US20120028819A1 - Method for the detection and characterization of a toxinogenic clostridium difficile strain - Google Patents

Method for the detection and characterization of a toxinogenic clostridium difficile strain Download PDF

Info

Publication number
US20120028819A1
US20120028819A1 US13/262,844 US201013262844A US2012028819A1 US 20120028819 A1 US20120028819 A1 US 20120028819A1 US 201013262844 A US201013262844 A US 201013262844A US 2012028819 A1 US2012028819 A1 US 2012028819A1
Authority
US
United States
Prior art keywords
deletion
nucleotide
seq
gene
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/262,844
Other languages
English (en)
Inventor
Patrick Theodoor Christian Van Den Bogaard
Astrid Elisabeth Visser
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Koninklijke Philips NV
Original Assignee
Koninklijke Philips Electronics NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=42224104&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20120028819(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Koninklijke Philips Electronics NV filed Critical Koninklijke Philips Electronics NV
Assigned to KONINKLIJKE PHILIPS ELECTRONICS N V reassignment KONINKLIJKE PHILIPS ELECTRONICS N V ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VAN DEN BOGAARD, PATRICK THEODOOR CHRISTIAN, VISSER, ASTRID ELISABETH
Publication of US20120028819A1 publication Critical patent/US20120028819A1/en
Abandoned legal-status Critical Current

Links

Images

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
    • C12Q1/6876—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
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium

