WO2012159060A2 - Methods, systems, and compositions for detection of microbial dna by pcr - Google Patents
Methods, systems, and compositions for detection of microbial dna by pcr Download PDFInfo
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- WO2012159060A2 WO2012159060A2 PCT/US2012/038649 US2012038649W WO2012159060A2 WO 2012159060 A2 WO2012159060 A2 WO 2012159060A2 US 2012038649 W US2012038649 W US 2012038649W WO 2012159060 A2 WO2012159060 A2 WO 2012159060A2
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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- 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
Definitions
- the invention pertains to the field of molecular assays. More particularly, the invention pertains to a PCR-based method for detecting minute amount of microbial DNA and compositions for performing the same.
- PCR polymerase chain reaction
- PCR reagents for performing PCR are obtained from bacterial sources.
- the reagents, in particular, the Taq DNA polymerases unavoidably contain some level of DNA contamination from the source bacteria.
- the contaminating DNA usually include more than one strain or species that cannot be identified as Thermus aquaticus or Escherichia coli, yet, they bear close homology to the species of Pseudomonas fluorescens, Pseduomonas aeruginosa, Alcaligenes faecalis, or Azotobacter vinelandii, all of which are clinically important.
- conventional PCR often co-amplifies these contaminants with the target microbial DNA, generating an exceedingly high rate of false positives, thereby, rendering PCR assays so unreliable that they are precluded from clinical applications.
- Some examples include UV irradiation, restriction endonuclease digestion, ultrafiltration, and pretreatment of reagents with DNase I (ref 20 - 23).
- DNase I DNase I
- PCR-based diagnostic assay to detect microbes in clinical specimens and other biological samples.
- PE-PCR a novel strategy, herein referred to as PE-PCR, that overcomes the long-standing problem of endogenous contamination in PCR reagents.
- the PE-PCR strategy recognizes that all previous efforts to solve the endogenous contamination problem had approached the problem heads-on by attempting to remove or destroy the contaminants. For nearly 20 years, such head-on approaches had yielded very little success.
- the inventors dispensed with conventional thinking and devised an indirect approach that ingeniously side-steps the problem of endogenous contamination.
- the PE-PCR strategy uses a DNA fusion probe to add a non-bacterial sequence to the 5 '-end of the target templates before any PCR reagent is added to the reaction mix. This way, the target templates are distinguished from the contaminating sequences by their 5 '-end non-bacterial sequences.
- the tagged templates can be selectively amplified by standard PCR. Because the non-bacterial fusion primer (a.k.a. fusion probe) will only amplify the 5 '-end tagged templates, this renders the bacterial-derived contaminants a non-issue.
- the non-bacterial fusion primer a.k.a. fusion probe
- a first aspect of the present invention is directed to a method for selectively amplifying one or more target microbial DNA in a sample.
- Methods in accordance with this aspect of the present invention will make use of one or more DNA fusion probe(s) consisting of a 5 '-end portion having a non-bacterial sequence and a 3'- end portion having a sequence complementary to a portion of the target microbial DNA.
- the methods will generally include the steps of hybridizing the fusion probe(s) to the target microbial DNA in the sample; removing the non-hybridized fusion probe(s) and unbound 3 '-end portion of the target microbial DNA; extending the 3 '-ends of the fusion probes and the target microbial DNA to form double stranded primer-extended templates; and performing a PCR method to selectively amplify the primer-extended templates with a primer set that includes at least one primer having a non-bacterial sequence complementary to the non-bacterial sequence of the fusion probes.
- a second aspect of the present invention is directed to a method for detecting microbial infection in a subject.
- Methods in accordance with this aspect of the invention will also require a plurality of DNA probes as described above. They will generally include the steps of adding the fusion probes to a sample taken from the subject; hybridizing the fusion probes to microbial DNA in the sample; removing non- hybridized fusion probes and any unbound 3 '-end portion of the microbial DNA; extending the 3 '-ends of the fusion probes and the microbial DNA to form double stranded primer-extended templates; amplifying the primer-extended templates by performing a PCR method with a primer set that includes at least one forward primer having a non-bacterial sequence complementary to the non-bacterial sequence of the fusion probe; and analyzing the amplified PCR products to determine the presence or absence of a microbe.
- a third aspect of the present invention is directed to a fusion probe for generating a primer-extended DNA template from a target microbial DNA in a sample for selective amplification by a PCR method.
- Fusion probes in accordance with this aspect of the invention generally consist of a 5 '-end portion having a non-bacterial sequence and a 3 '-end portion having a sequence complementary to a portion of the target microbial sample.
