Claims for Patent: 8,148,079

Summary for Patent: 8,148,079

Title:	Method of producing amplification product by PCR and usage thereof
Abstract:	A method of producing a PCR amplification product is provided that suppresses an effect of precipitate, turbidity, or the like derived from a whole blood sample on a detection in the detection of an amplified nucleic acid by an optical unit. The amplification product complementary to a target nucleic acid in the whole blood sample is produced by PCR in a condition where a ratio of the whole blood sample in a PCR reaction solution is in the range of 0.1 to 0.9% by volume or 0.01 to 1.8 g/L in term of hemoglobin content. When the PCR is carried out with such conditions, even with an untreated whole blood sample, a monitoring of the amplification product by the optical unit can be done while suppressing the effect of the precipitate or the turbidity.
Inventor(s):	Izumizawa; Yuji (Kyoto, JP), Majima; Satoshi (Kyoto, JP)
Assignee:	ARKRAY, Inc. (Kyoto, JP)
Application Number:	12/294,304
Patent Claims:	1. A method of analyzing an amplification product; performing qualitative or quantitative analysis of the amplification product prepared by PCR, wherein the method includes: (A) preparing the amplification product complementary to a target nucleic acid in a whole blood sample by PCR, wherein a ratio of the whole blood sample in a PCR reaction solution is in a range of 0.01 to 0.9% by volume; and (B) detecting the amplification product by an optical unit. 2. The method of analyzing an amplification product according to claim 1, wherein albumin is added to the PCR reaction solution in advance of start of PCR reaction. 3. The method of analyzing an amplification product according to claim 2, wherein a ratio of albumin in the PCR reaction solution is in a range of 0.1 to 1% by weight. 4. The method of analyzing an amplification product according to claim 2, wherein albumin is at least one selected from a group consisting of bovine serum albumin, human serum albumin, rat serum albumin, and horse serum albumin. 5. The method of analyzing an amplification product according to claim 1, further comprising applying a heat treatment to the whole blood sample in advance of start of PCR reaction. 6. The method of analyzing an amplification product according to claim 5, wherein the heat treatment is applied to a diluted sample of the whole blood sample in the process of the heat treatment, and a ratio of the whole blood sample in the diluted sample is in a range of 0.01 to 90% by volume. 7. The method of analyzing an amplification product according to claim 5, wherein a heating temperature in the process of the heat treatment is in a range of 80 to 99.degree. C. 8. The method of analyzing an amplification product according to claim 5, wherein a treating time in the process of the heat treatment is at least 30 seconds. 9. The method of analyzing an amplification product according to claim 1, wherein the target nucleic acid in the whole blood sample is DNA and the DNA is a template of PCR. 10. The method of analyzing an amplification product according to claim 1, wherein the target nucleic acid in the whole blood sample is RNA, and cDNA prepared from the RNA by a reverse transcription reaction is a template of PCR. 11. The method of analyzing an amplification product according to claim 1, wherein the number of amplifications of PCR is at least 30 cycles. 12. The method of analyzing the amplification product according to claim 1, wherein the amplification product is detected with time in the process (B). 13. The method of analyzing the amplification product according to claim 1, wherein the amplification product is detected by measuring a fluorescence generated from the amplification product. 14. The method of analyzing the amplification product according to claim 1, wherein the optical unit is a fluorometer. 15. The method of analyzing the amplification product according to claim 1, wherein the PCR reaction solution further contains intercalator which intercalates into double-stranded DNA, and in the process (B), the amplification product is detected by measuring a fluorescence generated in response to an irradiation of an excitation light to the intercalator. 16. The method of analyzing the amplification product according to claim 1, wherein the PCR reaction solution further contains fluorescent substance, quencher, and a probe having a partial sequence complementary to a template of PCR, and in the process (B), the amplification product is detected by measuring a fluorescence generated in response to an irradiation of an excitation light to the fluorescent substance. 17. The method of analyzing the amplification product according to claim 1, wherein, in the process (B), the method of detecting the amplification product is a Tm analysis. 18. A method of analyzing a target nucleic acid; performing quantitative analysis of the target nucleic acid contained in a sample, wherein the sample is a whole blood sample, and wherein the method includes: (A) preparing an amplification product complementary to the target nucleic acid in the whole blood sample by PCR, wherein a ratio of the whole blood sample in a PCR reaction solution is in a range of 0.01 to 0.9% by volume; (B) performing quantitative analysis of the amplification product by detecting the amplification product by an optical unit; and (C) performing quantitative analysis of the target nucleic acid contained in the whole blood sample that comprises confirming the number of cycles of PCR where the amplification product reaches a specified quantity. 19. The method of analyzing the target nucleic acid according to claim 18, wherein, in the process (B), the amplification product is detected with time. 20. The method of analyzing the target nucleic acid according to claim 18, wherein the amplification product is detected by measuring a fluorescence generated from the amplification product. 21. The method of analyzing the target nucleic acid according to claim 18, wherein the optical unit is a fluorometer. 22. The method of analyzing the target nucleic acid according to claim 18, wherein, in the process (B), the method of detecting the amplification product is a Tm analysis. 23. A method of suppressing the effect of precipitate and/or turbidity in PCR amplification of a whole blood sample, the method comprising: preparing an amplification product complementary to a target nucleic acid in the whole blood sample by PCR, and limiting a ratio of the whole blood sample in a PCR reaction solution to be in a range of 0.01 to 0.9% by volume. 24. The method of analyzing the amplification product according to claim 1, wherein the ratio of the whole blood sample in the PCR reaction solution is in a range of 0.01 to 1.8 g/L in term of hemoglobin content. 25. The method of analyzing the target nucleic acid according to claim 18, wherein the ratio of the whole blood sample in the PCR reaction solution is in a range of 0.01 to 1.8 g/L in term of hemoglobin content. 26. The method of suppressing the effect of precipitate and/or turbidity in PCR amplification of a whole blood sample according to claim 23, further comprising limiting a ratio of the whole blood sample in the PCR reaction solution to be in a range of 0.01 to 1.8 g/L in term of hemoglobin content.

Details for Patent 8,148,079

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Grifols Therapeutics Llc	ALBUKED, PLASBUMIN-20, PLASBUMIN-25, PLASBUMIN-5	albumin (human)	For Injection	101138	10/21/1942		2026-08-09
Baxalta Us Inc.	BUMINATE, FLEXBUMIN	albumin (human)	Injection	101452	03/03/1954		2026-08-09
Csl Behring Ag	ALBURX	albumin (human)	Injection	102366	07/23/1976		2026-08-09
Grifols Biologicals Llc	ALBUTEIN	albumin (human)	Injection	102478	08/15/1978		2026-08-09