WO2017204771A2 - Système et procédé d'identification de micro-organismes - Google Patents

Système et procédé d'identification de micro-organismes Download PDF

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Publication number
WO2017204771A2
WO2017204771A2 PCT/TR2017/050216 TR2017050216W WO2017204771A2 WO 2017204771 A2 WO2017204771 A2 WO 2017204771A2 TR 2017050216 W TR2017050216 W TR 2017050216W WO 2017204771 A2 WO2017204771 A2 WO 2017204771A2
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microorganism
data
microorganisms
processor unit
measurement data
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PCT/TR2017/050216
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English (en)
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WO2017204771A3 (fr
Inventor
Zulal Kesmen
Mete CELIK
Mine ERDEM
Esra OZBEKAR
Fatma Ozge OZKOK
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Erciyes Universitesi
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Publication of WO2017204771A2 publication Critical patent/WO2017204771A2/fr
Publication of WO2017204771A3 publication Critical patent/WO2017204771A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/141—Preventing contamination, tampering
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/06—Test-tube stands; Test-tube holders
    • 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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the present invention relates to a system and method for taxonomic classification/realization and identification of microorganisms (protista, fungus, bacteria, virus, etc.) at the level of strain/kind/species/family, etc.
  • Phenotypic methods are based on morphologic, physiologic and biochemical characteristics. The methods based on phenotypic methods are based on properties of traditional tests used for routine identification, microscopic inspection, assimilation of different carbon sources, development in different nitrogen sources, vitamin requirements, development in high concentration sugar or salt medium and in different temperature media, resistance to antibiotics and hydrolyzing various substrates.
  • phenotypic methods are frequently used, there are pluralities of technical problems in practice. For instance, in phenotypic identification methods, pluralities of tests shall be applied, in general, 60-90 tests shall be applied in order to realize accumulation in species level. Another problem is that most of the commercial systems like API, BIOLOG, Uni- YeastTec, Abbott Quantum II, VitekATB32, Automicrobic and similar commercial systems need additional analyses, and in cases where these analyses are not made, correctness levels of identifications stay at a very low level (50-80%). Moreover, since the morphologic properties, where the phenotypic methods are based, are substantially dependent on environmental conditions, their repetitiveness is substantially low.
  • Phenotypic methods do not give results which are as reliable as the genotypic tests since they are resistant to physiological and biochemical tests. This results in deficient or wrong estimation of the bio-variety in a specific habitat.
  • genotypic methods are frequently used in identification of the microorganisms. Genotypic methods used in microbial identification are separated into methods which are based and which are not based on polymerase chain reaction (PCR). Among the methods which are not based on PCR, DNA hybridization method and electrical field gel electrophoresis (PFGE) are provided.
  • genotypic methods comprise methods based on polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • methods like mitochondrial and ribosomal DNA sequence (array) analysis, restriction fragment length polymorphism analysis (PCR-RFLP), random augmented polymorphic DNA (PCR-RAPD) and repeating palindromic (rep-PCR) array analysis and real-time PCR analysis.
  • PCR-RFLP restriction fragment length polymorphism analysis
  • PCR-RAPD random augmented polymorphic DNA
  • rep-PCR repeating palindromic array analysis
  • the most frequently used method in identification of the microorganism species is the method based on sequence analysis of the ribosomal RNA (rRNA) genes and the internal transcribed spacers (ITS) and intergenic spacer regions (IGS).
  • rRNA ribosomal RNA
  • ITS internal transcribed spacers
  • IGS intergenic spacer regions
  • restriction endonucleases are used which identify specific DNA regions and which cut DNA from these regions.
  • the obtained fragments are separated by means of agarose gel electrophoresis.
  • the bands, painted with ethidium bromide, are imaged under ultraviolet light.
  • the whole genome cannot be analyzed by means of this method and the identification power of this method is lower than the identification power of array analysis.
  • PCR-RAPD analysis is one of fingerprint methods based on PCR. Fragments, which are in different dimensions and which are formed by using approximately 10 based short primers designed randomly without needing any preliminary information about the DNA array of the species to be analyzed, advance in different speeds in the electrophoresis gel, and they form species-specific fingerprints.
  • rep-PCR repetitive element palindromic PCR
  • rep-PCR repetitive element palindromic PCR
  • test kits are not provided where genotypic methods which are not based on PCR are used.