- a fourth aspect of the present invention is directed to a kit for generating primer- extended DNA template from a target microbial DNA for selective PCR amplification and detection.
- Kits in accordance with this aspect of the present invention will generally include a plurality of fusion probes as described above; and a mixture of 3 ' to 5 ' exonuclease and Klenow polymerase packaged in an easy to use format.
- a fifth aspect of the present invention is directed to a system for detecting a target microbe in a sample.
- Systems in accordance with this aspect of the invention generally include a sample receiving unit for receiving the sample; a sample processing unit for adding reagents to the sample and maintaining reaction conditions; and a sample analyzing unit for analyzing the processed sample.
- the sample processing unit is preferably automated and configured to perform the steps of adding a plurality of fusion probes to the sample and maintaining a condition to allow the fusion probes to hybridize with microbial DNA in the sample; adding a degradation/extension reagent to remove non-hybridized fusion probes and unbound 3 '-end portion of the hybridized target microbial DNA, and to extend the 3 '-ends of the hybridized fusion probes and the target microbial DNA in the 3' to 5' direction so as to generate double stranded primer- extended templates; and adding PCR reagents and maintain reaction conditions to perform amplification of the primer-extended templates; and forwarding the processed sample to the analyzing unit for analysis.
- Figure 1 shows a schematics view of a PE-PCR method in accordance with embodiments of the present invention.
- Figure 2 shows a gel electrophoresis image illustrating that commercially available polymerases are not sufficiently pure for sensitive and specific broad-range amplification of bacterial DNA.
- FIG. 3 shows gel electrophoresis images demonstrating that PE-PCR methods in accordance with embodiments of the present invention are capable of specifically amplifying target bacterial DNA without co-amplifying the contaminating bacterial DNA.
- A The indicated amount of S. aureus genomic DNA was subjected to PE-PCR using the fusion probe M13-TstaG422 and the primer set M13 and TstaG765.
- B The S. aureus genomic DNA (100 fg) was subjected to PE-PCR (upper panel) as described in panel A (lane 1), in the absence of Ml 3 primer (lane 2), and in the artificially contaminating condition by adding 100 fg S.
- aureus genomic DNA into the EK mix (lane 3) or PCR mixtures (lane 4). PE-PCR was also performed in the absence of template DNA (lane 5). The presence of S. aureus genomic DNA was confirmed by species-specific PCR that amplified a chromosomal DNA fragment specific for S. aureus (lower panel).
- C The indicated amount of S. aureus genomic DNA was used as the template for broad-range PE-PCR using the fusion probe M13-16S-p201F and the primer set Ml 3 and pi 370.
- D The S. aureus genomic DNA (100 fg) was subject to broad-range PE-PCR (upper panel) as described in panel C (lane 1), and in the artificially contaminating condition by adding 100 fg of S.
- aureus genomic DNA into the EK mix (lane 2) or PCR mixtures (lane 3). Broad-range PE-PCR was also performed in the absence of template DNA (lane 4). The presence of S. aureus genomic DNA was confirmed by species-specific PCR to amplify a chromosomal DNA fragment of S. aureus (lower panel). NTC stands for no template control.
- Figure 4 shows comparison of broad-range real-time PE-PCR and broad-range real-time PCR amplification of template bacterial DNA after pretreatment of PCR reagents with DNase I.
- A The indicated amounts of S. aureus genomic DNA were subject to broad-range real-time PE-PCR using the fusion probe M13-16S-p201F and the primer set Ml 3 and pi 370 in the presence of LCGreen I plus HRM dye.
- C Alternatively, the PCR reaction mixtures with or without pretreatment of DNase I (1 U and 2.5 U) were used for real-time PCR to amplify the indicated amounts of S. aureus genomic DNA.
- the PCR products were subject to HRMA using HR-1 instrument. The amplification (all figures on the left column) and derivative plots (all figures on the right column) were shown. NTC stands for no template control.
- Figure 5 shows the melting curve plots for broad-range real-time PE-PCR
- HRMA for 12 different bacterial species.
- the indicated amounts of genomic DNA from 12 different bacterial species were subject to broad-range real-time PE-PCR using the fusion probe M13-16S-p201F and the primer set M13 and pl370 in the presence of LCGreen I plus.
- the PCR product was subject to HRMA using HR-1 instrument and the derivative plots are shown.
- NTC stands for no template control.
- Figure 6 shows a schematics view of a system in accordance with embodiments of the present invention.