Definitions

  • the present invention is in the field of biology and chemistry.
  • the invention is in the field of molecular biology.
  • the invention is in the field of detection of nucleic acids and real-time PCR.
  • Most particularly the invention relates to the detection and characterization of a toxinogenic Clostridium difficile strain.
  • Clostridium difficile is a species of Gram-positive bacteria of the genus Clostridium. Clostridia are anaerobic, spore-forming rods ( bacillus ). C. difficile is the most serious cause of antibiotic-associated diarrhea (AAD) and can lead to pseudomembranous colitis, a severe infection of the colon, often resulting from eradication of the normal gut flora by antibiotics.
  • AAD antibiotic-associated diarrhea
  • the C. difficile bacteria which naturally reside in the body, become overgrown: The overgrowth is harmful because the bacterium releases toxins that can cause bloating, constipation, and diarrhea with abdominal pain, which may become severe. Latent symptoms often mimic some flu-like symptoms. Discontinuation of causative antibiotic treatment is often curative.
  • C. difficile infections can range in severity from asymptomatic to severe and life-threatening, especially among the elderly. People most often get infected in hospitals, nursing homes, or institutions, although C. difficile infection in the community, outpatient setting is increasing. The rate of C. difficile acquisition is estimated to be 13% in patients with hospital stays of up to 2 weeks, and 50% in those with hospital stays longer than 4 weeks. Frequency and severity of C. difficile colitis remains high and seems to be associated with increased death rates. Early intervention and aggressive management are key factors to recovery.
  • the inventors have found a pioneering method for the detection and characterization of a toxinogenic Clostridium difficile strain in a sample.
  • the advantage is that multiple diagnostic questions may be addressed in one single method.
  • This method now allows designation of a sample as comprising a hypervirulant Clostridium difficile strain. Further, it allows scoring of a sample as a non NAP1/BI/027 strain. Also the sample may be scored as NAP1/BI/027 strain. It may be also scored as ribotype 078 strain, or scored as 017 strain. Hence, in a single assay all of the above designations may be done.
  • the invention relates to a method for the detection and characterization of a toxinogenic Clostridium difficile strain in a sample, wherein the following steps are performed, (a) a sample is provided for, (b) in a multiplex PCR assay, (c) the sample is analyzed with respect to the presence or absence of the cytotoxin tcdB gene, (d) the sample is analyzed with respect to the presence or absence of one or more of the following deletions in the tcdC gene: (a) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347, (b) a 36 bp deletion in SEQ ID NO.
  • the invention also relates to a kit for performing the methods of the invention, comprising primers and or probes for amplifying and/or detecting (i) the cytotoxin tcdB gene, (ii) the 1.8 kb deletion in the tcdA gene, (iii) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347 of the tcdC gene, (iv) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 of the tcdC gene, (v) a single nucleotide deletion at position 117 of SEQ ID NO. 1 and primers and/or probes for (vi) the detection of the binary toxin cdtA/B gene.
  • PCR Polymerase chain reaction
  • PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
  • the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument.
  • a double stranded target nucleic acid may be denatured at a temperature >90° C., primers annealed at a temperature in the range 50-75° C., and primers extended at a temperature in the range 72-78° C.
  • PCR encompasses derivative forms of the reaction, including but not limited to, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like.
  • Reaction volumes range from a few hundred nanoliters, e.g. 200 nl, to a few hundred microlitres.
  • preferred volumes are 10-50 microliter more preferably about 25 microliters per reaction chamber.
  • Real-time PCR means a PCR for which the amount of reaction product, i.e. amplicon, is monitored as the reaction proceeds.
  • Detection chemistries for real-time PCR are reviewed in Mackay et aI, Nucleic Acids Research, 30: 1292-1305 (2002).
  • a two temperature stage reaction may also be used in which the polymerisation temperature equals the annealing temperature, even for typical hybridization probes like Scorpion primers or Pleiades probes.
  • Multiplexed PCR means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228 (1999) (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified. Typically, the number of target sequences in a multiplex PCR is in the range of from 2 to 10, or from 2 to 8, or more typically, from 3 to 6. The preferred number is 2-6 for the present invention.
  • Quantitative PCR means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relative quantitation of such target sequences. Quantitative measurements are made using one or more reference sequences that may be assayed separately or together with a target sequence.
  • the reference sequence may be endogenous or exogenous to a sample or specimen, and in the latter case, may comprise one or more competitor templates.
  • Typical endogenous reference sequences include segments of transcripts of the following genes: pactin, GAPDH, microglobulin, ribosomal RNA, and the like.
  • Primer means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed.
  • Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase.
  • the sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide.
  • primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides.
  • Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers.
  • Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following references: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2 nd Edition (Cold Spring Harbor Press, New York, 2003).
  • sample means a quantity of material from a biological, environmental, medical, or patient source in which detection or measurement of target nucleic acids is sought. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples.
  • a sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may include materials taken from a patient including, but not limited to cultures, blood, saliva, cerebral spinal fluid, pleural fluid, semen, needle aspirates, and the like.
  • Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, rodents, etc.
  • Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • the terms “sample” and “specimen” are used interchangeably.
  • target sequence 2 tcdB (amplicon 1) 120 CATTAGATGAAACTATAGACTTACTTCCTACATTATC TGAAGGATTACCTATAATTGCAACTATTATAGATGGT GTAAGTTTAGGTGCAGCAATCAAAGAGCTAAGTGAAA CGAGTGACC 3 tcdB (amplicon 2) 140 TTTTGCCCCAGCTAATACACTTGATGAAAACCTAGAA GGAGAAGCAATTGATTTTACTGGAAAATTAATTATTG ACGAAAATATTTATTATTTTGATGATAATTATAGAGG AGCTGTAGAATGGAAAGAATTAGATGGTG 4 tcdC_nt117 140 TGAAAGAAAAGGAAGCTCTAAGAAAATAATTAAATTC TTTAAGAGCACAAAGGATATTGCTCTACTGGCATTTA TTGGTGTTCTGAAGACCATGAGGAGGTCATTTC 5 tcdC
  • SEQ ID NO. 2 is the preferred tcdB amplicon.
  • SEQ ID NO. 3 is a further preferred tcdB amplicon.
  • SEQ ID NO. 4 discloses the tcdC nt117 deletion.
  • SEQ ID NO. 5 discloses the tcdC deletions 18 bp, 36 bp, 39 bp and 54 bp.
  • SEQ ID NO. 6 discloses the binary toxin.
  • 7 SEQ ID NO. 6 discloses the tcdA deletion(s).
  • 8 SEQ ID NO. 8 is the preferred tcdC 18-bp amplicon.
  • 9 SEQ ID NO. 9 is the preferred tcdC 36-bp. 10 SEQ ID NO.
