EP0873358A2 - Einfangen von mikroorganismen mittels komplement komponenten - Google Patents

Einfangen von mikroorganismen mittels komplement komponenten

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Publication number
EP0873358A2
EP0873358A2 EP96901269A EP96901269A EP0873358A2 EP 0873358 A2 EP0873358 A2 EP 0873358A2 EP 96901269 A EP96901269 A EP 96901269A EP 96901269 A EP96901269 A EP 96901269A EP 0873358 A2 EP0873358 A2 EP 0873358A2
Authority
EP
European Patent Office
Prior art keywords
sample
microorganisms
pcr
buffer
clq
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.)
Withdrawn
Application number
EP96901269A
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English (en)
French (fr)
Inventor
Paul Herbrink
Henk Van Den Munckhof
Wilhelmus Quint
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.)
Fujirebio Europe NV SA
DDL Diagnostic Laboratory BV
Original Assignee
Innogenetics NV SA
DDL Diagnostic Laboratory BV
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
Application filed by Innogenetics NV SA, DDL Diagnostic Laboratory BV filed Critical Innogenetics NV SA
Priority to EP96901269A priority Critical patent/EP0873358A2/de
Publication of EP0873358A2 publication Critical patent/EP0873358A2/de
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to the field of detection of pathogenic microorganisms in biological samples. More particularly it relates to a new method for capturing micro- organisms from a sample based on the use of complement components preferably bound to a solid support; it also relates to the complement components used as capturing agent in this new method, as well as to kits comprising the same.
  • Niesters et al.(1991) and Hedrun e al.(1992) described a specific antibody directed antigen capture followed by PCR, base on the specific capture of chlamydial particles by solid phase coated with chlamydi specific antibodies.
  • antibody directed antigen capture can be used for concentration of micro organisms from crude samples, each individual assay requires the coating o microorganism-specific antibodies to the solid phase.
  • a more universal capture system enabling the capture of different microorganisms simultaneously would simplify th recovery from clinical specimen and reduce the work load.
  • Microorganism-specifi detection could then be performed by PCR using specific primers, or any other type o specific bacterial assay.
  • Serum sensitive bacteria can bind high amounts of Clq from serum, independently of the presence of bacteria specific antibodies. Direct binding of Clq t serum sensitive bacteria has been reported by several laboratories (Betz et al. 1981 ; Cla et al. 1981; Loos et al. 1978; Tenner et al. 1981). It was shown that LPS and porin potentiate the binding of Clq in the absence of specific antibody (Loos et al. 1987; Cla et al. 1984).
  • Clq was shown to bind to retroviruses (Clas et al. 1984). It is the aim of this invention to provide for a method of capturing microorganisms from sample, said capturing method resulting in a concentration and/or purification of sai microorganisms from the sample.
  • the invention discloses the use of complement components or fragments thereof for capturing microorganisms from a sample.
  • complement refers to the complement system (human or animal), which is a system of serum proteins, activated by antibody-antigen complexes or by microorganisms, and active in the elimination of microorganisms invading the human body.
  • microorganisms refers most often to bacteria, but may also refer to viruses, or even to eucaryotic microorganisms (yeasts, fungi). The genera and species concerned are referred to below.
  • complement components refers to at least one of those serum proteins belonging to the complement system.
  • fragment thereof refers to any fragment of the complement components, with said fragments having retained the microorganism binding affinity of the complement component.
  • capturing refers to the binding of the microorganisms to the complement components resulting in a concentration and/or purification of said microorganisms from the sample.
  • sample may comprise any type of medium possibly containing micro- organisms, such as clinical specimens (faeces, sputum, broncheoalveolar lavage, cervical secretions, urine and possibly blood, cerebrospinal fluid, serum or tears%), food specimens, soil and water specimens, fermentation broth, etc.
  • the invention discloses the use of complement components as described above, wherein said complement components are bound to a solid phase.
  • solid phase refers to any type of solid substrate known in the art which allows the binding of complement components for the above-described use, and which allows subsequently an easy recovery of the (complement component)-(microorganism) complexes.
