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1. WO2020117059 - METHODS FOR DETECTING CELL WALL-DEFICIENT BACTERIA (CWDB) IN BIOLOGICAL SAMPLES

Publication Number WO/2020/117059
Publication Date 11.06.2020
International Application No. PCT/NL2019/050808
International Filing Date 05.12.2019
IPC
C12Q 1/6841 2018.01
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
6813Hybridisation assays
6841In situ hybridisation
C12Q 1/689 2018.01
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
6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
6888for detection or identification of organisms
689for bacteria
G06K 9/00 2006.01
GPHYSICS
06COMPUTING; CALCULATING OR COUNTING
KRECOGNITION OF DATA; PRESENTATION OF DATA; RECORD CARRIERS; HANDLING RECORD CARRIERS
9Methods or arrangements for reading or recognising printed or written characters or for recognising patterns, e.g. fingerprints
CPC
C12Q 1/6841
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
68involving nucleic acids
6813Hybridisation assays
6841In situ hybridisation
C12Q 1/689
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
68involving nucleic acids
6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
6888for detection or identification of organisms
689for bacteria
G06K 9/00127
GPHYSICS
06COMPUTING; CALCULATING; COUNTING
KRECOGNITION OF DATA; PRESENTATION OF DATA; RECORD CARRIERS; HANDLING RECORD CARRIERS
9Methods or arrangements for reading or recognising printed or written characters or for recognising patterns, e.g. fingerprints
00127Acquiring and recognising microscopic objects, e.g. biological cells and cellular parts
Applicants
  • BIOTRACK HOLDING B.V. [NL]/[NL]
Inventors
  • JANSEN, Gijsbert Johan
Agents
  • WITMANS, H.A.
Priority Data
202214706.12.2018NL
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) METHODS FOR DETECTING CELL WALL-DEFICIENT BACTERIA (CWDB) IN BIOLOGICAL SAMPLES
(FR) PROCÉDÉS DE DÉTECTION DE BACTÉRIES DÉFICIENTES EN PAROI CELLULAIRE (CWDB) DANS DES ÉCHANTILLONS BIOLOGIQUES
Abstract
(EN)
The invention relates to the field of microbiology, in particular to diagnosing cell wall deficient/defective bacteria (CWDB) in clinical samples. Provided is a method for determining the presence of CWDB in cells, comprising the steps of (i) subjecting a biological sample comprising cells to be analyzed for the presence of CWDB to fluorescence in-situ hybridization (FISH) analysis using a fluorescently labeled nucleic acid probe capable of specifically hybridizing with a target nucleic acid sequence of the predetermined bacterial target, (ii) collecting optical images of a plurality of cells, preferably using fluorescence-based detection fitted with the appropriate optical filter, to detect the fluorescently labeled nucleic acid probe, and digitally storing optical images; (iii) binarizing the digital optical images to discriminate fluorescent objects from an indifferent background; (iv) subjecting selected fluorescent objects on the binary image to a morphometric analysis comprising determining for a plurality of fluorescent objects at least the surface area and the roundness; and (v) correlating the results of the morphometric analysis with the presence of CWDB, wherein fluorescent objects having a surface area in the range of 5-50 µm2 and a roundness in the range of 0.800 to 1.000 are indicative of CWDB.
(FR)
L'invention concerne le diagnostic des bactéries déficientes en paroi cellulaire (CWDB) dans des échantillons cliniques. L'invention concerne un procédé pour déterminer la présence de CWDB dans des cellules consistant (i) à soumettre un échantillon biologique, comprenant des cellules à analyser pour la présence de CWDB, à une analyse d'hybridation in situ en fluorescence (FISH) à l'aide d'une sonde d'acide nucléique marquée par fluorescence capable de s'hybrider spécifiquement avec une séquence d'acide nucléique cible de la cible bactérienne prédéterminée, (ii) à collecter des images optiques d'une pluralité de cellules, de préférence à l'aide d'une détection basée sur la fluorescence ajustée avec le filtre optique approprié, pour détecter la sonde d'acide nucléique marquée par fluorescence, et stocker numériquement des images optiques; (iii) à binariser les images optiques numériques pour discriminer des objets fluorescents d'un arrière-plan indifférent; (iv) là soumettre les objets fluorescents sélectionnés sur l'image binaire à une analyse morphométrique consistant à déterminer pour une pluralité d'objets fluorescents au moins la surface et la rondeur; et (v) à corréler les résultats de l'analyse morphométrique avec la présence de CWDB, les objets fluorescents ayant une surface dans comprise entre 5 et 50 µm2 et une rondeur comprise entre 0.800 et 1.000 étant indicatifs de CWDB.
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