  • SEQ ID NO. 10 is the preferred tcdC 39-bp amplicon 11
  • SEQ ID NO. 11 is the preferred tcdC 54-bp amplicon 1
  • SEQ ID NO. 1 is the full length wt tcdC sequence (strain 630 ACCESSION AM180355 REGION: 804310 . . . 805008)
  • 12 is full length ribotype 027 tcdC sequence (incl nt117 and 18-bp deletion) ACCESSION DQ861412
  • SEQ ID NO. 13 is the full length tcdC sequence of 36-bp deletion variant (ACCESSION DQ861424)
  • 14 is the full length tcdC sequence of 39-bp deletion variant (ACCESSION EF470292)
  • SEQ ID NO. 15 is the full length tcdC sequence of 54-bp deletion variant.
  • FIG. 1 shows the preferred targets according to the present invention.
  • FIG. 2 shows a possible cartridge set-up according to the invention.
  • the invention relates to a method for the detection and characterization of a toxinogenic Clostridium difficile strain in a sample, wherein the following steps are performed, (a) a sample is provided for, (b) in a multiplex PCR assay, (c) the sample is analyzed with respect to the presence or absence of the cytotoxin tcdB gene, (d) the sample is analyzed with respect to the presence or absence of one or more of the following deletions in the tcdC gene: (a) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347, (b) a 36 bp deletion in SEQ ID NO.
  • the sample is additionally analyzed with respect to the presence or absence of the enterotoxin tcdA gene 1.8 kb deletion.
  • the sample is additionally analyzed with respect to the presence or absence of the binary toxin cdtA and/or cdtB.
  • the sample is analyzed with respect to, (i) the presence or absence of all of following the deletions, (a) a 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347, (b) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 and (c) a single nucleotide deletion at position 117 of SEQ ID NO.
  • the sample is analyzed with respect to the presence or absence of the cytotoxin tcdB gene, (iii) the sample is analyzed with respect to the presence or absence of the 1.8 kb tcdA deletion and (iv) the sample is analyzed with respect to the presence or absence of the cdtA/B binary toxin gene.
  • the sample is scored as toxinogenic Clostridium difficile, (b) if the tcdB gene sequence is present, the tcdA deletion is absent, the single nucleotide deletion at position 117 of SEQ ID NO.
  • the cdtA/B binary toxin gene is absent, then the sample is scored as a ribotype 017 Clostridium difficile strain and (d) if the tcdB gene sequence is present, the tcdA deletion is absent, the 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 is present, the cdtA/B binary toxin gene is present, then the sample is scored as a ribotype 078 Clostridium difficile strain.
  • targets associated with hypervirulence such as but not limited to tcdC ⁇ 36bp, tcdC ⁇ 54bp.
  • the strains carrying the 36-bp, 39-bp or 54-bp deletions all have additional specific mutations upstream in the tcdC gene that result in a truncated and non-functional
  • TcdC protein which is preferably part of the assay.
  • the amplification products in the multiplex PCR assay are between 60 and 200 bp in size.
  • the multiplex amplification reaction is done in a closed system in the presence of fluorescent indicators in the reaction mixture(s), the fluorescent indicators being capable of generating an optical signal related to a presence and/or quantity of each amplicon in the amplification reaction and monitoring the optical signal of the fluorescent indicators in the amplification reaction
  • the closed system gives an optical output for the user, indicating the scoring assignment outlined above.
  • the multiplex PCR amplification is quantitative real-time PCR.
  • the real-time PCR also designated herein as quantitative PCR or quantitative real-time PCR (qPCR)
  • qPCR quantitative real-time PCR
  • RT-qPCR quantitative real-time reverse transcription PCR
  • RT-qPCR quantitative real-time PCR method further comprising a reverse transcription of RNA into DNA, e.g. mRNA into cDNA.
  • the amplified nucleic acid is quantified as it accumulates.
  • fluorescent dyes that intercalate with double-stranded DNA e.g.
  • reporter probes modified nucleic acid probes that fluoresce when hybridized with a complementary nucleic acid (e.g. the accumulating DNA) are used for quantification in qPCR based methods.
  • fluorogenic primers e.g. LightCycler probes (Roche)
  • hydrolysis probes e.g. TaqMan probes (Roche)
  • hairpin probes such as molecular beacons, Scorpion primers (DxS), Sunrise primers (Oncor), LUX primers (Invitrogen), Amplifluor primers (Intergen) or the like can be used as reporter probes.
  • fluorogenic primers or probes may for example be primers or probes to which fluorescence dyes have been attached, e.g. covalently attached.
  • fluorescence dyes may for example be FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor, PET Biosearch BlueTM, Marina Blue®, Bothell Blue®, CAL Fluor® Gold, CAL Fluor® Red 610, QuasarTM 670, LightCycler Red640®, QuasarTM 705, LightCycler Red705® and the like.
  • Particular reporter probes may additionally comprise fluorescence quenchers.
  • selective primers can be used in quantitative real-time multiplex PCR.
  • a “primer” herein refers to an oligonucleotide comprising a sequence that is complementary to a nucleic acid to be transcribed (“template”). During replication polymerases attach nucleotides to the 3′ end of the primer complementary to the respective nucleotides of the template.
  • the polymerase used for quantitative real-time PCR is a polymerase from a thermophile organism or a thermostable polymerase or is selected from the group consisting of Thermus thermophilus (Tth) DNA polymerase, Thermus acquaticus (Taq) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus kodakaraensis KOD DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Sulfolobus solfataricus Dpo4 DNA polymerase, Thermus pacificus (Tpac) DNA polymerase, Thermus eggertssonii (Teg) DNA polymerase, Thermus pacificus (T
  • the fluorescently labelled probes are labelled with a dye selected from the group consisting of FAM, VIC, NED, Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor and PET.
  • a dye selected from the group consisting of FAM, VIC, NED, Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor and PET.
  • the hybridization probe is a LightCycler probe (Roche) or the hydrolysis probe is a TaqMan probe (Roche).
  • the hairpin probe is selected from the group consisting of molecular beacon, Scorpion primer, Sunrise primer, LUX primer and Amplifluor primer. The TaqMan probes are preferred.
  • the invention relates to a closed system amplification cartridge comprising one or more channels or chambers comprising primers and/or probes for amplifying and/or detecting (i) the cytotoxin tcdB gene, (ii) the 1.8 kb deletion in the tcdA gene, (iii) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347 of the tcdC gene, (iv) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 of the tcdC gene, (v) a single nucleotide deletion at position 117 of SEQ ID NO. 1 and primers and/or probes for the detection of the binary toxin cdtA/B gene.
  • the invention also relates to a kit for performing the methods of the invention, comprising primers and or probes for amplifying and/or detecting (i) the cytotoxin tcdB gene, (ii) the 1.8 kb deletion in the tcdA gene, (iii) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347 of the tcdC gene, (iv) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370 of the tcdC gene, (v) a single nucleotide deletion at position 117 of SEQ ID NO. 1 and primers and/or probes for (vi) the detection of the binary toxin cdtA/B gene.