  • solid phases include: beads, plates, dipsticks, membranes, tubes or others made of for example polystyrene, poly vinyl, latex, sepharose or other polymers, and most preferably the solid phase is composed of paramagnetic beads, e.g. tosylactivated Dynabeads as commercialised by Dynal AS, Oslo, Norway.
  • the type of binding of the complement components to the solid substrate can be covalent or passive binding, depending on the solid substrate used, according to any method known in the art.
  • the invention describes the use as described above, wherein said complement components are Cl or CIQ or C3b, or a fragment thereof, as long as said fragment is still able to bind microorganisms.
  • the abbreviations “Cl ", “CIQ” and “C3b” refer to serum proteins being components or subcomponents of the complement system.
  • the invention describes the use as described above, wherein said complement component is CIQ, or a fragment thereof, as long as said fragment is still able to bind microorganisms.
  • the current invention describes a system capable of capturing a broad range of micro- organisms, using solid phase immobilized Clq for concentration and purification of Clq binding microorganisms. Chlamydia trachomatis was used as a model system (see example I).
  • the capturing buffer should preferably be free of certain detergents like desoxycholate which lead to permeabilisation of the cell membrane (see example II).
  • the Clq-capturing procedure of the current invention enables the capturing of intact virus particles (virions) from a sample like serum or plasma, which reflects much more the infectivity of the sample than the free circulating viral nucleic acids, which are determined by the currently used methods of virus detection.
  • this method for capturing virus particles in a serum or plasma sample, in stead of free circulating nucleic acids may prove to be advantageous for differentiation between latent and non- latent viral infection phases.
  • the Clq-directed capture enables the concentration of microorganisms from large volumes of clinical specimens, with concomitant removal of inhibitors of PCR. This may lead to an increased sensitivity of capture-preceded PCR as compared to direct PCR.
  • An important feature of the Clq-directed capture however as compared to the antibody-directed PCR is its utility toward a broad range of micro ⁇ organisms (see example III).
  • the nature of the captured microorganisms can subsequently be determined by PCR using microorganisms specific primers, or any other type of microorganism detection assay. In certain applications a more universal capturing system is clearly advantageous to specific capturing.
  • pathogens causing gastero- enterological disorders in faeces (like Salmonella sp. , Shigella sp. , Enterococcus sp., Campy lobacter sp. etc.) or pathogens causing respiratory disorders in sputum or bronche- oalveolar lavages (like Streptococcus sp. , Mycobacteria sp. , Mycoplasma sp. , Moraxella sp. , Borde ⁇ ella sp. etc.) or pathogens causing sexually transmitted diseases in cervical secretions (like Neisseria sp.
  • pathogens causing gastero- enterological disorders in faeces like Salmonella sp. , Shigella sp. , Enterococcus sp., Campy lobacter sp. etc.
  • pathogens causing respiratory disorders in sputum or bronche- oalveolar lavages like Streptococc
  • complement components may be modified in order to make them more effective for the above-described use, e.g. in order to obtain a higher microorganism capture efficiency, and/or to obtain a capture specificity for a restricted group of microorganisms, and/or to enable a more efficient binding of the complement components to the solid substrate.
  • Said modifications to the complemen components which are comprised in the current invention may include e.g. fragmentatio of the molecules, addition of linker groups, binding to carrier molecules, mutation of th amino-acid sequence of said complement components, said mutation possibly includin addition, deletion or substitution of one or more amino acids.
  • the invention provides for the use of complement components fo the capturing of microorganisms in a sample, wherein said microorganisms belong to broad panel of different microorganisms, such as belonging to the group of viruses and/ o to eucaryotic microorganisms and/or to gram-negative and/or to gram-positive genera o bacteria, and more specifically belonging to at least one of the following genera: Chlamydia, Campy lobacter, Escherichia, Salmonella, Shigella, Enterococcus, Neisseria, Klebsiella, Pseudomonas, Mycoplasma, Streptococcus, Staphylococcus, Mycobacterium, Moraxella, Bordetella, Haemophilus, Branhamella, Legionella, Clostridium, Vibrio, Yersinia, Liste ⁇ a, Cryptococcus, Herpes simplex virus, Human Papilloma virus, Huma immunodeficiency virus, Hepatitis
  • the invention provides for the use as describe above, wherein said microorganisms possibly belong to the group of viruses. According to a more specific embodiment the invention provides for the use as describe above, wherein said microorganisms possibly belong to the group of eucaryotic micro ⁇ organisms. According to a more specific embodiment the invention provides for the use as describe above, wherein said microorganisms possibly belong to the group of gram-negative bacteria.
