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WO1999060009A1 - Procedes ameliores pour detecter un fragment d'acide nucleique cible - Google Patents

Procedes ameliores pour detecter un fragment d'acide nucleique cible Download PDF

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Publication number
WO1999060009A1
WO1999060009A1 PCT/US1999/010939 US9910939W WO9960009A1 WO 1999060009 A1 WO1999060009 A1 WO 1999060009A1 US 9910939 W US9910939 W US 9910939W WO 9960009 A1 WO9960009 A1 WO 9960009A1
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seq
nucleic acid
probe
sample
complex
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Jyotsna S. Shah
Nick S. Harris
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Igenex Inc
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Igenex Inc
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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

Definitions

  • Babesiosis resuls from infection by a protozoan sporozoa of the genus Babesia .
  • the internal parasites are transmitted by hard-bodied ticks, and invade and destroy erythrocytes .
  • Babesia infections occur with high frequency in many wild and domestic animals worldwide. Human infections are more rare, occurring mainly in Europe and North America, but are severe, resulting in death of more than half of the patients.
  • the Babesia species implicated in man are usually Babesia divergens and Babesia microti .
  • the spirochete Borrelia burgdorferi is the causative agent of Lyme disease. Infection is also transmitted through a tick vector host. Clinical diagnosis of infection relies on anatomical and pathological signs, with laboratory tests for confirmation based on immuno- biochemical detection of host antibodies to antigens present on the pathogen. Such tests are limited because they depend upon the reactivity of the host's immune system for sensitivity. In addition, false positive results can occur due to infection from a closely related infectious agent. Diagnostic tests which are based on the detection of nucleic acids specific for a particular pathogen are more accurate, more sensitive, and when properly controlled, provide less potential for false negative or false positive results than diagnostic tests based upon the immunological response of the host. The sensitivity and accuracy of detection of specific nucleic acids from a specimen is often complicated by the presence of nucleic acid binding proteins in the sample, and a limited sample amount with which to perform a wide array of tests.
  • the present invention relates to a method for detecting a target nucleic acid fragment in a clinical specimen.
  • the method is performed in several steps.
  • a sample of the clinical specimen is solubilized in a chaeotropic salt solution.
  • the solubilized sample is then treated by means to denature the nucleic acids contained therein.
  • the solubilized sample is then contacted the with at least one probe complex, the probe complex comprising a nucleic acid sequence which is complementary to a portion of the target nucleic acid fragment, the probe complex further comprising a first member of a specific binding pair.
  • the solubilized sample is incubated with probe complex under conditions appropriate for hybridization of the probe complex with the target nucleic acid fragment .
  • the probe complex in the incubated sample is then contacted with a solid substrate which is linked to the second member of the specific binding pair under conditions which promote binding of the specific binding pair, to isolate hybridized target nucleic acid fragment in a probe- target-solid substrate ternary complex.
  • the isolated probe-target-solid substrate complex is then separated from the solubilized sample.
  • the target nucleic acid and the probe complex are released into solution from the separated probe-target-solid substrate complex, and the released target nucleic acid fragment is then amplified by PCR or RT-PCR.
  • the presence of the target nucleic acid fragment in the clinical specimen is detected by comparison of the amplification products produced to amplification products produced in identically treated positive and negative control reactions.
  • Specific embodiments of the invention include the use of disclosed chaeotropic salt solutions, the use of disclosed wash buffers, and specific conditions for amplification.
  • Other embodiments include the use of the method on different specimens, including EDTA treated whole blood, serum, plasma, urine, cerebral spinal fluid, synovial fluid, and tissue.
  • Another embodiment of the present invention is the use of the method to detect the presence of a pathogen, such as Borrelia burgdorferi or species of Babesia .
  • Other embodiments include the use of the specific binding pair biotin: streptavidin.
  • Other embodiments are use of the method with specified probe complexes and PCR primers for the detection of pathogen nucleic acid sequences.
  • Another aspect of the present invention relates to specific oligonucleotide sequences disclosed which are used as probes or primers for the detection of either Babesia microti or Babesia WA-1 .
  • the present invention is based in part on the discovery of a rapid, sensitive, and accurate method for detecting one or more nucleic acid fragments from a specimen.
  • the method is a two part process, the first part being devoted to isolating one or more nucleic acid fragments from the specimen via hybridization with a probe complex, and the second part being detection of the isolated nucleic acid fragment.
  • the isolation removes unwanted nucleic acids from the desired nucleic acid fragment (herein referred to as the target nucleic acid fragment) , and also significantly eliminates PCR inhibitors from the target nucleic acid fragment . This allows for highly sensitive and accurate detection of the isolated target nucleic acid fragment .
  • the isolation portion of the method reduces or eliminates PCR inhibitors from the target nucleic acid fragment. This significantly increases the sensitivity and decreases background of amplification reactions performed on the isolated target nucleic acids, compared to standard methods of amplification of a target nucleic acid directly from a heterogenous sample.
  • the method is comprised of a series of steps outlined below: 1) A sample of the specimen is first solubilized in a chaeotropic salt solution.
  • Each probe complex contains a nucleic acid which can hybridize to the target nucleic acid fragment, linked to a member of a specific binding pair. The nature of the binding pair is such that binding of the two members can be used for physical isolation of the probe complex.