  • the kit additionally comprises enzymes such as a polymerase a buffer and other ingredients.
  • the method may take place in a cartridge which is designed for performing sample preparation and real-time multiplex PCR.
  • These are systems, methods, and apparatus for closed multi-stage nucleic acid amplification reactions wherein a portion of a prior-stage reaction mixture serves as the sample for the next stage reaction.
  • the invention provides a method as outlined above for controlling the amplification comprising the step of (i) amplifying said multiplex reaction in the presence of a fluorescent indicator in a reaction mixture, the fluorescent indicator being capable of generating an optical signal related to a quantity of an amplicon in the amplification reaction; (ii) monitoring the optical signal of the fluorescent indicator in the amplification reaction.
  • the invention also relates to a closed system amplification cartridge comprising one or more channels or chambers comprising primers and or probes for amplifying and/or detecting (i) the cytotoxin tcdB gene, (ii) the tcdC gene, (iii) an 18 bp deletion in SEQ ID NO. 1 from nucleotide 330 to nucleotide 347, (iv) a 36 bp deletion in SEQ ID NO. 1 from nucleotide 301 to nucleotide 336, (v) a 39 bp deletion in SEQ ID NO. 1 from nucleotide 341 to nucleotide 370, (vi) a 54 bp deletion in SEQ ID NO.
  • the cartridge may additionally comprise one or more chambers for sample preparation, e.g. cell lysis and/or nucleic acid extraction.
  • the PCR chambers comprise a optically transparent surface, such as glass, crystal or plastic that allows for online detection of the amplification product.
  • the DNA Once the DNA has been isolated in the cartridge it is resolubilized in the mastermix, which is stored in lyophilized form inside the cartridge. Homogenizing the eluate in the mastermix solution then takes place. Filling the at least 3 to 5 PCR chambers (or a subset if the application so requires), such that no air is entrapped in the chambers is performed in the cartridge. Less than 5 chambers may be required by the amplification, here a cartridge variant may be used. The required elution volume can be adapted via the assay protocol. Closing the chambers during amplification, so that no amplicons may escape into the environment, and no air is allowed into the chambers is performed. Temperature-cycling the chambers is done.
  • the cycling is synchronized between all chambers, with individual temperature set points. Fluorescence detection in up to 6 wavelengths is then done. Detection may be triggered at any moment in the cycle. Calculation of Ct values, initial concentrations and final test results, from the measurement data and, possibly, additional calibration data is performed. A test result is generated based on the Ct values for the targets of the invention.
  • the cartridge performs the PCR in a device.
  • a specimen container or also cartridge herein arrives at the console, and the user enters the identifier (e.g. barcode, identifier on a paper order form, etc.) associated to the order.
  • the console retrieves the associated order from the local console database.
  • the user scans the RFID tag of the cartridge.
  • the cartridge is checked on its validity (e.g. cartridge type corresponds with test type, expiration date, etc.). In case the cartridge is valid it is associated with the order in the console database.
  • the console main service requests an available slot of an instrument.
  • the instrument control subsystem returns an available slot after applying load-balancing. After notifying the user to insert the cartridge, the user inserts the cartridge in the suggested slot.
  • the instrument control subsystem is notified that a cartridge was inserted and the console main service checks whether the cartridge is associated with the order. In case the cartridge is associated with the order, the recipe database is accessed to retrieve the recipe matching the test type in the order. After retrieving the recipe, the instrument control subsystem is ordered to upload the recipe and start the test for the slot where the cartridge was inserted. After completion of the test, the test result is received by the instrument control subsystem and the following steps will be performed:
  • test result will be passed to the test result engine.
  • the test result engine will store the result in the console database.
  • the result will be send to the external IS via the external IS data exchange subsystem.
  • the user gets informed that the test is completed.
  • the user gets an optical and/or acoustical output concerning the score.
  • the cartridge is divided into 6 modules.
  • the modules are explained in detail in the following paragraphs.
  • a module within the cartridge is defined for its functionality. All functionality of the cartridge is integrated as much as possible and realized with a minimal number of parts.
  • the total process flow is as follows: The operator fills the lysis chamber manually with the sample and closes the input lid. The cartridge is placed on the tray of the slot and with the tray loaded into the slot. When loaded into the slot, the process starts with liquefying and lysing the sample. This is all done with the help of, e.g. HiFU energy in the lysis chamber. Different reagents are added after each other to the sample so that the final result can be flushed through the extraction membrane.
  • the transport of reagents to the chamber is done by the fluid transport module. Within this module the reagents are stored for the shelf life, are taken out of the reservoirs and transported to the lysis chamber. The module also transports the treated sample from the lysis chamber through the extraction membrane to the waste. In the same way the washing lysis module, fluid transport module, extraction module, waste handling module, PCR module, manual sample input, covering reagents are handled, taken from the reservoir and transported through the membrane to the waste.
  • the previous mentioned extraction membrane is embodied within the extraction module. This module ensures a good flow through the membrane and a good heat transfer to the membrane. This heat is needed for the ethanol removal and elution of the DNA.
  • the waste handling module is used to direct all fluids, except the eluate, to the waste this is done by the same type of valve as used in the fluid handling module. This is done to minimize the different techniques used within the cartridge for the same functionality.
  • the second function of this module is to suck the elute buffer through the extraction membrane. The actuation is done by this module to minimize the risk of contamination of the eluate.
  • the chambers are placed parallel within the fluidic structure to prevent any cross talk of primers and or probes that are specific per chamber. There is also a post-filling de-aeration filter.
  • Samples or specimens containing target polynucleotides may come from a wide variety of sources for use with the present invention, including cell cultures, animal or human tissues, patient biopsies, environmental samples, or the like. Samples are prepared for assays of the invention using conventional techniques, which typically depend on the source from which a sample or specimen is taken. Samples or specimens are collected so as to minimize the chance of contamination of the sample or specimen by external elements, or the environment by the sample or specimen if it contains hazardous components. Generally, this is carried out by introducing a sample for analysis, e.g., tissue, blood, saliva, etc., directly into a sample collection chamber within a fluidly closed system.
