Rapid one-step reverse transcriptase PCR
The present invention is directed to a method for performing a one-step RT-PCR for amplifying a target RNA comprising the steps (i) providing a sample which is supposed to contain said target RNA (ii) adding a reaction mixture comprising all reagents necessary to reverse transcribe said target RNA into cDNA and amplify at least a portion of said cDNA (iii) incubating said sample for a time interval of 0 seconds to 40 seconds at a temperature between 20° C. and 65° C., and (iv) subjecting said sample to multiple cycles of a thermocycling protocol wherein the temperature of said sample is varied between at least a first temperature between 37° C. and 72° C. and a second temperature between 85° C. and 100° C.
1. A method for amplifying a target RNA comprising the steps of:
providing a sample presumed to contain said target RNA,
forming a mixture of said sample and reagents necessary to reverse transcribe said, target RNA into cDNA and amplify at least a portion of said cDNA,
within 40 seconds or less of said forming, and
subjecting said sample to multiple cycles of a thermocycling protocol wherein the temperature of said sample is varied between at least a first temperature between 90° C. and 100° C. and a second temperature between 50° C. and 75° C., thereby amplifying wherein the thermocycling protocol includes increasing the temperature of the sample to the first temperature at a rate of 10° C. per second or less.
2. The method of claim 1 wherein the temperature prior to said subjecting is between 37° C. and 55° C.
3. The method of claim 1 wherein the reagents for reverse transcription and amplification comprise a thermostable polymerase comprising DNA dependent polymerase activity and reverse transcriptase activity.
4. The method according to claim 3 wherein said polymerase is from Thermus thermophilus.
5. The method of claim 1 wherein the reagents for reverse transcription and amplification comprise a mixture of a thermostable polymerase comprising DNA dependent polymerase activity and at least a reverse transcriptase.
6. The method of claim 1 wherein said cDNA is shorter than 0.2 kb.
7. The method of claim 1 wherein progress of the amplification of said target RNA is monitored in real time.