  • the invention provides for the use as described above, wherein said microorganisms belong to the group of gram-positive bacteria.
  • said sample is a clinical sample, such as faeces, sputum, broncheoalveola lavage, cervical secretion, urine, and possibly blood, cerebrospinal fluid, serum or tears.
  • the invention provides for the use as described above, wherein said sample is sputum or broncheoalveolar lavage, and wherein said microorganisms are at least one of the following species: Mycoplasma pneumoniae, Bordetella pertussis, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium sp. , Pseudomonas aeruginosa, Branhamella catarrhalis,
  • the invention provides for the use as described above, wherein said sample is a faecal sample, and wherein said micro ⁇ organisms are at least one of the following species: Salmonella sp., Shigella sp., Yersinia sp. , Campylobacter sp. , Clostridium difficile. Vibrio cholera or any other pathogenic microorganism possibly causing gastero-enteritic disorders.
  • the invention provides for the use as described above, wherein said sample is urine or cervical secretion, and wherein said microorganisms are at least one of the following species: Chlamydia thrachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma genitalium, Mycoplasma hominis, Garderella vaginalis, Haemophilus ducreyi, Streptococcus agalactiae, Trichomo ⁇ nas vaginalis, Candida albicans, Veilonella parvula, Mobiluncus sp. , Peptostreptococcus sp., Treponema pallidum, Herpes simplex virus, Human papilloma virus, or any other pathogenic microorganism possibly causing urogenital disorders.
  • said microorganisms are at least one of the following species: Chlamydia thrachomatis, Neisser
  • the invention provides for the use as described above, wherein said sample is blood, serum or cerebrospinal fluid, and wherein said microorganisms are at least one of the following species: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Listeria monocytogenes, Mycobacte ⁇ rium sp. , Streptococcus agalactiae, Cryptococcus neoformans, HIV, Hepatitis viruses, or any other pathogenic microorganism possibly present in the above-mentioned samples.
  • said sample is a food sample.
  • the invention provides for the use as described above, wherein said sample is a food sample, and wherein said microorganisms are at least one of the following species: Escherichia coli, Salmonella sp., Campylobacter sp. , Brucella sp. , Listeria sp. , Clostridium sp. , Staphylococcus sp. , Yersinia enterocolitica, or an other pathogenic microorganism possibly causing food intoxication.
  • said sample is a soil or water sample.
  • the invention provides for the use as described above, wherein said sample is a sample taken from a fermentation broth.
  • Another embodiment of the invention provides for the use as described above wherei said solid phase consists of paramagnetic beads.
  • the invention thus provides for a method for capturing microorganisms in a sample, comprising the use of complement components preferably bound to a solid phase.
  • the invention provides for a method for detecting microorganisms in sample, comprising the steps of
  • the detection of the microorganisms captured can be done according to any type of assay aiming at the detection and/or identification of microorganisms possibly present in sample.
  • assays are e.g. nucleic acid hybridisation, possibly preceded b any type of nucleic acid amplification, culture, staining techniques, or any othe microorganism characterization techniques.
  • said detection assay comprises a polymerase chain reaction (PCR, Saiki et al. 1988) or any other type of nucleic acid amplification, such as ligase chain reaction (LCR; Landgren et al. 1988; Wu and Wallace, 1989; Barany, 1991), nucleic acid sequence- based amplification (NASBA; Guatelli et al. 1990; Compton, 1991), transcription-base amplification system (TAS, Kwoh et al. 1989), strand displacement amplification (SDA, Duck, 1990; Walker et al. 1992) or amplification by means of Q ⁇ replicase (Lizardi et al. 1988; Lomeli et al.