  • the sample is then incubated with the probe complex under conditions appropriate for hybridization of the probe complex with the target nucleic acid fragment .
  • the probe complex which has been incubated with the sample is then contacted with a solid substrate which is linked to the second member of the specific binding pair used to create the probe complex. The probe complex, sample, and solid substrate are then incubated under conditions which promote binding of the specific binding pair.
  • ternary complex is then separated from the other components of the solubilized sample, generally by exploiting a property of the solid substrate.
  • target nucleic acid and probe complex are released from the ternary complex (e.g. through dissociation of the binding pair or disruption of the nucleic acid hybridization) .
  • Any released target nucleic acid fragment can then be detected by standard methods .
  • the preferred method of detection is amplification, either by PCR or RT-PCR.
  • Any clinical specimen or bodily secretion e.g. blood, serum, urine, synovial fluid, cerebral spinal fluid, tissue
  • Specimens obtained from any animal or human, and in some circumstances, from insect carriers are appropriate for use in this method.
  • Specific nucleic acid fragments can be isolated from any sample type, including, without limitation, insects such as ticks and mosquitoes. This is particularly useful when attempting to determine if a particular insect isolated from a patient has potentially transmitted a microbial pathogen to the patient.
  • the chaeotropic salt solution is GuSCN buffer.
  • 5 M GuSCN buffer (pH 7.4) is made of 100 mM Tris-HCl (pH 7.8), 40 mM EDTA, 5 M GuSCN, and 1%
  • a final concentration ranging from 1.5 M to 4.0 M GuSCN buffer is used to solubilize the sample, and also as solution in which probe complex is hybridized to the target nucleic acid fragment within sample.
  • the chaeotropic salt solution is
  • GuHCl buffer (pH 7.4). 8 M GuHCl buffer (pH 7.4) is made from 200 mM Tris-HCl (pH 7.4), 40 mM EDTA, 1% BSA, and 1% Sarkosyl. A final concentration ranging from 2.0 M to 6.0 M GuHCl buffer is used to solubilize the sample, and also as solution in which probe complex is hybridized to the target nucleic acid fragment within sample. The actual concentration varies with selection probe sequence and length. Because the chaotropic buffer used to solubilize the sample also serves as a hybridization buffer, the final concentration of chaeotropic buffer depends upon the probe sequence, probe length, and the hybridization temperature. The specificity of the hybridization depends on the salt conditions and the temperature.
  • the final concentration of the chaotropic buffer is adjusted for the different probe complexes used.
  • concentration of the buffer will depend on the Tm of the probe complex used.
  • the Tm of the probe in turn depends on the length and sequence of the probe.
  • concentration of GuSCN or GuHCl is directly related to the Tm of the probe. In general, shorter probes will require lower concentrations of GuSCN or GUHCl .
  • Determination of the exact concentrations to be used for a given probe complex is within the ability of one skilled in the art, through no more than routine experimentation. Vortexing the sample in either buffer for 30 seconds is sufficient to solubilize the sample.
  • a variety of means can be employed to denature the nucleic acids within the solubilized sample. Heating the sample to 85°C for 10 minutes is sufficient if either GuSCN buffer or GuHCl buffer describe above are used to solubilize the sample. Heating may also further facilitate the solubilization of the sample.
  • the sample should be equilibrated after heating, by reducing and holding the temperature at 65°C for 5 minutes.
  • a sample which is treated by other such means to denature nucleic acids may also require equilibration prior to further treatment.
  • the denatured (and equilibrated) sample is then contacted with probe complex.
  • the probe complex is comprised of an oligonucleotide linked to a member of a specific binding pair.
  • the oligonucleotide can be either RNA, DNA, an equivalent, or any combination thereof. Equivalents are described in detail below.
  • the oligonucleotide has a nucleic acid sequence of from 10 to 50 nucleotides which is sufficiently complementary to a portion of the target nucleic acid fragment to promote hybridization of the probe to the target. Additional, non-complementary sequences which do not interfere with hybridization to the target may also be present.
  • the probe complex is able to hybridize to the target nucleic fragment under stringent conditions.
  • specific binding pair is intended to encompass any binding pair which can be used to facilitate purification of an attached molecule from a heterogeneous mixture.
  • some examples are ligand binding pairs (e.g. streptavidin and biotin) , antibody and antigen, complementary polydeoxynucleotide tails (e.g. polydA and polydT, polydG or polyDeazo-G and polydC) .
  • ligand binding pairs e.g. streptavidin and biotin
  • antibody and antigen e.g. polydA and polydT, polydG or polyDeazo-G and polydC
  • the specific binding pair member can be attached to the oligonucleotide. Without limitation, attachment can be at the 5' end, the 3' end, at one or more internal sites, or any combination thereof.
  • a combination of probes which are each specific for a different target nucleic acid can be used. This allows for purification of multiple target nucleic acids from the same specimen sample. This ability to isolate multiple target nucleic acids at once from a single specimen sample is an important aspect of the present invention, and is discussed in greater detail below.
