JP-2026514286-A - Kits and methods for detecting targeted nucleic acids

Abstract

The subject of this invention is a kit for highly sensitive and robust detection of a target nucleic acid in a sample, and a sandwich method using the kit. This invention also relates to the use of the kit or method according to the present invention for target nucleic acids for the diagnosis of a disease or pathological condition. The kit and method according to the present invention are particularly useful for detecting point mutations in target nucleic acids. [Selection Diagram] Figure 1A-B

Inventors

• クリムチェンコ，アンドレイ
• クルス ダ シルヴァ，エリザベーテ
• ガキ，パラスケヴィ
• ライシュ，アンドレアス
• プフェッファー，セバスチャン

Assignees

• ユニヴェルシテ ド ストラスブール
• サントル ナショナル ドゥ ラ ルシャルシュ シアンティフィク

Dates

Publication Date

20260508

Application Date

20231027

Priority Date

20221027

Claims (15)

1. A kit for detecting target nucleic acids in a sample, wherein at least, i) A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to a functional unit F1, wherein NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, ii) Functional units F2 bonded to a solid surface, which exhibit high affinity for F1 or are covalently bonded to F1. iii) A kit comprising nucleic acid fragments NA2 bound to ultra-high brightness light-emitting particles exhibiting a brightness of at least 10⁷ M⁻¹ cm⁻¹ , preferably at least 2 × 10⁷ M⁻¹ cm⁻¹ , more preferably at least 5 × 10⁷ M⁻¹ cm⁻¹ , 10 × 10⁷ M⁻¹ cm⁻¹ , or at least 40 × 10⁷ M⁻¹ cm⁻¹, or a probe P2 comprising the same.
2. The kit according to claim 1, wherein the solid surface is selected from among the surfaces of an immobilized surface and solid particles, preferably magnetic beads or glass beads.
3. The kit according to claim 1 or 2, wherein the ultra-high brightness light-emitting particles are ultra-high brightness dye-retaining fluorescent polymer nanoparticles.
4. The kit according to any one of claims 1 to 3, wherein the nucleic acid fragment NA2 contains or consists of a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid.
5. The kit according to any one of claims 1 to 3, comprising or consisting of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein region T3 comprises a first portion distinct from region T1 and a second portion comprising or consisting of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and the first and second portions are linked via a nucleotide linker.
6. It includes at least two probes P1, each of which probes P1 is - The kit according to claim 5, comprising, or consisting of, at least one nucleic acid fragment NA1, named as NA1-1 or NA1-2, and complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid.
7. i) a kit according to claim 5 or 6, comprising at least two probes P3, each comprising a first part comprising or consisting thereof a nucleic acid fragment having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part comprising or consisting thereof a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are linked via a nucleotide linker; and ii) at least one probe P2 comprising or consisting thereof a nucleic acid fragment NA2 bound to an ultra-high brightness light-emitting particle.
8. A method for detecting a target nucleic acid molecule in a sample, the method comprising at least the following steps: a) Under conditions suitable for hybridization of complementary nucleic acid sequences and formation of non-covalent complexes of nucleic acids, at least, - A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to a functional unit F1, wherein NA1 comprises a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, - Functional unit F2 bonded to a solid surface, wherein F2 exhibits high affinity for F1 or is covalently bonded to F1, and - Probe P2 comprising or consisting of nucleic acid fragment NA2 bonded to ultra-high brightness light-emitting particles, wherein the particles exhibit brightness of at least 107 M⁻¹ cm⁻¹ , A step of bringing the two into contact to form a mixture, b) a method comprising the steps of applying an external physical force to the mixture of step a) in order to isolate the non-covalent complex of nucleic acids, and c) measuring the luminescence intensity of the non-covalent complex of nucleic acids.
9. The method according to claim 8, wherein the solid surface is the surface of solid particles, preferably magnetic beads or glass beads.
10. The method according to claim 8 or 9, wherein the ultra-high-brightness light-emitting particles are ultra-high-brightness dye-retaining fluorescent polymer nanoparticles.
11. The method according to any one of claims 8 to 10, wherein the nucleic acid fragment NA2 contains or consists of a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid.
12. Step a) is at least, - A probe P1 comprising or consisting of a nucleic acid fragment NA1 bound to functional unit F1, wherein NA1 contains or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid. - A functional unit F2 bound to a solid surface, wherein F2 exhibits high affinity for F1 or is covalently bound to F1; and - At least one probe P2 comprising or consisting of a nucleic acid fragment NA2 covalently bound to an ultra-high brightness fluorescent particle, wherein the nucleic acid fragment NA2 comprises or consists of a nucleotide sequence complementary to probe P3; and - At least one probe P3 comprising or consisting of a first part comprising or consisting of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part comprising or consisting of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are linked via a nucleotide linker. The method according to any one of claims 8 to 11, comprising bringing into contact with
13. Step a) is at least, The method according to claim 12, comprising contacting two probes P1, each of which probes P1 comprises or consists of a nucleic acid fragment NA1, each of which NA1 is named NA1-1 or NA1-2 and is complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid, respectively.
14. Step a) is, i) at least two probes P3 comprising, or comprising, a first part comprising, a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part comprising, or comprising, a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are linked via a nucleotide linker; and ii) at least one probe P2 comprising, or comprising, a nucleic acid fragment NA2 bound to an ultra-high brightness light-emitting particle.
15. Use of the kit according to any one of claims 1 to 7, or the method according to any one of claims 8 to 14, for the detection of a target nucleic acid.