  • a sample for analysis e.g., tissue, blood, saliva, etc.
  • the prevention of cross-contamination of the sample may be accomplished by directly injecting the sample into the sample collection chamber through a optionally sealable opening, e.g., an injection valve, or a septum.
  • sealable valves are preferred to reduce any potential threat of leakage during or after sample injection.
  • the sample collection portion of the device/cartridge may also include reagents and/or treatments for neutralization of infectious agents, stabilization of the specimen or sample, pH adjustments, and the like. Stabilization and pH adjustment treatments may include, e.g. introduction of heparin to prevent clotting of blood samples, addition of buffering agents, addition of protease, preservatives and the like.
  • Such reagents may generally be stored within the sample collection chamber of the device/cartridge or may be stored within a separately accessible chamber, wherein the reagents may be added to or mixed with the sample upon introduction of the sample into the device.
  • These reagents may be incorporated within the device in either liquid or lyophilized form, depending upon the nature and stability of the particular reagent used.
  • the preferred sample is human or animal feces.
  • sample preparation operations Prior to carrying out amplification reactions on a sample, it will often be desirable to perform one or more sample preparation operations upon the sample.
  • sample preparation operations will include such manipulations as extraction of intracellular material, e.g., nucleic acids from whole samples and the like.
  • these various operations may be readily incorporated into the fluidly closed systems contemplated by the present invention.
  • nucleic acids may be liberated from the collected cells, into a crude extract, followed by additional treatments to prepare the sample for subsequent operations, e.g., denaturation of contaminating (DNA binding) proteins, purification, filtration, desalting, and the like.
  • Liberation of nucleic acids from the sample cells, and denaturation of DNA binding proteins may generally be performed by chemical, physical, or electrolytic lysis methods.
  • chemical methods generally employ lysing agents to disrupt the cells and extract the nucleic acids from the cells, followed by treatment of the extract with chaotropic salts such as guanidinium isothiocyanate or urea to denature any contaminating and potentially interfering proteins.
  • chaotropic salts such as guanidinium isothiocyanate or urea
  • the appropriate reagents may be incorporated within a sample preparation chamber, a separate accessible chamber, or may be externally introduced.
  • the sample of cells is flowed through a microtubular array while an alternating electric current is applied across the fluid flow.
  • a variety of other methods may be utilized within the device of the present invention to perform cell lysis/extraction, including, e.g., subjecting cells to ultrasonic agitation, or forcing cells through small apertures, thereby subjecting the cells to high shear stress resulting in rupture.
  • nucleic acids Following extraction, it will often be desirable to separate the nucleic acids from other elements of the crude extract, e.g., denatured proteins, cell membrane particles, salts, and the like. Removal of particulate matter is generally accomplished by filtration, flocculation or the like. A variety of filter types may be readily incorporated into the device. Further, where chemical denaturing methods are used, it may be desirable to desalt the sample prior to proceeding to the next step. Desalting of the sample, and isolation of the nucleic acid may generally be carried out in a single step, e.g., by binding the nucleic acids to a solid phase and washing away the contaminating salts or performing gel filtration chromatography on the sample, passing salts through dialysis membranes, and the like.
  • Suitable solid supports for nucleic acid binding include, e.g., diatomaceous earth, silica (i.e., glass wool), or the like.
  • Suitable gel exclusion media also well known in the art, may also be readily incorporated into the device/cartridge of the present invention, and is commercially available from, e.g., Pharmacia and Sigma Chemical.
  • the isolation and/or gel filtration/desalting may be carried out in an additional chamber, or alternatively, the particular chromatographic media may be incorporated in a channel or fluid passage leading to a subsequent reaction chamber.
  • the interior surfaces of one or more fluid passages or chambers may themselves be derivatized to provide functional groups appropriate for the desired purification, e.g., charged groups, affinity binding groups and the like.
  • desalting methods may generally take advantage of the high electrophoretic mobility and negative charge of DNA compared to other elements.
  • Electrophoretic methods may also be utilized in the purification of nucleic acids from other cell contaminants and debris.
  • a separation channel or chamber of the device is fluidly connected to two separate “field” channels or chambers having electrodes, e.g., platinum electrodes, disposed therein.
  • the two field channels are separated from the separation channel using an appropriate barrier or “capture membrane” which allows for passage of current without allowing passage of nucleic acids or other large molecules.
  • the barrier generally serves two basic functions: first, the barrier acts to retain the nucleic acids which migrate toward the positive electrode within the separation chamber; and second, the barriers prevent the adverse effects associated with electrolysis at the electrode from entering into the reaction chamber (e.g., acting as a salt junction).
  • Such barriers may include dialysis membranes, dense gels, PEI filters, or other suitable materials.
  • the field channels may be disposed on the same or opposite sides or ends of a separation chamber or channel, and may be used in conjunction with mixing elements described herein, to ensure maximal efficiency of operation.
  • coarse filters may also be overlaid on the barriers to avoid any fouling of the barriers by particulate matter, proteins or nucleic acids, thereby permitting repeated use.
  • the high electrophoretic mobility of nucleic acids with their negative charges may be utilized to separate nucleic acids from contaminants by utilizing a short column of a gel or other appropriate matrix or gel which will slow or retard the flow of other contaminants while allowing the faster nucleic acids to pass.
  • the probes and/or primers are distributed in the channels or chambers as follows: A specific mix of primers and probes is stably stored as dried material in each individual PCR chamber. With the filling of the PCR chamber with the premixed template/PCR reactionmix the stored primers/probes form a homogeneous solution with concentrations optimal for their designated reactions.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
US13/262,844 2009-04-07 2010-03-31 Method for the detection and characterization of a toxinogenic clostridium difficile strain Abandoned US20120028819A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP09157547.2 2009-04-07
EP09157547 2009-04-07
PCT/IB2010/051396 WO2010116290A1 (en) 2009-04-07 2010-03-31 Method for the detection and characterization of a toxinogenic clostridium difficile strain