  • PCR polymerase chain reaction
  • LCR Landgren et al. 1988; Wu and Wallace, 1989
  • NASBA nucleic acid sequence- based amplification
  • SDA strand displacement amplification
  • Duck, 1990; Walker et al. 1992 or amplification by means of Q ⁇ replicase
  • the amplified products can be conventiently labele either using labeled primers or by incorporating labeled nucleotides.
  • Labels may b isotopic ( 32 P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
  • the amplificatio reaction is repeated between 20 and 70 times, advantageously between 25 and 45 times.
  • the invention thus provides for a method for capturing microorganisms from a sampl comprised in or for use in combination with any type of microorganism detection assay, said microorganism capturing method comprising the use of complement components possibly bound to a solid phase for capturing microorganisms from the sample, as described above.
  • the invention also provides for a capturing agent, consisting of complement components or fragments thereof preferably bound to a solid phase, said complement components being preferably Cl or CIQ or C3b, for use in the preparation of a diagnostic agent, kit or device for capturing microorganisms possibly present in a sample as described above.
  • complement components or fragments thereof may be purified from human or animal serum, or made by recombinant DNA techniques, or synthesised chemically, by any methods known in the art for polypeptide synthesis.
  • the invention also provides for a buffer for use in a capturing method as described above. More particularly, the invention provides for a buffer enabling efficient capturing of the microorganisms possibly present in a sample, said buffer being free from membrane permeabilizing agents such as desoxycholate.
  • the invention more particularly provides for a capturing buffer as described above, preferably comprising the following components:
  • Tris-HCl pH 8.0
  • BSA Bovine Serum albumin
  • Tris-HCl pH 8.0
  • DTT dithiothreitol
  • the mucus dissolver added to the capture buffer i DTT, at concentrations of 1 to 50 mM, and most preferably at a concentration of 1, 5 o 25 mM.
  • the invention also provides for a buffer suitable for lysin the captured cells, said lysing buffer being constituted preferably by at least the followin components: PBS, 50 ⁇ g/ml proteinase K and 0.75% Triton X-100.
  • the invention also provides for a composition comprising a capturing agent, as describe above, and a capturing buffer, or components necessary to produce the buffer, a described above.
  • the invention also provides for a kit for capturing microorganisms from a sample, said kit comprising at least the following elements:
  • a means for pretreatment of the sample possibly containing the micro organisms possibly containing the micro organisms; - a complement component or fragment thereof preferably bound to a solid phase fo capturing the microorganisms, as described above;
  • a capturing buffer or components necessary to make the buffer, as described above; - if necessary, a means for separating the captured microorganisms from the remainder of the sample.
  • the invention provides for a kit as described above, said kit being a sample preparation kit for any type of microorganism detection assay .
  • the invention provides for a kit as described above, said kit being a sample preparation kit for a PCR-based microorganism detection assay.
  • Figure 1 Clq capture of Chlamydia trachomatis using beads with different Clq coating concentrations.
  • lane 1 coating with 5 ⁇ g Clq/mg beads
  • lane 2 coating with 10 ⁇ g Clq/mg beads
  • lane 3 coating with 20 ⁇ g Clq/mg beads
  • lane 4 coating with 40 ⁇ g Clq/mg beads
  • Figure 2 Comparison of different capture buffers for Clq capture PCR of Salmonella lanes 1 ,3,5,7: captured fraction lanes 2,4,6,8: uncaptured fraction lanes 1 and 2: capture buffer PBS A lanes 3 and 4: capture buffer TA lanes 5 and 6: capture buffer TC lanes 7 and 8: capture buffer TEA
  • FIG. 3 Comparison of PBS and Tris-HCl as buffer component in the washing and lysis buffer in Clq capture PCR of Salmonella.
  • lanes 1 ,3,5 Tris-HCl in washing and lysis buffer lanes 2,4,6: PBS in washing and lysis buffer lanes 1 and 2: capture buffer PBSA lanes 3 and 4: capture buffer TA lanes 5 and 6: capture buffer TEA
  • FIG. 4 Comparison of different capture incubation times for Neisseria gonorrhoeae using Clq capture PCR, as well as the influence of addition of DTT or hyaluronidase to the capture buffer.
