JP-7856012-B2 - Nucleic acid amplification sensitizers, nucleic acid amplification compositions, and test kits

Inventors

• 鈴木 裕貴
• 近藤 聖奈
• 松田 将

Assignees

• 日油株式会社

Dates

Publication Date

20260511

Application Date

20220121

Priority Date

20210128

Claims (11)

1. Formula (1): (In the formula, X1 represents a (meth)acryloyloxy group or a (meth)acryloylamino group, L1 represents a C2-C4 alkylene group or a C2-C4 alkyleneoxyalkylene group, which may have one hydroxyl group, and R1 to R3 each independently represent a C1-C3 alkyl group. A nucleic acid amplification sensitizer that is a polymer containing constituent units derived from monomers represented by [the formula shown].
2. The nucleic acid amplification sensitizer according to claim 1, which is a homopolymer consisting of one type of structural unit derived from the monomer represented by formula (1) above.
3. Formula (2): (In the formula, R4 represents a hydrogen atom or a methyl group, and R5 represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms. The nucleic acid amplification sensitizer according to claim 1, which is a copolymer further comprising a constituent unit derived from a monomer represented by
4. The nucleic acid amplification sensitizer according to claim 3, wherein R5 is an alkyl group having 12 to 18 carbon atoms.
5. Formula (3): (In the formula, R6 represents a hydrogen atom or a methyl group, and R7 represents a C3-C6 alkyl group having two or more hydroxyl groups. A nucleic acid amplification sensitizer according to claim 1, 3, or 4, which is a copolymer further comprising a constituent unit derived from a monomer represented by .
6. A composition for nucleic acid amplification comprising a nucleic acid amplification sensitizer according to any one of claims 1 to 5.
7. A nucleic acid amplification composition according to claim 6, used in a reverse transcription polymerase chain reaction method.
8. A composition for nucleic acid amplification according to claim 6 or 7, used in a quantitative polymerase chain reaction method.
9. A test kit comprising the nucleic acid amplification composition according to any one of claims 6 to 8.
10. A test kit according to claim 9, for use in clinical testing.
11. A test kit according to claim 9 or 10, wherein the test subject is a virus.

Description

The present invention relates to a sensitizer for nucleic acid amplification, a composition for nucleic acid amplification, and a test kit. Nucleic acid amplification is a method of amplifying a target nucleic acid from a few copies to tens of thousands of times or more, and is used in a wide variety of fields for genetic testing, microbiological testing, and viral testing. A representative method for nucleic acid amplification is the polymerase chain reaction (PCR) method. In a typical PCR method, nucleic acid amplification is achieved by repeating three steps: (1) denaturation of template DNA (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primers to single-stranded template DNA, and (3) extension of primers by DNA polymerase. In classical endpoint PCR, the amplification product is detected at the reaction endpoint after a predetermined reaction cycle by visualizing it with a fluorescent compound or by measuring the turbidity of the solution. While typical PCR is a method for amplifying DNA, it can also be applied to amplify RNA. Such a PCR method is sometimes abbreviated as reverse transcription polymerase chain reaction (RT-PCR). In RT-PCR, complementary DNA (cDNA) is synthesized from RNA using an enzymatic reaction with reverse transcriptase, and this cDNA is used as a template to perform PCR, thereby amplifying the RNA. A method for determining the initial amount of nucleic acid based on the amount of amplification product obtained by PCR is also known, and such a PCR method is called quantitative polymerase chain reaction (hereinafter sometimes abbreviated as "qPCR method"). In a narrow sense, the qPCR method refers to real-time PCR, which is a method of visualizing the amount of DNA amplified in each PCR cycle using a fluorescent DNA staining reagent or fluorescent probe. Among these, the method using a fluorescent probe is known to be a particularly reliable method because it can specifically detect the amplification of the target nucleic acid. A method combining RT-PCR and qPCR, known as reverse transcription-quantitative PCR (hereinafter sometimes abbreviated as "RT-qPCR"), is also known. RT-qPCR can be further divided into a two-step method, in which cDNA synthesis and qPCR are performed in separate containers, and a one-step method, in which these are performed as a series of reactions in the same container. Of these, the one-step method is superior in terms of ease of operation, minimal contamination from outside the system, and high detection sensitivity (hereinafter sometimes abbreviated as "sensitivity") of the nucleic acid being measured. Furthermore, in recent years, a method called digital PCR (sometimes abbreviated as "dPCR method") has been developed, which applies microfluidic flow path formation technology and other techniques to distribute the reaction solution into tens to tens of thousands of extremely small compartments, perform PCR simultaneously on all of them, and determine the initial nucleic acid concentration using a statistical model based on the proportion of compartments where amplification occurred. The dPCR method is an advanced version of the endpoint PCR method. In addition to determining the presence and/or quantity of target nucleic acids as described above, PCR is also used to determine the base sequence of target nucleic acids (sequencing). While there are various sequencing PCR methods, such as the Sanger method, all are based on the endpoint PCR method. In the use of nucleic acid amplification methods, sensitivity is often a concern, whether for qualitative, quantitative, or sequencing analysis. Therefore, various techniques to accelerate PCR have been investigated. For example, it is common practice to increase the sensitivity of PCR reaction solutions by adding salts (e.g., potassium chloride, ammonium sulfate, etc.), betaine, polyhydric alcohols, etc. However, there are limits to the sensitivity improvement achieved by adding these components, and there has been a need for a higher level of sensitivity improvement. For example, a method of destabilizing double-stranded DNA by adding single-stranded DNA-binding proteins (SSBs) is known. Furthermore, Patent Document 1 discloses mutant PCNA (proliferation nucleus antigen) monomers as a highly versatile DNA replication promoter (additive) for promoting DNA elongation. However, additives composed of these proteins have drawbacks, including difficulty in mass production, storage stability issues, and high manufacturing costs. International Publication No. 2007/004654 The present invention will be described in detail below. In this specification, "(meth)acryloyloxy group" basically means "acryloyloxy group or methacryloyloxy group." If multiple (meth)acryloyloxy groups are present, "(meth)acryloyloxy group" means "acryloyloxy group and/or methacryloyloxy group." Other terms similar to "(meth)acryloyloxy group" also have the same meaning as "(meth)acryloyloxy group." Furthermore, whe