NZ736174B2 - Detection of nucleic acid molecules - Google Patents
Detection of nucleic acid molecules Download PDFInfo
- Publication number
- NZ736174B2 NZ736174B2 NZ736174A NZ73617416A NZ736174B2 NZ 736174 B2 NZ736174 B2 NZ 736174B2 NZ 736174 A NZ736174 A NZ 736174A NZ 73617416 A NZ73617416 A NZ 73617416A NZ 736174 B2 NZ736174 B2 NZ 736174B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- nucleic acid
- target nucleic
- target
- biological sample
- batch
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2531/00—Reactions of nucleic acids characterised by
- C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
- C12Q2531/113—PCR
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2545/00—Reactions characterised by their quantitative nature
- C12Q2545/10—Reactions characterised by their quantitative nature the purpose being quantitative analysis
- C12Q2545/114—Reactions characterised by their quantitative nature the purpose being quantitative analysis involving a quantitation step
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The present invention relates to methods for the detection of target nucleic acids at the lower detection limit, and use of these methods in detecting a disease or disorder in a subject or of determining the extent or progression of a disease or disorder. The target nucleic acid may be DNA or cDNA. In one embodiment, the same target nucleic acid is amplified in parallel preamplification reactions. Equal amounts of the products of the reactions are combined and the level of the target nucleic acid is determined by performing a single quantitative real-time PCR (qRT-PCR).
Claims (15)
1. A method of determining the level of a target nucleic acid in a biological sample, 5 wherein the target nucleic acid is DNA obtained from the biological sample or cDNA obtained from RNA obtained from the biological sample, the method comprising the steps of: (i) providing a batch A sing the target nucleic acid; (ii) providing three or more aliquots of batch A provided in step (i) and performing an independent polymerase chain on (PCR) with each of the three or more aliquots 10 in order to amplify the same single target nucleic acid in each of the three or more aliquots, thereby providing three or more batches B comprising the same amplified target nucleic acid; and (iii) mixing equal amounts of the three or more batches B, thereby providing a batch C, and determining the level of the target nucleic acid in batch C by performing a 15 single quantitative real-time PCR (qRT-PCR), n the level determined in step (iii) corresponds to the expression level of the target nucleic acid in the biological sample.
2. The method of claim 1, wherein the concentration of the target c acid in 20 the biological sample is < 1x10-11 M, or < 1x10-12 M, or < 1x10-13 M, or < 1x10-14 M, or < 1x10-15 M, or < 1x10-16 M.
3. The method of claim 2, wherein the concentration of the target nucleic acid in the biological sample is between 1 M and 7 M, or 2 M and 1x10-17 M, or 25 1x10-13 M and 1x10-17 M, or 1x10-14 and 1x10-17 M, or 1x10-15 and 1x10-17 M, or 1x10-16 and 1x10-17 M.
4. The method of any one of claims 1 to 3, n the target nucleic acid is selected from the group consisting of a target miRNA, a target cell-free circulating DNA, a 30 target mRNA, a target siRNA and a target snRNA.
5. The method of claim 4, wherein the target nucleic acid is a target miRNA.
6. The method of claim 5, n the target miRNA is selected from the group consisting of miR-371a-3p, miR5p, miR-372, miR-373, miR-367 and a-5p.
7. The method of any one of claims 1 to 6, wherein the biological sample is 5 selected from the group consisting of body fluid, tissue, cells, cell lysate and cell culture supernatant.
8. The method of claim 7, wherein the body fluid is selected from the group consisting of blood serum, blood plasma, seminal plasma, hydrocele fluid, spermatocele fluid, 10 whole blood, urine, amniotic fluid, exudate, sputum, saliva and cerebrospinal fluid.
9. The method of claim 7, n the tissue is selected from the group consisting of native tissue, snap-frozen tissue and formalin-fixed and paraffin-embedded (FFPE) tissue. 15
10. The method of claim 7 or 9, wherein the tissue is tumor tissue.
11. The method of any one of claims 1 to 10, wherein batch A comprises cDNA.
12. The method of claim 11, wherein step (i) comprises the steps of: 20 (ia) isolating RNA from the biological sample; and (ib) converting the RNA isolated in step (ia) into cDNA, y providing batch A comprising the cDNA.
13. The method of any one of claims 1 to 12, wherein, in step (ii), three aliquots of 25 batch A are provided.
14. A method of ing a disease or disorder in a subject or of determining the extent or progression of a disease or disorder in a subject, the method comprising the step of: determining the level of a target nucleic acid in biological sample obtained from the 30 subject with the method of any one of claims 1 to 13, n the level of the target nucleic acid in the biological sample is tive of the presence, absence and/or extent or progression of the disease or disorder in the subject.
15. The method of claim 4, wherein the target nucleic acid is a target cell-free circulating DNA, wherein the target cell-free circulating DNA is a target ree circulating tumor DNA. RNA—isciation cDNA-synthesis lification Preampli?caticn Preamplificati‘on‘ l 1 l Real-time PCR Real-time PCR Real-time PCR
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102015106646 | 2015-04-29 | ||
| EP15195182 | 2015-11-18 | ||
| PCT/EP2016/059604 WO2016174199A1 (en) | 2015-04-29 | 2016-04-29 | Detection of nucleic acid molecules |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NZ736174A NZ736174A (en) | 2024-10-25 |
| NZ736174B2 true NZ736174B2 (en) | 2025-01-28 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111108220B (en) | CRISPR effector system-based diagnostics for virus detection | |
| CN112020562B (en) | CRISPR effector system-based diagnostics | |
| CN111448311B (en) | Multi-effector CRISPR-based diagnostic system | |
| AU2014293096B2 (en) | Method and device for collection and amplification of circulating nucleic acids | |
| Enderle et al. | Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method | |
| US20250043366A1 (en) | Tiled assays using crispr-cas based detection | |
| JP2023011606A (en) | Crispr effector system based diagnostics | |
| JP6821531B2 (en) | A method for determining the amount of nucleic acid of interest in an unprocessed sample | |
| CN111836903A (en) | Multiplex diagnostics based on CRISPR effector system | |
| US8936909B2 (en) | Method for determining the origin of a sample | |
| Li et al. | A strategy for co-analysis of microRNAs and DNA | |
| US20240102115A1 (en) | Crispr effector system based diagnostics for virus detection | |
| CN105039321B (en) | A kind of modified constant temperature exponential amplification techniques and its application in microRNA detections | |
| US9617606B2 (en) | Oligonucleotide for HIV detection, HIV detection kit, and HIV detection method | |
| CN103555848B (en) | 3'-5'-qPCR (quantitative polymerase chain reaction) quantitative detection technology for small RNA (ribonucleic acid) | |
| US20210139968A1 (en) | Rna amplification method, rna detection method and assay kit | |
| JP2018514218A5 (en) | ||
| Bhattacharya et al. | Experimental toolkit to study RNA level regulation | |
| Ioannidis et al. | Profiling of MicroRNAs in the biofluids of livestock species | |
| NZ736174B2 (en) | Detection of nucleic acid molecules | |
| Qiao et al. | A split crRNA with CRISPR-Cas12a enables highly sensitive, selective, and multiplexed detection of RNA and DNA | |
| Norhazlin et al. | Effect of DNase treatment on RNA extraction from preimplantation murine embryos | |
| Asaga et al. | Direct serum assay for microRNA in cancer patients | |
| NZ736174A (en) | Detection of nucleic acid molecules | |
| Yang et al. | Enriched high-throughput reverse transcription-quantitative PCR template preparation without pre-amplification |