  • lanes 1,2,3,4,8 capture time 90 min lanes 1,2,3: with DTT in capture buffer, in increasing amounts: 1 mM, 5mM and 25 mM respectively
  • lane 4 with hyaluronidase in capture buffer
  • lane 5 capture time 30 min lane 67: capture time 60 min lane 7: double amount of beads added (20 ⁇ l in stead of 10 ⁇ l)
  • lane 9 capture time 120 min lanes
  • 5,6,7,8,9 no additives in capture buffer
  • Figure 5 Influence of N-acetyl cysteine and Nal in capture buffer on efficacy of Clq capture PCR of Chlamydia trachomatis (lanes 1 to 7) and Neisseria gonorrhoeae (lanes 11 to 17).
  • lanes 1 and 11 no additives in capture buffer lanes 2 and 12: 3 mM DTT in capture buffer lanes 3 and 13: 6 mM Nal in capture buffer lanes 4 and 14: 6 mM Nal and 3 mM DTT in capture buffer lanes 5 and 15: 0.6 mM Nal in capture buffer lanes 6 and 16: 0.6 mM Nal and 3 mM DTT in capture buffer lanes 7 and 17: 15 mM N-acetyl cysteine in capture buffer lane 8: positive PCR control: 100 ng of isolated endogeneous plasmid DNA of Chlamydia trachomatis lane 9: MW marker lane 10: positive PCR control: 100 ng of isolated genomic DNA of Neisseria gonorrhoeae
  • Figure 6 Agarose gel electrophoresis of the PCR-amplified rRNA spacer region after capturing of the microorganisms by Clq-coated beads (example III).
  • lane 1 Salmonella D lane 2: Neisseria gonorrhoeae lane 3: negative Clq-capture control: addition of blanc capture buffer to Clq-coated beads lane 4
  • Marker 1 kbp DNA ladder lane 5 Staphylococcus aureus lane 6 Staphylococcus epidermidis lane 7
  • Escherichia coli lane 9 Pseudomonas aeruginosa lane 10 Enterobacter cloacae lane 11
  • Citrobacter diversus lane 12 Haemophilus influenzae lane 13 Klebsiella pneumoniae lane 14: Streptococcus pneumoniae lane 15: marker 1 kbp DNA ladder lane 16: negative PCR
  • Figure 7 Clq capture PCR of Campylobacter jejuni in the presence of increasing amounts of E. coli.
  • a constant amount of C. jejuni cells (1,5 JO 5 cells) is mixed with decreasing amount of E. coli cells (lanes 1-9).
  • lane 1 2J0 8 E. coli cells
  • lane 2 2 JO 7 E. coli cells
  • lane 3 2J0 6
  • lane 4 2J0 5
  • E. coli cells lane 6 2J0 3
  • Fi g ure 8 HCV RNA detection in duplo by 5'UTR PCR with an internal assay control after virion capture with CIQ.
  • Clinical specimen Clinical specimens used were samples submitted to the routine microbiology laborator for detection of Chlamydia trachomatis. The samples were taken with an ENT swab Boehringer, Mannheim ) and placed in 2 ml transport medium (0.2 M sucrose i phosphate buffer). The specimens for chlamydial culture were stored at 4°C or, when no tested within 24 hours after collection, at -70°C. All specimens were processed within days. The remainder of the samples was stored at -70°C untill further testing.
  • Chlamydia trachomatis was cultured in cycloheximide-treated McCoy cells, grown in 9 wells microtiter plates as described by Thewessen et al.(27). Briefly, 2 wells per plat were each inoculated with 0.2 ml of patient sample.
  • Human Clq was isolated as described by Tenner et al.(1981). Briefly, fresh human seru was adjusted to 5 mM EDTA and applied to a Biorex 70 column, equilibrated wit starting buffer ( 82 mM NaCl, 2 mM EDTA, 50 mM sodium phosphate, pH 7.2 ). Afte washing with 1000 ml of starting buffer, the column was eluted with an ionic strengt gradient composed of 600 ml starting buffer and 600 ml buffer pH 7.2 containing 30 mM NaCl, 2 mM EDTA and 50 mM sodium phosphate.