  • the probe complex is generally contacted to the nucleic acids in the sample by adding the probe complex to the sample to about 20 ng/ml to 100 ng/ml final concentration. The exact final concentration of probe depends upon the hybridizing oligonucleotide sequence (s), length and the target nucleic acid(s) . The sensitivity depends on the efficiency of hybridization, which in turn is driven by the molar concentration of the probe and time of hybridization.
  • the mass of the probe used depends directly on the length and sequence of the probe. Therefore, in order to provide the same number of molecules for the different probes, the shorter the probe, the lower the mass required for use. Likewise, for longer probes, more overall probe mass is required. Hybridization can be driven by either increasing the time hybridized, or the probe concentration.
  • the probe complex is incubated with the sample under conditions appropriate for hybridization of the probe complex with the target nucleic acid fragment. These conditions will depend upon the solution in which the incubation occurs, the length and GC content of the sequences to be hybridized, etc. Such conditions can be determined by one of skill in the art through routine experimentation.
  • a solid substrate which is derivatized with a member of a specific binding pair which matches that of the probe complex is used to isolate the probe complex and any hybridized nucleic acids.
  • the solid substrate is composed of one or more materials which have one or more properties which are useful in isolation of the substrate from a heterogeneous mixture. Without limitation, some examples are insoluble monomers or polymers such as agarose beads , polyacrylamide, polystyrene beads, paramagnetic or magnetic particles. In one embodiment, the solid substrate is comprised of paramagnetic particles.
  • the paramagnetic particles are preferably derivatized with from 10 ⁇ g/ml to 100 ⁇ g/ml streptavidin, wherein the probe complex is linked to biotin.
  • a solid substrate which is dispersable within the liquid sample (e.g. fine particles) is particularly advantageous since dispersion facilitates rapid binding of the probe complex.
  • the solid substrate can be localized (e.g. a column) and the liquid sample can be exposed to the surface (e.g. poured over the column) to accomplish binding of probe complex to the substrate.
  • the solid substrate is contacted to the probe complex within the incubated sample under conditions which promote binding of the respective members of the specific binding pair. These conditions will vary with respect to the particular binding pair used.
  • the binding conditions used should preserve the hybridization between probe and target, such that binding of the specific binding pair isolates any hybridized target in a probe- target-solid substrate ternary complex.
  • such favorable binding conditions for streptavidin and biotin can be created when using the GuSCN buffer or GuHCl buffer described above by diluting the sample containing the probe complex and target (e.g. with deionized water) to 1.25 M GuSCN buffer or 2 M GuHCl buffer, respectively, prior to or during contact of the solid substrate to the probe complex.
  • the solid substrate is in dispersable form, contact is achieved by adding the solid substrate to the incubated sample solution. If in stationary form, such as a column, the incubated sample solution is contacted to the surface of the solid substrate.
  • the complex Upon formation of the ternary complex, the complex is physically separated from the other components of the solubilized sample, generally by exploiting properties of the solid substrate. For example, gravity, centrifugation, or filtration can be used to separate a dispersed solid substrate from solution. If the dispersed solid substrate is of an appropriate composition, magnetism can be used to physically isolate the substrate with the attached complex.
  • gravity, centrifugation, or filtration can be used to separate a dispersed solid substrate from solution.
  • magnetism can be used to physically isolate the substrate with the attached complex.
  • washes may be performed to completely eliminate residual sample components from the complex. Such washes are performed using a wash buffer which does not disrupt the ternary complex.
  • wash buffers will contain one or more detergents to facilitate elimination of unwanted cellular components.
  • a suitable wash regimen is a first wash with chaeotropic wash buffer, a second and third wash with 0.1 X SSCN buffer (pH 7.4) (0.015 M NaCl , 0.0015 M Na Citrate, 0.1% BSA, 0.1 % NP40) and a fourth wash with 0.1 X SSC buffer (pH 7.4) (0.015 M NaCl , 0.0015 M Na Citrate) .
  • chaeotropic wash buffers are GuSCN wash buffer (1.25 M GuSCN buffer, 1% BSA) and GuHCl buffer (2 M GuHCl buffer, 0.1% BSA).
  • the target nucleic acid is released from the ternary complex into solution.
  • This release of probe and target may or may not also release the probe complex. Separation can be accomplished by contacting the solid substrate with a solution of deionized water, or Tris buffer, which will elute the target into the water or buffer, allowing physical separation from the solid substrate by means used to isolate the solid substrate from solution in the above described wash steps.
  • the target nucleic acid is now significantly enriched, and heterogeneous nucleic acids and other contaminants such as polymerase inhibitors are minimized, if not completely removed.
  • This purification significantly enhances the ability to sensitively and accurately detect the target by a variety of standard methods.
  • a preferred method of detection is amplification by either PCR or RT-PCR.
  • the elimination of inhibitors from the target greatly enhances the sensitivity and accuracy of amplification of isolated target nucleic acid.
  • PCR or RT-PCR can be used to simultaneously detect several different target nucleic acids from a single sample in the same reaction.
  • Another aspect of the present invention relates to the identification of PCR conditions which produce significantly superior results to standard conditions.
  • a buffered solution of from 10 mM to 30 mM Tris-HCl (pH 8.4), 50 mM potassium chloride, and from 1.0 mM to 5.0 mM magnesium chloride, for the PCR or RT-PCR reaction produces significantly lower background and higher sensitivity compared to that produced by standard PCR conditions.