  • the test kits based on PCR, are kits which are related to the specific detection of only specific microorganism species. For instance, there are kits based on the real-time PCR method developed for identification of the species of Escherichia coli 0157:H7, Listeria monocytogenes, Salmonella Typhymurium, etc. among the food-sourced pathogen bacteria and of the species of Candida albicans and Cryptococcus neoformans which are some opportunist pathogen yeast species. These kits are kits which are only for identifying the target microorganism species in a specific manner.
  • genotypic methods is a direct identification method, and most of the methods require long and difficult processes after PCR. In this step, particularly the usage of toxic and cancerogenic chemicals like ethidium bromide is required.
  • High Resolution Melting (HRM) and Melting Temperature (Tm) analysis method is a substantially new method which has been begun to be used in mutation scanning and methylation profile analysis in the recent years.
  • HRM High Resolution Melting
  • Tm Melting Temperature
  • the present invention relates to a system, method and software and a multiple tube medium like plate/strip for use in this system, for eliminating the above mentioned disadvantages and for bringing new advantages to the related technical field.
  • Another object of the present invention is to provide a microorganism identification system whose identification range (spectrum) is substantially wide.
  • Another object of the present invention is to provide a method where the results are not affected due to the changes like analyzer, device or laboratory conditions, etc. when the method is standardized.
  • Another object of the present invention is to provide a system and method for identification of genotypic microorganisms, which is not affected by the environmental conditions like phenotypic methods.
  • Another object of the present invention is to provide a system and method for identification of microorganisms, whose identification power and sensitivity are increased.
  • the present invention is a system for identification of microorganisms and comprising a processor unit which receives as input the HRM curves and Tm values of the amplification products of DNA regions, whose heterogeneity between different kind/species microorganisms is high, through a device which makes fluorometric/spectroscopic measurement combined with a real-time PCR device or thermal cycler. Accordingly, the improvement of the present invention is to simultaneously analyze pluralities of DNA regions which show difference between different kind/species/family, etc. of microorganisms.
  • the improvement of the present invention is that the processor unit is configured such that the fluorometric/spectroscopic measurement device combined with the real time PCR device or thermal cycler receives as input the measurement data received from at least one reference tube comprising the DNA of the reference microorganism and simultaneously the measurement data received from the tubes comprising DNAs of the other microorganisms, the processor unit comprises a known microorganism database within this embodiment which accommodates recorded data comprising at least one of HRM curves and Tm values and information of strain/kind/species/family, etc.
  • the processor unit is configured in a manner comparing the recorded data of the reference microorganism and the measurement data of the reference microorganism and in a manner correcting by pre-processing the data of the unknown microorganisms by taking into consideration the deviation values in between, the processor unit is configured in a manner comparing the recorded data and the corrected measurement data related to each unknown microorganism and in a manner assigning at least one of information of strain/kind/species/family, etc. of the known microorganisms having the recorded data to matched unknown microorganisms in case the measurement data and the recorded data are matched.
  • the microorganisms can be identified in a sensitive manner based on information of different radiations of different species/kinds.
  • measurement data of pluralities of microorganisms is identified simultaneously by scanning the database.
  • the principle of identification is based on HRM and Tm analysis which can even distinguish single nucleotide polymorphism and whose distinguishing power is substantially high. Accordingly, the principle of identification is based on matching by means of comparing recorded HRM and Tm data of the known microorganisms in the database with the measurement results obtained from HRM and Tm analyses of the amplification products after amplification of pluralities of (at least two of) different DNA regions of an unknown microorganism simultaneously in different tubes. In order to harmonize the measurement results in accordance with the changing measurement/environment conditions, the same target DNA regions of the reference microorganism are analyzed simultaneously, and the measurement results of the reference microorganism and the recorded data of the reference microorganism are compared, and the deviation data is obtained.
  • the measurement data of the unknown microorganism is passed through a preliminary process and corrected, and afterwards, said measurement data is compared with the recorded data of the known microorganisms in the database.
  • the known microorganism database comprises the recorded data comprising at least one of the HRM and Tm curves of different DNA regions of the known microorganisms,
  • the processor unit is configured in a manner receiving as input the measurement data of the HRM curves and Tm values for the different DNA regions in different tubes of an unknown (first) microorganism,
  • the processor unit is configured in a manner comparing the received measurement data and the recorded data and in a manner assigning to the unknown (first) microorganism at least one of the identification information of the known microorganism with the recorded data in case there is matching with a pre-calculated closeness.
  • the processor unit is configured in a manner comparing the received measurement data and the recorded data and in a manner assigning to the unknown (first) microorganism at least one of the identification information of the known microorganism with the recorded data in case there is matching with a pre-calculated closeness.