  • Coating of paramagnetic beads with Clq was performed according to the manufacturers 's instructions. Tosylactivated Dynabeads ( 500 ⁇ l; M-280, Dynal AS, Oslo, Norway, 6-7 x 10 8 beads/ml ) were pelleted by placing the tube in the powerful magnetic field of a magnetic particle concentrator ( Dynal MPC-6 ). Following removal of storage buffer, the beads were washed once with 1 ml of coating buffer ( 0.05 M borate pH 9.5 ). After a final concentration, 250 ⁇ l coating buffer was added, the beads were suspended and 250 ⁇ l human Clq ( 0.4 mg/ml coating buffer ) was added. Coupling of Clq was performed by gentle rotation for 24 hr at 37°C.
  • the beads were washed 3 times with PBS pH 7.2 containing 0J % BSA (PBS A). Following an overnight wash at 4°C with the same buffer, the beads were suspended in 0.5 ml of 0J % PBSA and stored at 4°C.
  • lysis buffer 50 ⁇ g/ml prote ⁇ nase-K, 0.75 % Triton X-100 .
  • the beads were incubated for 60 min at 37°C and the prote ⁇ nase-K was inactivated by a 15 min incubation at 100 °C.
  • the sample was cooled to room temperature for 5 min in the air.
  • Ten ⁇ l was used for the PCR. From samples with low inclusion numbers 50 ⁇ l was used for PCR.
  • the polymerase chain reaction (Saiki et al. , 1988) was used for the amplification of Chlamydia trachomatis target DNA.
  • the primer sequences were selected from the common endogenous plasmid of Chlamydia trachomatis (Claas et al. , 1990, 1991) which generates a species specific fragment of 517 basepairs with all known C. trachomatis serovars ( PI : 5' GGACAAATCGTATCTCGG 3'; P2: 5' GAAACCAACTCTACGCTG 3' ).
  • This PCR product was positively identified by using an internal labelled oligonucleo- tide probe ( 5' CGCAGCGCTAGAGGCCGGTCTATTTATGAT 3').
  • the primers and probes were synthesized on an Applied Biosystem 381 A DNA synthesizer using the ⁇ - cyanoethyl phosporamidite method. Specificity has been determined as described (Claas et al. 1990). A spatial separation of the different steps of the technique was routinely used to prevent contamination of the samples.
  • the reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl, pH 8.3,
  • Hybridization was performed as described by Claas et al. (Claas et al. 1990, 1991). Briefly, prehybridization was performed at 37 °C for 15 min in a solution containing 5x SSC [75 mM sodiumcitra- te, 750 mM NaCl], 5x Denhardt [0.1 % bovine serum albumin, 0.1 % Ficoll, 0.1 % poly vinyl pyrrolidone], 0.5% SDS, 5 mM EDTA and 0J mg/ml denatured, sonicated herring sperm DNA. Hybridization was performed in the same mixture by adding the probe to the prehybridization mixture.
  • Hybridization was routinely done for 16 hrs. After hybridization, the blots were washed twice for 15 min at 42°C in 2x SSC containing 0J % SDS. Autoradiography was performed for 4 hr on a Kodak Royal X-Omat film using 2 intensifying screens at -70 °C.
  • PCR and Clq directed antigen capture PCR was identical to cell culture.
  • the sensitivity of Clq directed antigen capture PCR in relation to direct PCR was determined using 34 patient specimens, positive by direct PCR. All samples, positive by direct PCR, were also positive by Clq directed antigen capture PCR (results not shown).
  • 71 consecutive clinical specimens were tested. The results are shown in table 1. Of these 71 clinical specimens, 11 samples were positive by cell culture, direct PCR and Clq directed antigen capture PCR.
  • Clq directed antigen capture PCR revealed a sensitivity equal to direct PCR and cell culture.
