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1. WO2022093127 - DIGITAL CRISPR-BASED METHOD FOR THE RAPID DETECTION AND ABSOLUTE QUANTIFICATION OF NUCLEIC ACIDS

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Publication Number WO/2022/093127
Publication Date 05.05.2022
International Application No. PCT/SG2021/050661
International Filing Date 28.10.2021
IPC
G01N 33/50 2006.1
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
C12Q 1/6844 2018.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING 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
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
68involving nucleic acids
6844Nucleic acid amplification reactions
C12Q 1/70 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING 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
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
70involving virus or bacteriophage
Applicants
  • MASSACHUSETTS INSTITUTE OF TECHNOLOGY [US]/[US]
  • NATIONAL UNIVERSITY OF SINGAPORE [SG]/[SG]
Inventors
  • WU, Xiaolin
  • YU, Hanry
  • LU, Timothy Kuan-Ta
Agents
  • DOWSING, Bruce, John
Priority Data
63/106,98029.10.2020US
Publication Language English (en)
Filing Language English (EN)
Designated States
Title
(EN) DIGITAL CRISPR-BASED METHOD FOR THE RAPID DETECTION AND ABSOLUTE QUANTIFICATION OF NUCLEIC ACIDS
(FR) PROCÉDÉ À BASE DE CRISPR NUMÉRIQUE POUR LA DÉTECTION RAPIDE ET LA QUANTIFICATION ABSOLUE D'ACIDES NUCLÉIQUES
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Abstract
(EN) The present invention relates to a digital CRISPR-based method for detecting and quantitating target nucleic acids in a sample comprising: forming a mixture comprising sample nucleic acids; isothermal amplification reaction reagents for amplifying one or target nucleic acid sequences; partitioning the mixture into a plurality of compartments; incubating the partitioned mixture at a temperature for isothermal amplification and Cas effector cleavage of an amplified DNA strand, detecting a signal from cleavage of the non-target sequence, thereby detecting the one or more target sequences in the sample, and determining the copy number of the target nucleic acid based on a Poisson distribution of the proportion of positive-to-negative compartments. The invention also relates to a method for detecting presence and/or of a disease in a subject, and a kit to quantitate nucleic acids in a sample. In certain embodiments, the target nucleic acids are SARS-CoV-2, human adenovirus, herpes simplex virus, or Epstein-Barr virus nucleic acids.
(FR) La présente invention concerne un procédé à base de CRISPR numérique pour détecter et quantifier des acides nucléiques cibles dans un échantillon, comprenant les étapes consistant à : former un mélange comprenant des acides nucléiques d'échantillon; des réactifs de réaction d'amplification isotherme pour amplifier une ou plusieurs séquences d'acides nucléiques cibles; diviser le mélange dans une pluralité de compartiments; incuber le mélange cloisonné à une température pour une amplification isotherme et un clivage d'effecteur Cas d'un brin d'ADN amplifié, détecter un signal à partir du clivage de la séquence non cible, ce qui permet de détecter les une ou plusieurs séquences cibles dans l'échantillon, et déterminer le nombre de copies de l'acide nucléique cible sur la base d'une distribution de Poisson de la proportion de compartiments positifs à négatifs. L'invention concerne également un procédé de détection de la présence et/ou d'une maladie chez un sujet, et un kit pour quantifier des acides nucléiques dans un échantillon. Dans certains modes de réalisation, les acides nucléiques cibles sont des acides nucléiques de SARS-CoV-2, d'adénovirus humain, de virus d'herpès simplex ou de virus d'Epstein-Barr.
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