (b) without first subjecting the target nucleic acid to a thermal denaturation step associated with amplification of the target nucleotide sequence, combining, in a single step, the obtained sample directly with an amplification reagent mixture to form a reaction mixture or diluting the obtained sample and combining, in a single step, the diluted sample with an amplification reagent mixture to form a reaction mixture, in either case, the amplification reagent mixture being free of bumper primers and comprising:
(i) a polymerase,(ii) a forward template nucleic acid comprising a 5′ portion that is non-complementary to the target nucleic acid sequence and contains a nicking site and a 3′ template recognition region that hybridizes to the target nucleic acid sequence,(iii) a reverse template nucleic acid comprising a 5′ portion that is non-complementary to the target nucleic acid sequence and contains a nicking site and a 3′ recognition region that hybridizes to the target nucleic acid sequence, and(iv) one or more nicking enzymes; (c) subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions to amplify the target nucleotide sequence without the assistance of bumper primers, wherein amplification is performed, in the presence of the one or more nicking enzymes, by multiple cycles of the polymerase extending the reverse template nucleic acid along the target nucleotide sequence and extending the forward template nucleic acid along the complement of the target nucleotide sequence, thereby producing a double-stranded nucleic acid amplification product comprising first and second double-stranded nicking sites spaced apart by the target nucleotide sequence; and (d) detecting the amplified target nucleotide sequence in real time within 10 minutes of subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions, wherein: (i) the target nucleotide sequence is between 20 and 40 nucleotides in length; and (ii) the target nucleotide sequence is amplified 1E+6-fold or more in about ten minutes. |