  • the processor unit is configured in a manner comparing the measurement data and the recorded data by using the Euclid algorithm.
  • the processor unit is configured in a manner comparing the recorded data of the reference microorganism and the measurement data of the reference microorganism and in a manner forming the deviation data.
  • the known microorganism database can be turned into a dynamic library comprising the updated measurements in accordance to the deviation values of the HRM and Tm values.
  • said processor unit is configured in a manner passing the measurement data through the interpolation process prior to the comparing process.
  • the intermediate measurement values are obtained in an estimated manner.
  • the present invention moreover comprises an analysis medium like a plate/strip and multiple tube made of a material resistant to high temperature and having light transmittance and having dimensions compliant to the measurement device, characterized by comprising primer sets (forward and backward primer) positioned in a lyophilized manner in each tube.
  • primer sets forward and backward primer
  • a ready-for-use plate/strip/multiple tube medium is provided for HRM and Tm analysis.
  • there are different primer sets which separate different DNA regions of at least two tubes provided in multiple tube medium like a plate/strip.
  • HRM and Tm analysis of the different DNA regions of the microbial isolate can be realized simultaneously on a single plate/strip/multiple tube medium.
  • the present invention is compliant with the devices which can realize fluorometric/spectroscopic measurement combined with real-time PCR devices or thermal cycler.
  • all primers can give reaction at a very close annealing degree with respect to each other.
  • the plate comprises 8x12 tubes such that there are 12 tubes in a row and there are 8 tubes in a column.
  • the primers provided at each row of the plate act on a different DNA region and all primers provided in the same row act on the same target DNA region.
  • the present invention is a method for identification of microorganisms, where pluralities of microorganism DNAs are placed to pluralities of tubes in order to be analyzed in fluorometric/spectroscopic measurement device combined with a real-time PCR device or thermal cycler.
  • the present invention is a system comprising a processor unit which realizes identification and which comprises an analysis medium like ready-for-use plate/strip/multiple tube medium and which receives as input the database consisting of HRM curves and Tm values of the known microorganisms and measurement data of HRM curves and Tm values of unknown microorganism and which compares this data with the data of the known microorganisms provided in the database, in order to realize HRM and Tm analysis of at least 2 different DNA regions of the unknown microorganisms.
  • pluralities of different DNA regions (at least two) of the unknown microbial isolates simultaneously realize HRM and Tm analysis of the amplification products in an analysis medium like a single plate/strip/multiple tube.
  • At least one reference microorganism and at least one unknown microorganism are analyzed by means of fluorometric/spectroscopic measurement device combined with real-time PCR device or thermal cycler, and measurement data, including at least one of HRM and Tm values, is obtained,
  • a known microorganism database which includes strain/kind/species/family, etc. information of pluralities of known microorganisms including the reference microorganism therein and recorded data which comprises at least one of HRM and
  • Tm data is accessed by the processor unit
  • the measurement data of the reference microorganism and the recorded data of the reference microorganism are compared and the measurement data of the unknown microorganisms are corrected by the processor unit by means of a pre-process taking the deviation data in between into consideration,
  • the corrected measurement data of each unknown microorganism and the recorded data are compared and in case the measurement data and the recorded data match at a pre-calculated closeness, at least one of strain/kind/species/family, etc. information of the known microorganisms which is the owner of the recorded data is assigned to the matched unknown microorganisms by the processor unit.
  • the principle of the microorganism identification system in the present invention is based on the analysis of HRM curves and Tm values.
  • the principle of the measurement is based on melting curve analysis which analyses the decomposition characteristics of double chain DNA together with temperature increase.
  • HRM analysis the change graphic in the fluorescent intensity is formed depending on the temperature of this melting curve.
  • Tm melting temperature
  • the subject matter system realizes HRM and Tm analysis simultaneously after more than one target DNA region, which shows variety between microorganisms, is amplified by means of an amplification process like PCR.
  • the present invention in general compares the average HRM curves and Tm values, obtained for each DNA region for the microorganism species to be defined, with the data of the species recorded in the database, and the present invention moreover realizes identification with respect to the similarity ratio.
  • the microorganism identification system is used in identification of unknown microorganisms, isolated and purified from various environmental and biological materials like soil, water, foodstuff and feed, etc., at family/species/kind/strain, etc. level.
  • DNAs, analyzed in the microorganism identification system are high purity DNAs extracted from the unknown microorganisms by means of a suitable method.