  • the listed buffer concentrations are particularly suited for amplification of target nucleic acid fragment (s) isolated by the above methods.
  • identification of a nucleic acid of interest using PCR amplification of nucleic acids from a clinical sample suffers from several drawbacks.
  • Standard PCR from clinical specimens is effected by the presence of inhibitors of PCR. Even after extensive purification, using labor intensive purification methods, inhibitors are not always completely removed.
  • the present invention provides methods which significantly eliminates PCR inhibitors from the sample nucleic acids.
  • Another drawback of the standard PCR is that it is generally performed on a single component of the sample (e.g. from blood, either serum, plasma, or cells) .
  • Applicants have observed that only testing a single component of a sample may provide inaccurate results .
  • serum may test positive whereas plasma or cellular elements may test negative.
  • tests must be run on each component of the sample, increasing cost and time spent on the assay.
  • Another advantage of the present invention is that the purification methods described above can be performed on intact specimens, without the need for dividing the specimen into components, thus alleviating the need for the performance of multiple tests.
  • the present invention provides methods which can extensively purify and concentrate several different DNA fragments from a sample in the same reaction, thus decreasing the total size of a sample required for the performance of multiple analyses.
  • the present invention also provides specific oligonucleotide sequences which are useful in the identification of Borrelia burgdorferi nucleic acid sequences by the above methods.
  • biotin is the specific binding pair which is used in the method, and the probe complex which is made from the above listed sequences is derivatized with biotin.
  • Amplification of target nucleic acid fragments isolated using the above described probe complex is performed using the following primer sets:
  • oligonucleotide sequences have been determined to provide superior results compared to the use of other complementary B . burgdorferi sequences, when used as described. This determination was made by empirical comparison of a wide range of oligonucleotides which are complementary to B. burgdorferi specific sequences.
  • the present invention also provides specific oligonucleotide sequences which provide superior results when used in hybridization based assays to detect the tick-born protozoan parasites, Babesia microti , Babesia - WA-1 , and other closely related Babesia species which cause human and veterinary disease.
  • B3 5 -GTTATAGTTTATTTGATGTTCGTT-3' (SEQ ID NO: 12)
  • B8 - 1 5 ' -AATAAACGCAGCCAAGAC-3 ' (SEQ ID NO : 17)
  • Oligonucleotides with the above listed sequences hybridize, under specific conditions, to the ribosomal RNA molecules (rRNA) or rRNA genes (rDNA) of Babesia but which do not hybridize under the same conditions to the rRNA or rDNA of other parasites, bacteria and humans, commonly present in a clinical sample. Therefore, oligonucleotides with these specific sequences provide the basis for the development of valuable nucleic acid hybridization assays for the specific detection of , the etiological agent of Babesiosis in a clinical sample (e.g, blood, urine, cerebrospinal fluid, skin biopsy or other tissues or fluid samples from human patients) .
  • a clinical sample e.g, blood, urine, cerebrospinal fluid, skin biopsy or other tissues or fluid samples from human patients.
  • the oligonucleotides also provide the basis for testing tick vectors of Babesiosis to assess infectivity rates or endemic range.
  • the rRNAs which are detected with the above oligonucleotides constitute a significant component of cellular mass. Although estimates of cellular ribosomal vary, actively growing Babesia may contain upwards of 10,000 ribosomes per cell, and therefore 10,000 copies of each of the rRNAs (present in 1:1:1 stoichiometry in ribosomes) . In contrast, other potential cellular target molecules such as genes or RNA transcripts thereof, are less ideal especially for non-isotopic direct in si tu hybridization assays and non-amplified assays.
  • the rRNAs are not subject to lateral transfer between contemporary organisms.
  • the rRNA primary structure provides an organism-specific molecular target, rather than a gene-specific target as would be the case, for example, of a plasmid-borne gene or product thereof, which might be subject to lateral transmission between contemporary organism.
  • Table 4 in the Exemplification below lists organisms which specifically contain nucleic acid sequences to which these individual nucleotides will hybridize under stringent conditions. These organisms can be specifically identified through detection of specific nucleic acid fragments using these oligonucleotide sequences. From the information provided, one of skill in the art can determine various combinations of primer sequences for use in amplification assays in the identification of the listed organisms. One of skill in the art will also recognize that sequences which are exact complements to the above listed sequences, designated herein and referred to as C-Bl through C-B8-1, function equally well when used in place of the above listed sequences.
  • a probe or primer which contains at least 10 consecutive bases of one of the above listed nucleotide sequences Bl through B8-2 is also expected to function adequately in the detection of DNA from the species listed in Table 4, when used in the methods disclosed herein, or in other standard hybridization assays. Such a probe or primer and the 1 exact complement thereof, is also encompassed by the present invention. Under certain conditions, a probe or primer which contains at least 6 consecutive bases of one of the above listed nucleotide sequences Bl through B8-2, is expected to function adequately in the hybridization and detection of DNA from the species listed in Table 4, when used in the method of the present invention and also in standard methods.
  • oligonucleotides can be either deoxyribonucleotides or ribonucleotides, or the equivalents thereof.