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2010/051396 A-371-Of-International WO2010116290A1 (en) 2009-04-04 2010-03-31 Method for the detection and characterization of a toxinogenic clostridium difficile strain

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/617,010 Division US10876174B2 (en) 2009-04-07 2017-06-08 Method for the detection and characterization of a toxinogenic Clostridium difficile strain

Publications (1)

Publication Number Publication Date
US20120028819A1 true US20120028819A1 (en) 2012-02-02

Family

ID=42224104

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/262,844 Abandoned US20120028819A1 (en) 2009-04-07 2010-03-31 Method for the detection and characterization of a toxinogenic clostridium difficile strain
US15/617,010 Active US10876174B2 (en) 2009-04-07 2017-06-08 Method for the detection and characterization of a toxinogenic Clostridium difficile strain

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/617,010 Active US10876174B2 (en) 2009-04-07 2017-06-08 Method for the detection and characterization of a toxinogenic Clostridium difficile strain

Country Status (8)

Country Link
US (2) US20120028819A1 (de)
EP (1) EP2419527B1 (de)
JP (1) JP5859424B2 (de)
KR (1) KR101798211B1 (de)
CN (1) CN102378817B (de)
BR (1) BRPI1006757A2 (de)
RU (1) RU2612789C2 (de)
WO (1) WO2010116290A1 (de)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9133526B2 (en) 2012-06-08 2015-09-15 Samsung Electronics Co., Ltd. Composition and kit for detection and analysis of strains of Clostridium difficile and method of detecting strains of Clostridium difficile by using the same
US10876174B2 (en) 2009-04-07 2020-12-29 Koninklijke Philips N.V. Method for the detection and characterization of a toxinogenic Clostridium difficile strain