- the term "gene” refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
- the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained.
- the term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences.
- the term "gene” encompasses both cDNA and genomic forms of a gene.
- a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
- Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
- mRNA messenger RNA
- Probes and/or primers can be RNA or DNA.
- DNA can be either cDNA or genomic DNA.
- Polynucleotide probes and primers are single or double- stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements.
- Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
- PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is targeted for analysis.
- a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescence, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, Oreg., and Amersham Corp., Arlington Heights, 111., using techniques that are well known in the art.
- DNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage.
- an end of an oligonucleotide or polynucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
- a nucleic acid sequence even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends.
- discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand.
- the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence “A-G-T,” is complementary to the sequence “T-C- A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
- hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T.sub.m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized.”
- Amplification is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.
- PCR polymerase chain reaction
- the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
- the primers are extended with a polymerase so as to form a new pair of complementary strands.
- the steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
- the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
- PCR polymerase chain reaction
- PCR assay is monitored during the reaction as an indicator of amplicon production during each PCR amplification cycle (i.e., in "real time"), as opposed to conventional
- Real time PCR is generally based on the detection and quantitation of a fluorescent reporter.
- the signal increases in direct proportion to the amount of PCR product in a reaction.
- By recording the amount of fluorescence emission at each cycle it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template.
- real time PCR see Dehee et al. J. Virol. Meth. 102:37-51 (2002); and Aldea et al. J. Clin. Microbiol. 40: 1060-1062 (2002) (referring to the "LightCycler," where real-time, kinetic quantification allows measurements to be made during the log-linear phase of a PCR).
- PCR With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of .sup.32P -labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
- any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules.
- the amplified segments created by the PCR process are, themselves, efficient templates for subsequent PCR amplifications.
- PCR product refers to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
- sample is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention. General Description of the PE-PCR strategy
- Figure 1 illustrates schematically how an exemplary PE-PCR experiment works.
- a sample analyte containing target DNA templates such as the genomic DNA of a microbe is mixed with an excess amount of fusion probes.
- Each fusion probe consist of a 5 '-end portion having a non-bacterial sequence followed by a 3 '-end portion having a sequence complementary to a site on the target DNA template.
- step lb the sample mixture is heated to denature the DNA templates so that the fusion probes may be hybridized to the target DNA templates (step 2a).
- step 2b a mixture of 3'- end exonuclease and Klenow DNA polymerase (the EK mix) is added to the sample (step 2b).
- the EK mix 3'- end exonuclease and Klenow DNA polymerase
- step 3a the non-bonded portion of the target DNA's 3 '-end (the frayed end) along with the unbound excess fusion probes are digested away by the 3 '-end exonuclease. After exonuclease digestion, a 5' overhang on the fusion probe strand is created.
- step 3b the Klenow DNA polymerase will then extend both the fusion probe strand and the template strand in the 5 '- 3' direction to form blunt ended double strand templates.
- the strand that extended from the fusion probe will have the non-bacterial tagging sequence located at its 5 '-end whereas the strand that extended from the target DNA template will have the non-bacterial tagging sequence located at its 3 '-end.
- This primer extension step can be terminated by heat inactivation of the EK mix.
- the templates have been tagged with distinguishing sequences and ready to be amplified by PCR.
- the primer set used to run the PCR in step 4 must include at least one forward primer (designated as non-bac-F in Figure 1) that is complementary to the non-bacterial tagging sequence and a reverse primer (designated as bac-R in Figure 1) that is complementary to a sequence on the target DNA template downstream of the fusion probe.
- the PE-PCR strategy outlined above is simple and can be performed in a single reaction vessel without requiring any other pretreatments of the reagents. It provides a turnkey solution that solves the long-standing problem of endogenous bacterial DNA contamination and is compatible with existing PCR protocols. Based on this strategy, the inventors have devised various methods, systems, compositions, and kits. Those skilled in the art will appreciate that the PE-PCR strategy may be advantageously used in conjunction with any analytical procedures to further quantify or characterize the PCR products. Some exemplary applications of this strategy may include molecular diagnostics of clinical specimens for early detection of bacterial, viral, or fungal infection. Other applications may include forensic applications, bioweapon detection, or archeo logical sample quantification, but not limited thereto.
- a fusion probe according to embodiments of the present invention is preferably a forward primer. It will have two components, one encoding a non-bacterial sequence, the other encoding a sequence complementary to a portion of the target DNA.