  • oligonucleotide refers to polynucleotides comprising nucleotide units formed with naturally occurring bases and pentofuranosyl sugars joined by phosphodiester linkages (e.g. DNA and RNA) . Equivalents thereof include structurally related molecules formed from non-naturally occurring or modified subunits of oligonucletides . These modifications occur either on the base portion of a nucleotide, on the sugar portion of a nucleotide, or on the internucleotide linkage groups.
  • Bases of the oligonucleotide can be modified providing that this does not interfere with Watson-Crick base pairing. Such modifications are commonly utilized to increase hybrid stability. Examples are 5-substituted cytosine or uracil, especially 5-propynyl cytosine and 5- propynyl uracil, to replace C or U and T respectively, or 2 , 6-diaminopurine to replace A (Freier & Altmann, Nucleic Acids Res . 25: 4429-4443 (1997)).
  • Complementarity or complementary is meant a sufficient number in the oligonucleotide of complementary base pairs in its sequence to interact specifically (hybridize) with the target nucleic acid sequence to be amplified or detected.
  • Exact complementarity is assumed to mean 100% base pair hybridization. As known to those skilled in the art, a very high degree of complementarity is needed for specificity and sensitivity involving hybridization, although it need not be 100%. Thus, for example, an oligonucleotide which is identical in nucleotide sequence to an oligonucleotide disclosed herein, except for one base change or substitution, may function equivalently to the disclosed oligonucleotide.
  • Oligonucleotides which have sufficient sequence complementarity to the above listed oligonucleotides to function similarly to the above listed oligonucleotide sequences can be determined by one of skill in the art using no more than routine experimentation in combination with prior art teachings, from the disclosed sequences using the information available in Persing et al . , Target Selection and Optimization of Amplification Reactions, in Diagnostic Molecular Microbiology: Principles and Applications, pp. 88-104, Edited by Persing et al . , Mayo Foundation, Rochester, MN (1993) , the contents of which are incorporated herein by reference.
  • oligonucleotides of the present invention may also be derivatized or labeled with a chemical moiety used for detection or isolation.
  • a chemical moiety used for detection or isolation.
  • moieties and methods for attachment or incorporation into oligonucleotides are well known in the art. Some examples include, members of specific binding pairs, described above .
  • the identified oligonucleotide sequences Bl through B8-2 are preferably used in the above described detection method of the present invention.
  • probe complex created using the indicated oligonucleotides are labeled with biotin, and streptavidin:biotin is the specific binding pair.
  • the above described method of isolation of target nucleic acid fragment is performed with a probe complex which contains one of the sequences Bl, B2 , or B4 , the target nucleic acid being specific for B . icroti .
  • all three probe complexes, made from either Bl, B2 , or B4 respectively, can be used together.
  • a mixture containing of probe complex made from Bl and probe complex made from B2 is used.
  • a mixture of probe complex made from Bl and probe complex made from B4 is used.
  • Target nucleic acid fragment isolated using any of these combinations of probe complexes can be specifically amplified by PCR or RT-PCR using the following oligonucleotide pairs, or the exact complements thereof, as primers: B3 and B5 ; B3 and B6; B3 and B8-1; B3 and B8-2; Bl and B5; Bl and B6 ; Bl and B8-1; Bl and B8-2.
  • these specific primer pairs used in standard amplification methods e.g. PCR or RT-PCR
  • to specifically amplify B . microti sequence from a heterogeneous mixture also provide superior results, compared to other primer pairs containing B . microti specific sequences.
  • the method of isolation of target nucleic acid fragment of the present invention is performed with a probe complex which contains one of the sequences B5, B6, B7, B8-1, or B8-2, or the exact complements thereof.
  • the probe complex is derivatized with biotin, and streptavidin: biotin is the specific binding pair used in the method.
  • BaJbesia specific nucleic acids are detected from a sample containing a heterogeneous mixture by hybridizing to oligonucleotides with the sequence of B5, B6, B7, B8-1 or B8-2, under conditions appropriate for hybridization to Babesia nucleic acids, but not for hybridization to non-Babesia nucleic acids. These conditions would be considered stringent conditions, a variety of which are known to those of skill in the art. Use of these oligonucleotide sequences or their exact complements will identify nucleic acids which are specific to the species listed in Table 4 in the following Exemplification section. Use of these oligonucleotides to detect these specific nucleic acid sequences provides an accurate assay for the presence of the corresponding pathogen, as discussed in the Exemplification section below.
  • Example 1 - Detection of Borrelia burcrdorferi Directly from Whole Blood 177 whole blood samples obtained from patients suspected of suffering from Lyme Borreliosis were tested for the presence of B. burgdorferi nucleic acids by a two step procedure. The procedure first isolates any specific sequences present and then detects the presence of those sequences via PCR. 50 ⁇ l samples of EDTA treated whole blood were first solubilized in a chaotropic salt solution. Following DNA denaturation, biotin derivatized selection probes specific for B . burgdorferi nucleic acid sequences were added to the sample and hybridized to any B . burgdorferi sequences present.