  • the measurement device is a fluorometric/spectroscopic detection system combined with real-time PCR device (4) or a thermal cycler, and it is a device which applies cyclic thermal process to the microorganisms provided in a tube (31 ) and thereby which amplifies the microorganisms and which passes the microorganisms through rapid heating and stepped cooling processes and which turns the change, occurring in the radiation intensity, into measurement data.
  • the thermal cycle reactions and fluorometric measurements are realized with tubes (31 ) having dimension and transmittance (heat and light) compliant to the device.
  • each tube (31 ) there is a forward and a backward primer (primer set (5)) which provide amplification of the microorganism when the microorganism is subjected to thermal cycles in the related DNA region, together with the DNA of the microorganism desired to be tested, and there is a colorant whose radiation intensity changes as a result of decomposition of the double chain DNA.
  • the measurement device may apply light in various wavelengths, in order to excite fluorescent or corresponding colorant inside the tubes (31 ), and afterwards, the measurement device may measure the emission occurring.
  • the subject matter system comprises a processor unit (1 ) which can communicate with the measurement device and a memory unit (2) connected to the processor unit (1 ).
  • the processor unit (1 ) may be any general-purpose or special-purpose processor.
  • the memory unit (2) may comprise RAM, ROM or any data storage device combination which is readable by a fixed disc or computer which has magnetic or optic properties.
  • the measurement data, received from the measurement device comprises high resolution melting curve (HRM curve) and melting degree (Tm).
  • HRM curve comprises high resolution melting curves (HRM analysis) of different DNA fragments of each microorganism species analyzed, and the Tm values comprise the temperature degree values which are required for turning 50% of the DNA fragments, provided in a tube (31 ), into single chain form.
  • the target DNA regions can be ribosomal RNA genes used frequently in phylogenic and molecular systematic studies and which are accessible through the gene bank for approximately all species to the nucleotide arrays or ITS and ISR regions provided in between or another DNA region whose heterogeneity is high.
  • the database of the known microorganisms of the memory unit (2) comprises the data of pluralities of known microorganisms. In more details, the database comprises the identity information covering the family, species, kind and strain information of the known microorganisms and recorded data covering the HRM curves and Tm values.
  • HRM curves and Tm values can be pre-known or pre-tested data. HRM curves and Tm values are provided for pluralities of different DNA regions (at least two different regions) of each microorganism recorded in the database. For instance, the recorded data for a first DNA region connected to the kind and species information of a known microorganism, the recorded information for a second DNA region, recorded data for a third DNA region, etc. are provided in the database. In an exemplary embodiment, microorganisms can be provided externally to the database (23).
  • the database (23) of the known microorganisms of the microorganism identification system is formed, at least two strains of each species are used.
  • the database (23) of the known microorganisms comprises HRM curves and Tm values obtained for at least two different DNA regions of each species.
  • PCR reactions of the DNAs of the microorganisms and the physical media, where HRM and Tm analyses are realized afterwards, can be multiple tube, plate (3) or strip compliant to the measurement device.
  • the subject matter analysis system is explained below through the plate (3) example.
  • a plate (3) or strip/multiple tube medium is provided in order to analyze the target regions by the measurement device for the DNAs of the unknown microorganisms and desired to be identified.
  • Said plate (3) and said strip are reaction media where multiple tubes (if strip, 8 or 12 tubes (31 )) are adjacent to each other.
  • Said tubes (31 ) have a form whose one each ends are open and which can hold liquid therein.
  • Said plate (3) comprises tubes (31 ) arranged in matrix form.
  • Said plate (3) comprises 8 tubes (31 ) in a column and 12 tubes (31 ) in a row.
  • a column of said plate is defined as a control column (32).
  • DNA is not placed to the tubes (31 ) in this column, and thus, the radiations, which are to be sensed in the tubes (31 ) in this column, provide advantage in determination of the contamination in the measurement medium or various errors.
  • the plate (3) also comprises a column defined as a reference column (33). Said reference column (33) is provided so as to place the DNA of a microorganism whose identity information is also known together with HRM and/or Tm values in order to be used as reference.
  • a column defined as a first column (34) is provided for placement of DNAs of a first microorganism
  • a column defined as a second column (35) is provided for placement of DNAs of a second microorganism.
  • DNAs of pluralities of microorganisms can be placed to the other columns.
