Patent
Composition for detection of RNA
| العنوان: | Composition for detection of RNA |
|---|---|
| Patent Number: | 10760,074 |
| تاريخ النشر: | September 01, 2020 |
| Appl. No: | 13/059703 |
| Application Filed: | August 31, 2009 |
| مستخلص: | 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. |
| Inventors: | Usui, Kanako (Otsu, JP); Uemori, Takashi (Otsu, JP); Mukai, Hiroyuki (Otsu, JP); Kato, Ikunoshin (Otsu, JP) |
| Assignees: | TAKARA BIO INC. (Kusatsu-Shi, JP) |
| Claim: | 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). |
| Claim: | 2. The method according to claim 1 , wherein the step (E) is carried out during the polymerase chain reaction in the step (D). |
| Claim: | 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). |
| Claim: | 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. |
| Claim: | 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. |
| Claim: | 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. |
| Claim: | 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. |
| Claim: | 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. |
| Claim: | 9. The method according to claim 8 , wherein step (C) is carried out during the polymerase chain reaction in the step (B). |
| Claim: | 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). |
| Claim: | 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). |
| Patent References Cited: | 5268289 December 1993 Dahl 5310652 May 1994 Gelfand 5436149 July 1995 Barnes 5561058 October 1996 Gelfand et al. 5712127 January 1998 Malek 5853990 December 1998 Winger et al. 5972607 October 1999 Kondo 5981183 November 1999 Takarada 6251637 June 2001 Blusch 6303306 October 2001 Takarada et al. 7354742 April 2008 Kamme et al. 2004/0038366 February 2004 Uemori 2004/0137451 July 2004 Sagawa 2005/0059000 March 2005 Sagawa 2006/0019366 January 2006 Bi 2009/0269745 October 2009 Tonoike et al. 2009/0269809 October 2009 Hokazono et al. 2010/0016250 January 2010 Nagata et al. 2013/0302794 November 2013 Li 2007-228977 September 2007 4022522 October 2007 2008-17777 January 2008 2008-17787 January 2008 2009-50217 March 2009 WO 03/072798 September 2003 WO 03/074696 September 2003 WO 03072798 September 2003 WO 2007/052765 May 2007 WO 2007/119815 October 2007 |
| Other References: | Myers TW, Gelfand DH. Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry. 1991. 30(31):7661-6. cited by examiner BcaBESTManual.pdf. cited by examiner Maeda et al., Quantitative real-time PCR using TagMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunol Med Microbiol. 2003. 39(1):81-6. cited by examiner Callison SA, Hilt DA, Jackwood MW. Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction. J Virol Methods. Mar. 2005;124(1-2):183-90. cited by examiner Qiaquick spin handbook (Mar. 2008). cited by examiner Epicentre manual (1993). cited by examiner SuperScript First Strand Synthesis System Manual (Invitrogen/Life Technologies) 2003. cited by examiner Maeda et al. (2003). FEMS Immunology and Medical Microbiol 39: 81-86. cited by examiner Schneeberger et al. (1995) PCR methods Appl. 4: 234-238. cited by examiner Wang et al. (2003) Nuc. Acid Research. vol. 31 No. 24 E154: p. 1-8. cited by examiner Gasparic M, Cankar K, Zel J, Gruden K. Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms. BMC Biotechnol. Mar. 6, 2008;8:26. cited by examiner Epicentre manual (1995) Hybridase Thermostable RNase H, (also disclosed in U.S. Pat. No. 5,268,289, Dec. 7, 1993), retrieved from http://www.epibio.com on Jan 6, 2013. cited by examiner Epicentre manual (2012) E. coli RNase H, retrieved from http://www.epibio.com on Jan. 6, 2013. cited by examiner Polumuri SK, Ruknudin A, Schulze DH. RNase H and its effects on PCR. Biotechniques. Jun. 2002; 32(6):1224-5. (Year: 2002). cited by examiner Kitabayashi M, Esaka M. Improvement of reverse transcription PCR by RNase H. Biosci Biotechnol Biochem. Nov. 2003; 67(11): 2474-6. (Year: 2003). cited by examiner Uemori T, Mukai H, Takeda O, Moriyama M, Sato Y, Hokazono S, Takatsu N, Asada K, Kato I. Investigation of the molecular mechanism of ICAN, a novel gene amplification method. J Biochem. Aug. 2007; 142(2):283-92. Epub Aug. 24, 2007. (Year: 2007). cited by examiner Higuchi et al., “Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions”, Bio/Technology, vol. 11, Sep. 1993, pp. 1026-1030. cited by applicant International Search Report dated Sep. 29, 2009 for International Application No. PCT/JP2009/065151. cited by applicant Kitabayashi et al., “Improvement of Reverse Transcription PCR by RNase H”, Biosci. Biotechnol. Biochem., vol. 67, No. 11, 2003, pp. 2474-2476. cited by applicant Japanese Office Action for Japanese Application No. 2014-224007, dated May 17, 2016. cited by applicant Extended European Search Report dated Oct. 31, 2011, for European Application No. 09811459.8. cited by applicant Harvey et al., “Characterization and applications of CataCleave probe in real-time detection assays”, Analytical Biochemistry, vol. 333, 2004, pp. 246-255, XP004573012, (Available online Jul. 20, 2004). cited by applicant Mukai et al., “Highly Efficient Isothermal DNA Amplification System Using Three Elements of 5′-DNA-RNA-3′ Chimeric Primers, RNaseH and Strand-Displacing DNA Polymerase”, Journal of Biochemistry, vol. 142, No. 2, 2007, pp. 273-281, XP002661810. cited by applicant |
| Assistant Examiner: | Oyeyemi, Olayinka A |
| Primary Examiner: | Benzion, Gary |
| Attorney, Agent or Firm: | Birch, Stewart, Kolasch & Birch, LLP |
| رقم الانضمام: | edspgr.10760074 |
| قاعدة البيانات: | USPTO Patent Grants |
| الوصف غير متاح. |