  • the selection probes with hybridized sequences were then isolated from the sample using streptavidin derivatized paramagnetic particles. The probe bound particles were then washed and the hybridized sequences eluted into deionized water (note: the probe was not eluted) . The isolated JS. burgdorferi sequences were then amplified by PCR.
  • Positive controls were produced by adding a different amounts B. burgdorferi organisms to whole blood, serum, or plasma samples obtained from a Borreliosis negative patient. 29/177 samples were determined to be positive for B . burgdorferi DNA by these methods (Table 1) . The detection limit was observed to be one B. burgdorferi organism per sample tested with the positive controls. 148/177 of these whole blood clinical samples which were negative by PCR had no detectable B . burgdorferi DNA present in the test samples. These results indicate that the sample processing protocol used can concentrate and purify DNA of interest from whole blood. The presence of hemoglobin may have inhibitory effects on PCR in whole blood samples. To determine if the sample processing method used for concentration of B . burgdorferi extensively purifies and removes PCR inhibitors, 15 whole blood samples, determined to be negative by the above methods, were tested for the presence of PCR inhibitors .
  • Urine Samples A total of 182 urine samples obtained from patients suspected of Borreliosis were processed as described above. The processed samples were PCR amplified with B . burgdorferi specific primers and the amplification products were analysed on a 2% gel, with the products of positive and negative controls. Seventeen samples were found positive. The other 165 urine samples were negative (Table 1) .
  • Clinial samples from patients preliminarily diagnosed with Lyme Borreliosis were processed in the following volumes: 50 ⁇ l EDTA treated whole blood, 100 ⁇ l serum and plasma, 3-6 ml urine, 200 ⁇ l cerebral fluid, 200 ⁇ l synovial fluid.
  • Sample plus probe was left at 65°C for another 5 minutes. The sample was then incubated at 37°C for more than three hours to allow hybridization of the probes to the complementary B . burgdorferi specific nucleic acids. The probe and any hybridized nucleic acids was then captured onto paramagnetic particles derivatized with streptavidin (10 ⁇ g/ml to 100 ⁇ g/ml) by diluting the GuSCN concentration to 1.25 M GuSCN.
  • the probe bound particles were washed once with GuSCN wash buffer (1.25 M GuSCN buffer, 0.1% BSA), followed by 2 washes with 0.1 x SSCN buffer (0.015 M NaCl, 0.0015 M Na Citrate, 0.1% BSA, 0.1% NP40, pH 7.4), and then once with 0.1 X SSC buffer (0.015 M NaCl, 0.0015 M Na Citrate, pH 7.4) .
  • the nucleic acids hybridized to the probe was released by the addition of 100 ⁇ l deionized water.
  • the released nucleic acids in 10 ⁇ l of the water were PCR amplified by the standard PCR procedure using the following 10 X PCR buffer.
  • the selection probes used were:
  • Plasmid Primers 5' -AAG-CAA-AAT-GTT-AGC-AGC-CTT-GA-3' SEQ ID NO: 6
  • Dilutions of the culture were made in PBS such that 5 ⁇ l contained either 1, 10, or 100 B . burgdorferi organisms. 5 ⁇ l volume of these dilutions were then added to the control samples. Positive and negative controls were otherwise processed identically to the other samples.
  • Probes used were the same as in Example 1. It should be noted that the probe concentration can vary between 10 ng/ml to 100 ng/ml, depending on the probe sequence, length and the specific DNA to be selected.
  • Example 4 Comparision of PCR Performed on Samples processed by (1) "New Sample Processing" Protocol and (2) Standard PCR for Borrelia burgdorferi .
  • a mini-study was conducted on 28 clinical specimens from patients with Borreliosis-like symptoms. This included 24 serum samples, 3 whole blood samples and one tick.
  • DNA from the clinical samples was prepared by two different methods, 1) a standard method using Qiagen DNA extraction protocol (Qiagen Inc, CA) , and 2) the sample processing protocol described above in Examples 1, 2 and 3. Purified DNAs were amplified by PCR and the amplification products compared to those of both positive and negative controls to detect the presence of B. burgdorferi nucleic acid sequences. The results are listed below in Table 2.
  • the two step isolation and PCR detection method of the present invention was compared to Fluorescence in si tu hybridization analysis (FISH) and indirect immunofluorescence analysis (IFA) in the detection of the pathogen B . microti .
  • FISH Fluorescence in si tu hybridization analysis
  • IFA indirect immunofluorescence analysis
  • a FISH assay which detects B . microti specific ribosomal RNA on a whole blood smear was used.
  • IFAs for detection of B . microti IgG and IgM antibodies were performed on serum, from all the patients according to the manufacturer's recommendations.
  • the isolation and PCR assay was used to detect B . microti specific DNA from whole blood, using B . microti specific primers. Tests were performed on a total of 221 whole blood samples from patients suspected of Babesiosis.
  • a FISH assay result was considered positive if either the ring form or the merozoite form or both forms were detected in the red cell .
  • Samples were considered positive by IFA if the B . microti antibody titers were greater than 1:64.
  • the FISH method of detection is producing false positive results. Since these samples were also determined negative by IFA, these 15 results may be false positives. Assuming that these 15 samples were "true negatives", the FISH assay still has a specificity of 93%.
  • the discrepancy between the FISH assay results and the PCR results is also not surprising.