  • Each row of tubes (31 ) wherein DNAs of microorganisms are to be placed comprises different forward and backward primers (primer set (5)) which can amplify different DNA regions. Said primers are positioned in lyophilized tubes (31 ). While a first row (36) comprises a first primer set (51 ) in order to provide amplification of the first DNA region, a second row (37) (second DNA region row) comprises a second primer set (52) in order to provide amplification of the second DNA region.
  • the plate (3) which is to be used in identification of microorganism, is made in dimensions and of a material compliant to another fluorometric/spectroscopic measurement system or real-time PCR device (4) with 8x12 wells, and PCR reaction is realized in tubes (31 ) (wells) and afterwards, HRM and Tm analysis is realized.
  • simultaneous analysis of a different DNA region provided in each row can be realized.
  • each row of the plate (3) comprises 8 different primer sets (5) which will provide amplification of 8 different DNA regions.
  • the primer sets consist of forward and backward primers which will amplify the target DNA region, and each one is provided in a lyophilized manner and in suitable concentration inside the plate (3).
  • One of the tubes (31 ) provided in the first column (34) existing on the plate (3) is defined as the first tube (313), and one of the tubes (31 ) placed in the second column (35) is defined as the second tube (314), and one of the tubes (31 ) placed in the reference column (33) is defined as the reference tube (312), and one of the tubes (31 ) placed in the control column (32) is defined as the control tube (31 1 ).
  • DNA is not placed to the control column (32), and the microorganism DNAs are placed to the tubes (31 ) provided in the other columns.
  • DNA of a reference microorganism is placed to the reference column (33).
  • DNA of a first microorganism is placed to the tubes (31 ) of the first column (34).
  • Said first microorganism kind, species, etc. is a microorganism which is desired to be learned.
  • DNA of a second microorganism is placed to the tubes (31 ) of the second column (35). Amplification of the target DNA region is added and mixtures providing HRM and Tm analysis of the amplification products are added to the tubes (31 ) comprising DNAs of the microorganisms.
  • a fluorescent colorant and PCR master mixture are added.
  • the first DNA region of the microorganisms provided in each row is amplified
  • the second DNA regions of the microorganisms provided in each row are amplified.
  • 8 different DNA regions at 8 rows are amplified.
  • the plate (3) of the microorganism identification system is ready-for-use, and PCR master mixture and DNA are added to each well, and the reaction is realized inside suitable volumes.
  • PCR master mixture may comprise reaction buffer, de- oxy-nucleotide triphosphates (dATP, dCTP, dGTP, dUTP), MgCI 2 , fluorescent or equivalent paint which can intercalate to the double chain DNA, DNA polymerase enzyme and PCR grade water.
  • dATP de- oxy-nucleotide triphosphates
  • dCTP de- oxy-nucleotide triphosphates
  • dGTP dGTP
  • dUTP de- oxy-nucleotide triphosphates
  • the reference microorganism means a strain which reacts with all primers used in the (positive control) identification system and whose species is exactly known and provided from ATTC or other culture collections.
  • the fluorescence measurement system which can be combined with the real-time PCR device (4) or a thermal cycler, obtains the HRM and Tm curves of the target DNA fragments as a result of thermal application to the tubes (31 ).
  • the processor unit (1 ) receives as input the measurement data comprising the HRM and/or Tm curves.
  • the processor unit (1 ) subjects the measurement data to the interpolation process and records the measurement data in said first database (21 ). Accordingly, while the database is formed, the incremental values between 0.01 -0.1 °C in the range of 60-99°C are accepted as standard.
  • the measurement data obtained from the measurement device for the fluorescence intensity against the increasing temperature of the PCR products, is transformed into RDML (Real-time PCR Data Markup Language) files.
  • RDML Real-time PCR Data Markup Language
  • the obtained RDML files are analyzed by means of language R and the HRM and Tm values are obtained.
  • the processor unit (1 ) compares the measurement data of the known microorganisms (for instance, the first microorganism or the second microorganism, etc.) with the recorded data of the known microorganisms provided in the known microorganism database (23). When all of the known microorganism data, provided in the known microorganism database (23), are matched with the data obtained from the unknown microorganisms, at least one (strain/kind/species/family, etc.) of the identity information of the known microorganisms is assigned to the unknown microorganism.
  • the measurement data (data of 8 different DNA regions), obtained from all rows in a column of the plate (3) where the DNA of the first microorganism is placed, matches with the data of a microorganism A provided in the known microorganisms database (23), at least one of the identity information (strain/kind/species, etc.) of the microorganism A is assigned to the first microorganism.