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012087135A1 (en) 2010-12-22 2012-06-28 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Genetic markers specific for clostridium difficile ribotypes 027 (nap01/b1; rt 027) and 078 (nap7/8; rt 078) and their use
GB201110712D0 (en) 2011-06-23 2011-08-10 Univ Ulster Diagnostic methods
WO2013006793A2 (en) * 2011-07-06 2013-01-10 Quest Diagnostics Investments Incorporated Direct amplification and detection of viral and bacterial pathogens
GB201207998D0 (en) * 2012-05-08 2012-06-20 Univ Cardiff A screening method for the detection of clostridium difficle
US9133527B2 (en) * 2012-05-11 2015-09-15 Techlab, Inc. Cell wall protein CwpV (CD0514) as a diagnostic marker for Clostridium difficile ribotype 027
CN102952886A (zh) * 2012-11-28 2013-03-06 中华人民共和国张家港出入境检验检疫局 艰难梭菌肠毒素a、b双重荧光定量pcr检测方法及检测用试剂盒
WO2014164563A1 (en) 2013-03-13 2014-10-09 Quidel Corporation Pcr assays and reagents for molecular detection of infectious agents
TWI504751B (zh) * 2013-09-25 2015-10-21 Univ Nat Cheng Kung 檢測困難梭狀桿菌核醣分型027之序列、技術平台及其方法
FI126289B (en) * 2014-12-19 2016-09-15 Mobidiag Ltd Method for detecting the presence of a hypervirulent Clostridium difficile strain
US20190211377A1 (en) * 2016-12-22 2019-07-11 Roche Molecular Systems, Inc. Cobra probes to detect a marker for epidemic ribotypes of clostridium difficile
EP3724354A1 (de) * 2017-12-15 2020-10-21 Gen-Probe Incorporated Zusammensetzungen und verfahren zum nachweis von toxigenem clostridium difficile
EP3822370A1 (de) 2019-11-15 2021-05-19 Curiosity Diagnostics Sp. z o.o. Verfahren zur bestimmung der anwesenheit eines hypervirulenten clostridioides difficile-stammes der gruppe b1/nap1/027 in einer probe
CN111705150B (zh) * 2020-08-19 2020-12-25 中南大学湘雅医院 用于检测艰难梭菌核糖体027型毒力调节基因tcdC的方法、引物和试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050026277A1 (en) * 2000-07-28 2005-02-03 Genesystems Device for thermo-dependent chain reaction amplification of target nucleic acid sequences, measured in real-time
US20070026426A1 (en) * 2005-04-26 2007-02-01 Applera Corporation System for genetic surveillance and analysis

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5298421A (en) 1990-04-26 1994-03-29 Calgene, Inc. Plant medium-chain-preferring acyl-ACP thioesterases and related methods
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
AU726501B2 (en) 1996-06-04 2000-11-09 University Of Utah Research Foundation Monitoring hybridization during PCR
CN101068932B (zh) * 2004-10-27 2013-02-13 塞弗德公司 封闭系统多阶段核酸扩增反应
RU2348695C2 (ru) * 2006-05-23 2009-03-10 Закрытое акционерное общество "Молекулярно-медицинские технологии" Дифференцирующий и специфический олигонуклеотиды для идентификации последовательностей днк инфекционных агентов в биологических материалах, способ видовой идентификации инфекционных агентов, биочип и набор для осуществления этого способа
JP5596893B2 (ja) 2006-11-10 2014-09-24 キヤノン株式会社 プローブセット、プローブ固定担体及び遺伝子検査方法
US7687084B2 (en) 2007-08-06 2010-03-30 Tien Linsheng W Cool-pet system
US9096638B2 (en) * 2007-09-06 2015-08-04 Geneohm Sciences Canada, Inc. Detection of toxigenic strains of Clostridium difficile
WO2009100215A2 (en) * 2008-02-08 2009-08-13 Mayo Foundation For Medical Education And Research Detection of clostridium difficile
JP5859424B2 (ja) 2009-04-07 2016-02-10 コーニンクレッカ フィリップス エヌ ヴェＫｏｎｉｎｋｌｉｊｋｅ Ｐｈｉｌｉｐｓ Ｎ．Ｖ． 毒素産生クロストリジウムディフィシル菌の検出及び特性解析方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050026277A1 (en) * 2000-07-28 2005-02-03 Genesystems Device for thermo-dependent chain reaction amplification of target nucleic acid sequences, measured in real-time
US20070026426A1 (en) * 2005-04-26 2007-02-01 Applera Corporation System for genetic surveillance and analysis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Antikainen et al., APMIS 2009; 117: 607-613. *
Belanger et al., Journal of Clinical Microbiology, vol. 41, No. 2, pages 730-734, Feb. 2003. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10876174B2 (en) 2009-04-07 2020-12-29 Koninklijke Philips N.V. Method for the detection and characterization of a toxinogenic Clostridium difficile strain
US9133526B2 (en) 2012-06-08 2015-09-15 Samsung Electronics Co., Ltd. Composition and kit for detection and analysis of strains of Clostridium difficile and method of detecting strains of Clostridium difficile by using the same