  • the FISH assay is a non-amplified assay that detects B . microti rRNA within red blood cells
  • the PCR analysis is an amplified assay that detects minute quantities of parasite DNA, independent of the host immune response and the viability of the organism. Therefore, PCR analysis is more sensitive than FISH. This explains why 32 samples tested positive by PCR but tested negative by FISH, including the six samples positive by IFA and 26 samples negative by IFA. Based on the data presented above, 89 samples (40%) were considered true positives assuming a PCR assay specificity as 100% and FISH assay specificity as 93%.
  • the FISH assay monitors active infection, and has sensitivity of 67% as compared to the PCR assay. In addition, these results indicate that FISH assay is a more sensitive and specific than the IFA.
  • the smears were air dried and stored at room temperature until the day of the experiment.
  • the air dried smears were treated by a mixture of methanol : acetic acid (95:5) for 10 minutes at room temperature. Excess solution was removed, and the slides were air dried.
  • 25 ⁇ l of the hybridization fluid (50% formamide, 2 x SSC, pH 7.4, 1% NP40 and 1-2 ⁇ g/ml of the oligomer probes/primer B6, labeled with fluorescin at 3' and 5' ends) was applied per slide.
  • the slide was then covered with a cover-slip and incubated in a humid chamber at 42 °C for 30 minutes.
  • cover slips are removed and slides are washed three times individually with a wash buffer, pH 7.4, (0.3 M sodium chloride, 0.03 M sodium citrate, 0.5% NP40) at room temperature, for 2 minutes each.
  • a wash buffer pH 7.4, (0.3 M sodium chloride, 0.03 M sodium citrate, 0.5% NP40) at room temperature, for 2 minutes each.
  • slides were rinsed in phosphate buffered saline, pH 7.2 (PBS) for 2 minutes at room temperature.
  • PBS phosphate buffered saline
  • washed sides were placed in a Coplin jar containing PBS and Evans Blue (30 ml PBS + 3 drops of 0.1% Evans Blue in PBS) .
  • the slides were blotted dry and 2 drops of fixative added per slides and covered with a cover slip.
  • the slides were stored for about 10 minutes in darkness at room temperature and then viewed under dark field at 40x. FISH was considered positive if either the ring form or the merozoite form or both forms
  • microti IgG and IgM antibodies were performed on serum from all patients according to the manufacturer's recommendations (MRL Diagnostics, CA) .
  • B2 was used as selection probe (5'-Biotin-ATAGGTCAGAAACTTGAATGATACATCGCCGGC-3' (SEQ ID NO: 11)).
  • oligonucleotides were determined to detect specific pathogens when used as either selection probe or primer in the methods described above . These oligonucleotides were derived from a comparison of known nucleotide sequences specific for the pathogens of interest. The nine oligonucleotides listed below were determined to provide superior specificity in the detection of nucleic acids specific to a subset of pathogens through a process of trial and error. The complements of these oligonucleotides can also be used in place of each respective oligonucleotide.
  • Probe/primers B5, B6, B7 B8-1 and B8-2 were designed from known pathogen sequences (Genbank, Accession #BBONSSR M93660, Babesia microti ribosomal RNA small subunit gene sequence (1992); Genbank, Accession #BBU09833 U09833, Babesia microti 16S-like small subunit rRNA (1994) ; Herwaldt et al., J " . Infect Dis . 175 : 1259-1262 (1997)).
  • Table 4 lists the organisms which are specifically detected by each oligonucleotide, or its complement, when used in the above described methods .
  • At least two oligonucleotides listed as being inclusive of that organism must be used as primers in PCR or RT-PCR amplification of the target nucleic acid fragment.
  • oligonucleotides can be used in the identification of the pathogen of interest. These oligonucleotides were determined to be optimal PCR primers after extensive testing of many different oligonucleotides.
  • At least one of probe B5 , B6 , B7 or the complements thereof can be used as selection probe to detect B. microti in combination with the PCR primers Bl or B3.
  • At least one of probe B8-1 or B8-2 or the complements thereof can be used as selection probe to detect Babesia WA-1 in combination with the PCR primers Bl or B3.
  • Detection of B . microti 18S rDNA can be accomplished using either Bl, B2 or B4 as selection probe, followed by PCR amplification using B3 in combination with either B5 or B6 as primers. Alternatively, PCR amplification using Bl in combination with either B5, B6, B8-1 or B8-2 as primers, following selection with Bl, B2 , or B4 selection probe will also detect 18S rDNA. Detection of Babesia WA-1 18S rDNA can be accomplished using either Bl, B2 or B4 as selection probe, followed by PCR amplification using B3 in combination with either B8-1 or B8-2 as primers. Alternatively, PCR amplification using Bl in combination with either B8-1 or B8-2 as primers, following selection with Bl, B2 , or B4 selection probe will also detect 18S rDNA of some Theileria species.
  • PCR amplification using either Bl and B2 as primers, or B3 and B4 as primers will specifically amplify Babesia 18S rDNA.
  • RT-PCR amplification using these primer sets will specifically amplify B . microti 18s rRNA.
  • the B . microti specific products of these amplifications should hybridize to B5 , B6, B7.