Description

The subject of this invention is a kit for highly sensitive and robust detection of target nucleic acids in a sample, and a sandwich method using the kit. This invention also relates to the use of the kit or method according to the present invention, for example, for molecular diagnosis of disease or pathological conditions. This invention relates to the field of molecular biological testing methods. Nucleic acids are considered important markers for diseases such as cancer and infectious diseases, but their detection can be difficult due to their extremely low concentrations. Because disease-related nucleic acid biomarkers often exist in extremely low abundances, accurate and robust detection of analytes, primarily in large volumes of biological fluids, is crucial for biological research, precision medicine, and early diagnosis. The COVID-19 pandemic highlighted the critical need for rapid, sensitive, and specific detection methods to control rapidly evolving pandemics. In clinical diagnosis, direct nucleic acid testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been applied due to the difficulty in detecting extremely low concentrations of RNA molecules in suspension. Direct detection is limited by strong background noise from nonspecific proteins, nucleic acids, or other biomolecules. In molecular diagnostics, there are two types of testing methods, each with its own unique advantages: (1) targeted amplification tests and (2) signal amplification tests. Targeted amplification tests, such as PCR, are highly sensitive, but require target extraction and purification, as well as enzymatic reactions, making them prone to false positives. In contrast, signal amplification tests tend to omit nucleic acid purification and enzymatic amplification, making them simpler. Therefore, they result in less material loss and a lower rate of false positives. However, signal amplification tests are less sensitive than PCR-based targeted amplification tests. Much effort has been dedicated to developing ultra-sensitive signal amplification tests to detect unamplified nucleic acids. Portable microfluidic devices have demonstrated specific and highly sensitive detection of Ebola nucleic acids compared to RT-PCR (Cai, H. et al., 2015). Interestingly, some of these studies were able to directly detect target nucleic acids from whole blood lysates (Ngo, H. T. et al., 2018) (Zheng, Z. et al., 2006). To date, the recommended method for diagnosing COVID-19 is based on quantitative reverse transcription polymerase chain reaction (qRT-PCR), which requires nucleic acid extraction from pharyngeal swabs and targeted amplification procedures. Such procedures require skilled individuals, specialized equipment, and long processing times (over 2 hours), making the implementation of SARS-CoV-2 nucleic acid testing in the field difficult. Furthermore, due to high demand for commercially available RNA extraction kits and resulting shortages of these reagents, several diagnostic workflows have been developed to bypass the lengthy intermediate RNA extraction step. Over the past decade, the performance of magnetic beads in nucleic acid extraction from biological fluids before molecular detection has been demonstrated in several studies. Magnetic beads consist of iron oxide nanoparticles embedded in a polymer matrix, allowing for the separate isolation of nucleic acids through surface functionalization. This functionalization affects binding rates and compatibility with molecular detection strategies. For example, silica-coated beads non-selectively bind to all nucleic acids via electrostatic interactions and are therefore primarily used in detection methods resistant to high concentrations of non-targeted background nucleic acids, such as RT-PCR. Oligonucleotide-conjugated beads, such as oligo(dT) beads, or beads surface-functionalized with specific sequences, are used for mRNA target extraction and exhibit high recovery rates. This purification can be performed manually, in a microfluidic chip, or using automated robotic equipment. Beyond nucleic acid isolation and elution, several specific nucleic acid sequences have been directly detected on the bead surface using hybridization methods with various materials (biotin-avidin, protein-enzyme, fluorescent dyes, quantum dots, etc.). However, most of these methods suffer from limitations such as low signal intensity or rapid photodecolorization (Lim et al., 2009). EP3536806, "Oligonucleotide-functionalized hydrophobic polymer nanoparticles," describes nanoparticles comprising a hydrophobic polymer and a luminescent component as an energy donor element, which can be used to detect target nucleic acids. These nanoparticles can be conjugated to oligonucleotides complementary to a specific target or a non-specific sequence of the target. The target-specific oligonucleotides conjugate to the energy donor element, and their nucleotide sequences are complementary to another nucl