Composition for detection of RNA
A composition for use in amplifying cDNA synthesized by a reverse transcription reaction and detecting RNA that serves as a template of the reverse transcription reaction, the composition containing a thermostable DNA polymerase, a thermostable ribonuclease H, and an intercalating dye. Since the composition of the present invention can suppress the influences to the nucleic acid amplification reaction by RNA that serves as a template for cDNA synthesis, the composition is useful in the detection of RNA, and more useful in quantification of RNA having a desired sequence by real-time RT-PCR.
1. A method for detecting RNA, comprising the steps of:
(A) preparing a first composition comprising a reverse transcriptase, at least a first oligonucleotide primer, at least one deoxyribonucleotide, and RNA that serves as a template of a reverse transcription reaction;
(B) incubating the first composition prepared in the step (A) to synthesize cDNA, wherein the cDNA after the reverse transcription reaction forms a DNA/RNA hybrid with RNA that serves as a template for a reverse transcriptase;
(C) preparing a second composition comprising the cDNA synthesized in the step (B), a thermostable DNA polymerase, a thermostable ribonuclease H, wherein the thermostable ribonuclease H is contained in the second composition at a concentration of 0.001 to 100 U/25 μL, at least a second oligonucleotide primer, at least one deoxyribonucleotide, a buffer for polymerase chain reaction, and an intercalating dye;
(D) carrying out a heat treatment of the second composition prepared in the step (C) at 90° C. or more for 15 seconds to 10 minutes, and carrying out polymerase chain reaction using the product obtained by the heat treatment, and amplifying nucleic acids; and
(E) measuring a fluorescent signal intensity from the intercalating dye, thereby detecting the nucleic acids amplified in the step (D).
2. The method according to claim 1 , wherein the step (E) is carried out during the polymerase chain reaction in the step (D).
3. The method according to claim 1 or 2 , further comprising the step (F) of quantifying RNA that serves as a template from the signal intensity measured in the step (E).
4. The method according to claim 1 , wherein the thermostable ribonuclease H is contained in the second composition at a concentration of 0.01 to 10 U/25 μL.
5. The method according to claim 1 , wherein the thermostable ribonuclease H is contained in the second composition at a concentration of 0.1 to 10 U/25 μL.
6. The method according to claim 1 , wherein the thermostable ribonuclease H is contained in the second composition at a concentration of 0.1 to 1 U/25 μL.
7. The method according to claim 1 , wherein the thermostable ribonuclease H is selected from a ribonuclease H from bacteria belonging to the genus Thermus or Thermococcus.
8. A method for detecting RNA, comprising the steps of:
(A) preparing a composition comprising a DNA/RNA hybrid, a thermostable DNA polymerase, a thermostable ribonuclease H, at least one oligonucleotide primer, at least one deoxyribonucleotide, a buffer for polymerase chain reaction, and an intercalating dye,
(B) subjecting the composition prepared in step (A) to polymerase chain reaction continuously with heat treatment at 90° C. or more for 15 seconds to 10 minutes to amplify the nucleic acid, and
(C) detecting the nucleic acid amplified in step (B) by measuring the intensity of the fluorescent signal from the intercalating dye,
wherein the thermostable DNA polymerase is selected from the group consisting of Thermus aquaticus, Thermus thermophiles, Thermus flavus, Thermus filiformis, Pyrococcus furiosus, Pyrococcus woesei, Pyrococcus horikoshii, Thermococcus litoralis, Thermococcus celer, Thermococcus siculi, Thermococcus sp. KS-1 and Thermococcus kodakaraensis; and
wherein the thermostable ribonuclease H is selected from the group consisting of Thermus thermophiles, Thermus flavus and Thermococcus litoralis.
9. The method according to claim 8 , wherein step (C) is carried out during the polymerase chain reaction in the step (B).
10. The method according to claim 8 , further comprising the step (D) of quantifying RNA that serves as a template from the signal intensity measured in the step (C).
11. The method according to claim 9 , further comprising the step (D) of quantifying RNA that serves as a template from the signal intensity measured in the step (C).