Also Published As

Publication number Publication date
JP2012528567A (ja) 2012-11-15
KR20120005019A (ko) 2012-01-13
BRPI1006757A2 (pt) 2020-03-10
US20180237827A1 (en) 2018-08-23
JP5859424B2 (ja) 2016-02-10
CN102378817A (zh) 2012-03-14
CN102378817B (zh) 2015-09-30
RU2011144853A (ru) 2013-05-20
WO2010116290A1 (en) 2010-10-14
US10876174B2 (en) 2020-12-29
EP2419527B1 (de) 2013-06-26
EP2419527A1 (de) 2012-02-22
RU2612789C2 (ru) 2017-03-13
KR101798211B1 (ko) 2017-11-15

Similar Documents

Publication Publication Date Title
US10876174B2 (en) Method for the detection and characterization of a toxinogenic Clostridium difficile strain
AU2013312191C1 (en) Multiple amplification cycle detection
TWI697560B (zh) 用於偵測細菌污染之方法及組合物
JP2022028837A (ja) 薬剤に対する生物の応答のマイクロ流体測定
CA2895945C (en) Target capture system
Amita et al. Qualitative evaluation of mycobacterial DNA extraction protocols for polymerase chain reaction
BRPI0517131B1 (pt) Métodos para simplificar ácidos nucléicos microbiais por modificação química de citosinas
EP2902506B1 (de) Nachweis von Listerien in Lebensmittel- und Umweltproben, Verfahren und Zusammensetzungen dafür
Fatima et al. Microbial DNA extraction from soil by different methods and its PCR amplification
Grúas et al. Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR
WO2009061752A1 (en) Methods for detecting toxigenic microbes
CN106715718A (zh) 用于检测和分析结核分枝杆菌的组合物和方法
EP2889382A1 (de) Oligonukleotiden, deren Verwendung, Verfahren und Kit zur DNS Detektion von Borrelia burgdorferi, Bartonella henselae, Babesia divergens, Anaplasma phagocytophilum
EP3438280A1 (de) Hämoplasmadetektionsverfahren
Tripathi A simplified and cheapest method for the diagnosis of sickle cell using whole blood PCR and RFLP in Nepal
WO2011008942A2 (en) Simultaneous quantitative multiple primer detection of clostridium difficile
CN104032000A (zh) 蜡样芽孢杆菌的检测方法及试剂盒
Mohammed et al. Review on polymerase chain reaction and its diagnostic merit over conventional techniques in animal disease
US20240035077A1 (en) Methods for performing multiplexed real-time pcr with the use of large stokes shift fluorescent dyes
KR101911017B1 (ko) 타일로렐라 에퀴제니탈리스의 검출용 조성물, 타일로렐라 에퀴제니탈리스 감염에 의한 말전염성자궁염의 진단용 조성물, 타일로렐라 에퀴제니탈리스의 검출방법, 및 타일로렐라 에퀴제니탈리스 감염에 의한 말전염성자궁염의 진단방법
WO2016209938A1 (en) Compositions and methods for processing a biological sample
RU2289627C2 (ru) СПОСОБ И НАБОР ДЛЯ ОБНАРУЖЕНИЯ ДНК БАКТЕРИИ ЗАБОЛЕВАНИЯ ЛАЙМСКОГО БОРРЕЛИОЗА-Borrelia burgdorferi
Point 16.1 Polymerase chain reaction (PCR)
Scialfa et al. Isolation of saprophytic Leptospira spp. from a selected environmental water source of Argentina= Aislamiento de Leptospira spp. saprófitas desde fuentes de agua en un ambiente seleccionado de Argentina
De Bruin et al. Molecular detection and typing of Coxiella burnetii

Legal Events

Date Code Title Description
AS Assignment

Owner name: KONINKLIJKE PHILIPS ELECTRONICS N V, NETHERLANDS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VAN DEN BOGAARD, PATRICK THEODOOR CHRISTIAN;VISSER, ASTRID ELISABETH;SIGNING DATES FROM 20100401 TO 20100406;REEL/FRAME:027010/0121

STCB Information on status: application discontinuation

Free format text: ABANDONED -- AFTER EXAMINER'S ANSWER OR BOARD OF APPEALS DECISION