  • the Babesia WA-1 specific products of these amplifications should hybridize to B8-1 or B8-2.
  • Either derivatized Bl, B2 or B4 can be used as selection probe to isolate Babesia nucleic acids by the methods described in Example 1.
  • B . microti specific PCR amplification of the purified rDNA target can then be accomplished using primer sets B3:B5, B3:B6, or the complements thereof, either alone or in combination.
  • RT-PCR performed using these selection probes and primer sets will identify rRNA fragments of Babesia.
  • Babesia WA-1 specific PCR amplification of the purified rDNA target can then be accomplished using primer sets B3:B8-1, B3:B8-2, B1:B5, B1:B6, B1:B8-1, Bl:B8-2, or the complements thereof, either alone or in combination.
  • RT-PCR performed using these selection probes and primer sets will identify rRNA fragments of Babesia WA-1 .
  • Table 4 Babesia Primer/Probes inclusivity and Exclusivity Based on Sequence Data
  • Probe/primer B6 is specific for B. microti and primer/probe B8-1 and B8-2 are specific for Babesia WA-1
  • test oligo Two test oligomers were synthesized, test oligo (1) has a sequence identical to B. microti rDNA at the 5' end and test oligo (2) has a sequence identical to Babesia WA-1 , rDNA at the 5' end. These two oligomers were mixed in a sample and then PCR amplified with either primer set B3:B6 or primer set B3:B8-1.
  • primer Set B3:B6 produced a DNA fragment which corresponded to the size of test oligo
  • Probe/Primer B3 can be used in the identification of either B. microti or Babesia-WA-1 , depending upon the other primer used in the amplification reaction, with B6 being specific for B. microti , and B8-1 being specific for Babesia-WA-1 .

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Abstract

L'invention concerne un procédé pour détecter un fragment d'acide nucléique cible dans un prélèvement clinique. On solubilise un échantillon du prélèvement et on le traite de manière à dénaturer les acides nucléiques qu'il contient. On met ensuite l'échantillon en contact avec au moins un complexe de sondes qui comprend une séquence complémentaire à une partie du fragment cible ainsi qu'un premier élément d'une paire de liaison spécifique. Après l'hybridation des sondes et de la cible, on met le complexe de sondes en contact avec un substrat solide lié au deuxième élément de la paire de liaison spécifique, et ce afin d'isoler le fragment de la cible hybridée dans un complexe ternaire sonde/cible/substrat solide. Le complexe ternaire isolé est séparé de l'échantillon, et le fragment cible avec le complexe de sondes sont libérés dans la solution. On amplifie le fragment de cible libéré par la technique PCR ou celle PCR à transcription inverse (RT-PCR), et l'on détecte la présence du fragment cible dans le prélèvement clinique. Le procédé peut être utilisé avec divers types de prélèvements et se montre utile dans la détection de pathogènes tels que Borrelia burgdorferi et des espèces de Babesia. L'invention concerne aussi des solutions de sels chaeotropiques spécifiques, des tampons de lavage et des conditions pour l'amplification ainsi que des complexes de sondes et des amorces servant à la détection des pathogènes tels que Borrelia et Babesia.
PCT/US1999/010939 1998-05-21 1999-05-18 Procedes ameliores pour detecter un fragment d'acide nucleique cible Ceased WO1999060009A1 (fr)

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US20120015360A1 (en) * 2009-12-07 2012-01-19 Ibis Biosciences, Inc. Compositions for use in identification of babesia bioagents
EP2534259A1 (fr) * 2010-02-12 2012-12-19 Vereniging voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek en Patiëntenzorg Procédé pour la surveillance combinée de la détection d'au moins deux cibles moléculaires et trousse pour la mise en uvre de ce procédé
CN108486224A (zh) * 2018-06-04 2018-09-04 华中农业大学 一种田鼠巴贝斯虫rpa分子检测方法
WO2018226798A1 (fr) * 2017-06-07 2018-12-13 Gen-Probe Incorporated Détection d'acide nucléique d'espèce babesia dans un échantillon

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120015360A1 (en) * 2009-12-07 2012-01-19 Ibis Biosciences, Inc. Compositions for use in identification of babesia bioagents
EP2534259A1 (fr) * 2010-02-12 2012-12-19 Vereniging voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek en Patiëntenzorg Procédé pour la surveillance combinée de la détection d'au moins deux cibles moléculaires et trousse pour la mise en uvre de ce procédé
WO2018226798A1 (fr) * 2017-06-07 2018-12-13 Gen-Probe Incorporated Détection d'acide nucléique d'espèce babesia dans un échantillon
US11667978B2 (en) 2017-06-07 2023-06-06 Gen-Probe Incorporated Detecting Babesia species nucleic acid in a sample
EP4276201A3 (fr) * 2017-06-07 2024-01-17 Gen-Probe Incorporated Détection d'acide nucléique d'espèce babesia dans un échantillon
US12404559B2 (en) 2017-06-07 2025-09-02 Gen-Probe Incorporated Detecting Babesia species nucleic acid in a sample
CN108486224A (zh) * 2018-06-04 2018-09-04 华中农业大学 一种田鼠巴贝斯虫rpa分子检测方法

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