  • Clq binding Chlamydia cells can be concentrated from large volumes with concomitant removal of inhibitors of PCR, enabling the use of large volumes of clinical samples for clinical testing. Since Clq has been shown to bind to a range of gram-negative and gram-positive bacteria (see example III), the newly developed techniques have utility toward a broad range of bacteria. Table 1. Comparison between cell culture, direct PCR and Clq directed antigen capture PCR for detection of Chlamydia trachomatis in 71 consecutive clinical samples.
  • Bacterial strain As test microorganisms Salmonella D, Neisseria gonorrhoeae and Chlamydia trachomatis LGV-2 were used. The strains were obtained from the Diagnostics:
  • DTT dithiotreitol
  • NALC N-Acetyl-L-cysteine
  • hyaluronidase 0.6 M or 6 M
  • Sodium Iodide Naodide
  • the suspension was incubated for 1.5 h at room temperature with gentle rotation.
  • the beads were pelleted by placing the tube in a powerful magnetic field of a magnetic particle concentrator (Dynal MPC-6).
  • the capture buffer with not-captured bacteria was transfer ⁇ red to a new 1.5-ml tube. After centrifugation at 12,000 x g, the pellet was washed twice with PBS.
  • the beads with captured bacteria were washed four times by addition of 500 ⁇ l PBS or 0.05 M Tris-HCl (pH 7.3), followed by resuspension, pelletation with the magnetic particle concentrator and removal of the supernatant.
  • 50 ⁇ l of lysis buffer 50 ⁇ g proteinase K per ml PBS or 0.05 M Tris-HCl (pH 7.3), 0.75% Triton X-100 was added.
  • the samples were incubated for 60 min. at 37 °C, and the proteinase K was inactivated by a 15-min. incubation at 100°C.
  • the samples were cooled to room temperature for 5 min. in air and 10 ⁇ l was used for PCR.
  • PCR amplification PCR was used for the amplification with general primers (5' 16S primer : 5' ATATTGGATCCGAGAGTTTGATCCTGGCTCAG 3'; 3' 16S primer : 5' AAAGGATCCTGCAGACCTTGTTACGACTTCACCCCA 3') obtained from the 16S ribosomal region of bacterial DNA (Giesendorf et al. 1992), respectively C.
  • trachomatis specific primers as descripted in example 1 and Neisseria gonorrhoeae specific primers (primer 1 : 5' CGCTACCAAGCAATCAAGTTGCCC 3' (SEQ ID NO 1); primer 2 : 5' GACGGCAGCACAGGGAAGCTTGCTTCTCGGG 3' (SEQ ID NO 2).
  • the reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl (pH 9.0), 50 mM KC1, 2.5 mM MgCl 2 , 0.01 % gelatin, 200 ⁇ M (each) deoxynucleoside triphosphates, 50 pmol of both primers, and 0.25 U SuperTaq DNA polymerase (Sphaero Q, Leiden, The Netherlands). Ten microliters of sample was used. The reaction mixture was overlayed with 3 drops of mineral oil (Sigma M-3516) and subjected to 40 cycles of amplification in a thermocycler (Bio-med, Theres, Germany). Each cycle consisted of a 1-min DNA denaturation step at 94°C, a 2-min primer annealing step at 42°C, and a 3-min extension step at 74°C. Water was used as negative control for the PCR.
  • DTT has no significant effect on the efficacy of Clq capture PCR of N. gonorrhoeae whilst hyaluronidase showed a deleterious effect.
  • N-acetyl-cysteine and Nal The effect of addition of N-acetyl-cysteine and Nal to the capture buffer is shown in fig. 5. N- acetyl-cysteine does not effect the efficacy of capmre. Addition of Nal showed a deleterious effect on the capture PCR efficacy. It should be noted that the effect of additives to the capture buffer may be dependent on the type of microorganism to be captured.
  • Tris H.C1 pH 8.0
  • Triton X-100 Optimal capturing incubation time is in the range of 30 to 90 minutes.
  • Tris-HCl in washing and lysis buffer has a deleterious effect on Clq capmre PCR.
  • PBS should be used.
  • Addition of N-acetyl cysteine has no significant effect on the efficacy of Clq capmre PCR.