  • the measurement system is standardized in every measurement by means of the measurement data of a reference microorganism.
  • the processor unit (1 ) compares the measurement data of the reference microorganism with the recorded data provided on the known microorganism database (23) of the reference microorganism.
  • the processor unit (1 ) records the deviation between the measurement data of the reference microorganism and the recorded data of the same microorganism in a second database (22) as deviation data.
  • the processor unit (1 ) takes the deviation data into account and arranges the data of the unknown microorganisms in accordance with these deviation values.
  • the processor unit (1 ) then compares the data, recorded in the known microorganism database (23), with the corrected measurement data of the unknown microorganisms.
  • the identity information of the microorganisms known to be matched is assigned to the unknown microorganisms matched with the known microorganisms.
  • the deviation data may comprise the difference between the measurement data and the recorded data or the deviation data may comprise an average between the measurement data and the recorded data.
  • the basically important factor is to normalize the recorded data by means of the data received from the measurement medium and to provide obtaining of a well-directed match.
  • the recorded data and the measurement data of the reference microorganism are compared and the measurement data of the unknown microorganisms is pre-processed in accordance with the obtained deviation data and the corrected HRM curves and Tm values can be formed.
  • the obtained HRM and Tm data are passed through pre-process and recorded. This process is repeated for the data of all unknown microorganisms and it is compared with the data of the known microorganisms recorded in the database. By means of this method, the errors resulting from the environmental factors and the measurement conditions are minimized.
  • the known microorganisms where the HRM and Tm data are defined have flexible database (23) structure, and this structure is open for updates.
  • the processor unit (1 ) compares the measurement data, belonging to HRM and Tm analysis of the amplification products obtained by amplifying different DNA regions in different rows for a microorganism DNA placed to all tubes (31 ) in the same column, and the recorded data of the related DNA regions of the known microorganisms provided in the database. Said comparison can be realized by means of Euclid algorithm. Thus, the measurement data received from different DNA regions of a microorganism is compared with the recorded data, and a more sensitive matching is provided.
  • the processor unit (1 ) assigns to the unknown microorganism the species information of a known microorganism which is substantially close to the unknown microorganism in cases where the data of all the analyzed DNA regions and the data of the recorded species in the database do not match in the strain or species level.
  • the comparison process is realized by means of HRM curves and Tm value which are recorded in the database. "Corrected HRM curve" is the curve obtained by updating by means of pre-processing the HRM curve obtained for the same DNA region of the unknown microorganism (of the analyzed microbial isolate) with respect to the deviation values of the HRM curve obtained for any DNA region of the reference microorganism.
  • Corrected Tm value is the value obtained by updating by means of preprocessing the Tm value of the same DNA region of the unknown microorganism (of the analyzed microbial isolate) with respect to the deviation values of the Tm value obtained for any DNA region of the reference microorganism.

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Abstract

La présente invention concerne un système développé pour l'identification de micro-organismes à l'échelon de la souche/type/espèce/famille, etc. et pour la classification de manière taxonomique. La présente invention est notamment une plaque de type milieu d'analyse (3) ou une barrette/pluralité de tubes prêts à l'emploi pour l'analyse HRM et Tm développée pour la classification d'un micro-organisme inconnu obtenu à partir de n'importe quel milieu et pour l'identification à l'échelon de la souche/type/espèce/famille, etc. et un système informatique ainsi qu'un procédé supportant ledit support d'analyse.
PCT/TR2017/050216 2016-05-27 2017-05-24 Système et procédé d'identification de micro-organismes WO2017204771A2 (fr)

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CN117116351A (zh) * 2022-10-21 2023-11-24 青岛欧易生物科技有限公司 基于机器学习算法的物种鉴定模型、物种鉴定方法和物种鉴定系统

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US6844158B1 (en) * 1997-12-22 2005-01-18 Hitachi Chemical Co., Ltd. Direct RT-PCR on oligonucleotide-immobilized PCR microplates
GB201401584D0 (en) * 2014-01-29 2014-03-19 Bg Res Ltd Intelligent detection of biological entities
WO2015164517A1 (fr) * 2014-04-22 2015-10-29 The Johns Hopkins University Procédés et systèmes d'analyse entrainée de profil de fusion pour le génotypage fiable de variants de séquence

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CN117116351B (zh) * 2022-10-21 2024-02-27 青岛欧易生物科技有限公司 基于机器学习算法的物种鉴定模型的构建方法、物种鉴定方法和物种鉴定系统

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