  • Addition of DTT may have a beneficial effect on the efficacy of the Clq capmre PCR.
  • Addition of hyaluronidase or Nal has a deleterious effect on the efficacy of Clq capmre PCR.
  • the capture buffer should be free of certain detergents like desoxycholate, which lead to permeabillisation of the cell membrane (results not shown).
  • the system was tested on a variety of gram-negative and gram-positive bacteria.
  • Salmonella D Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus epidermi- dis, Streptococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter diversus, Haemophilus influenzae, Klebsiella pneumoniae, Strepto ⁇ coccus pneumoniae.
  • Detection of the presence of microbial DNA after capmre was performed by PCR using universal primers obtained from the 16S and 23S ribosomal RNA genes.
  • Bacterial strains All bacterial strains were obtained from the Diagnostic Centre SSDZ, Delft, The Netherlands, and the State Institute for Quality Control of Agricultural Products (RIKILT-DLO), Wageningen, The Netherlands. Clq coating of paramagnetic beads / cell capture / DNA isolation. This was performed exactly as described in example I. PCR performance. PCR was used for the amplification of microbial DNA present in the capmred sample. Use was made of the delta/omega universal primers derived from the 16S and 23S ribosomal RNA genes as described by Rossau et al. (WO 91/16454).
  • the reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl [pH8.3], 50 mM KC1, 1.5 mM MgC12, 0.01 % gelatin, 200 ⁇ M (each) deoxynucleoside triphosp- hates, 100 pmol of both primers, and 0.25 U SuperTaq DNA polymerase (Sphaera Q, Leiden, the Netherlands). Ten microliters of sample was used.
  • the reaction mixture was overlayed with 3 drops of mineral oil (Sigma M-3516) and subjected to 30 cycles of amplification in a thermocycler (Bio-med, Theres, Germany). Each cycle consisted of a 1-min DNA denaturation step at 95 °C, a 1-min primer annealing step at 50 °C, and a 1-min extension step at 72 °C. The final extension was performed 10 min. at 72 °C.
  • PCR products were separated on a 2% agarose gel and stained with ethidium bromide.
  • Example IV Sensitivity of Clq capture PCR for detection of Campylobacter jejuni in the presence of excess E. coli.
  • Faecal samples contain large amounts of enterobacteria like E. coli. These bacteria can also bind Clq coated solid phases possibly leading to a diminished binding, due to competition, of the bacteria to be detected.
  • C. jejuni cells (1 ,5 x 10 5 ) was mixed with a dilution series of E. coli and processed for Clq capmre PCR.
  • Campylobacter jejuni strain was obtained from the State Instimte for Quality Control of Agricultural Products (RIKILT-DLO), Wageningen, The Netherlands. Clq coating of magnetic beads/antigen capture/DNA isolation/PCR/amplimer analysis were performed exactly as described in example I.
  • HCV viremia is determined by detection of the viral RNA genome isolated from serum of plasma by the proteinase K or guanidiniumthiocyanate method. By these methods circulating virus particles as well as free HCV RNA are detected. The detected HCV RNA by RT-PCR does not really reflect infectivity of a sample. Therefore, an assay was developed that captures the infectious particles in serum.
  • Serum from one chronically infected chimpanzee, one acute infected patient and three chronically infected patients were analysed in this study.
  • the four serum samples from the acute and chronically infected patients and the chimpanzee serum were HCV RNA positive using the guanidiniumthiocyanate isolation method and standard 5'UTR RT-PCR (with internal assay control) (results not shown).
  • Clq coated beads for virus isolation, the HCV RNA amplicon of 296bp was detected in the acute infected patient whereas HCV RNA was hardly or not detectable in three chronically infected patients and the chimpanzee ( Figure 8). In the three chronic samples only the internal assay control amplification of 346bp can be seen.
  • beads coated with BSA or antibodies directed to the E2 antigen of HCV no amplicons of 296bp were detected after capturing.
  • it was not possible to detect synthetic HCV RNA by capturing it was not possible to detect synthetic HCV RNA by capturing (data not shown).
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO

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