- the non-bacterial sequence serves as a tagging sequence to distinguish from endogenous bacterial contaminants in PCR reagents.
- the complementary sequence serves as a targeting sequence that bonds the fusion probe to the target DNA under hybridization conditions.
- the fusion probe consists of non-bacterial DNA sequence at the 5 '-end and the sequences complementary to the microbial target sequence. Due to the possible presence of human genomic DNA during PCR amplification, the 5 '-end non-bacterial DNA sequence preferentially does not complementary to the human genomic DNA sequence. Similar strategy applies to the applications related to bacterial DNA detection in the samples from other species.
- the 3 '-end bacterial DNA sequences are complementary to the target gene sequences that universally present in the microbial DNA.
- the Tm of the 3'- bacteiral sequence is at least 37 °C and at most 95 °C.
- General rule of primer design such as non hairpin sequence is applied for sequence selection.
- the tagging sequence may be a sequence derived from a non-bacterial source, or it may be an artificially designed sequence.
- the tagging sequence is a viral sequence, preferably one derived from an Ml 3 phage.
- the targeting sequence can be a unique sequence that binds the fusion probe to a specific target template or a conversed sequence that binds the fusion probe to a broad- range of microbial DNA.
- the targeting sequence is preferably a conserved sequence from the 16S rRNA gene.
- the fusion probes may be manufactured by any methods commonly known in the art, including, but not limited to chemical synthesis.
- Microbial DNA that exist in low abundance in a sample are difficult to detect because the limitations of detection methods and instruments.
- Using standard PCR to detect low abundance DNA in a sample generally involves selectively amplifying target DNA templates against a sea of noisy background. By replicating the target DNA templates to a large enough quantity, they can then be easily detected. Fusion probes as described above provides a way to effectively distinguish the target DNA from the background noise and undesirable contaminations, thereby, allowing the target DNA to be selectively amplified. Once amplified, a number of analytical tools may then be applied to characterize the amplified nucleic acid products.
- methods for detecting microbial infection in a subject will generally include the steps of adding the fusion probes to a sample taken from the subject; hybridizing the fusion probes to microbial DNA in the sample; removing non-hybridized fusion probes and any unbound 3 '-end portion of the microbial DNA; extending the 3 '-ends of the fusion probes and the microbial DNA to form double stranded primer-extended templates; amplifying the primer-extended templates by performing a PCR method with a primer set that includes at least one forward primer having a non-bacterial sequence complementary to the nonbacterial sequence of the fusion probe; and analyzing the amplified PCR products to determine the presence or absence of a microbe.
- the sample is taken from the subject's bodily flood, such as blood, urine, cerebral spinal fluid, saliva, sputum, or the like.
- the type of sample taken from the subject is not particularly limited, but will generally correspond to the type of clinical condition the subject is suspected of suffering.
- the sample is a blood sample from a subject suspected of suffering from bacteremia.
- the sample is a cerebral spinal fluid from a subject suspected of suffering from a bacterial infection that is difficult to culture in vitro (e.g. Chlamydia pneumoniae).
- Preparation of the sample are typically needed to release the DNA from the microbes.
- Methods for releasing/extracting microbial DNA are generally known in the art and can be advantageously used to prepare the samples. However, such preparatory steps are not always necessary, depending on the application and specific objectives of the assay.
- Hybridization of the fusion probes to the target DNA is generally achieved by heating the sample to above melting temperature (T m ) for the target DNA first so that they denature into single stranded DNA and then gradually cool to below Tm to allow binding and annealing to occur.
- T m melting temperature
- Suitable hybridization conditions are also known in the art and can be routinely determined by those skilled in the art.
- Removal of excess fusion probes and 3 '-end unbound portion of the target DNA can be achieved by a 3' exonuclease.
- the 3' exonuclease is E. coli exo I.
- Other 3' exonuclease may also be suitably used.
- Extension of the templates can be achieved by the use of 5' to 3' polymerases.
- a 5 ' to 3 ' Klenow polymerase is used.
- the 3' exonuclease and the 3' to 5' polymerase can be provided in a single mixture (the EK mix) for convenience.
- the optimal ratio of exonuclease and polymerase will depend on the design of the fusion probes as well as the particular exonuclease and polymerase used. Such ratios can be determined by routine optimization experiments and should preferably be done prior to deploying the detection method in clinical settings.
- a quenching agent can be added or the sample can simply be heated to inactivate the enzymes.
- PCR methods are preferably standard PCR using commercially available Taq
- DNA polymerase and a set of primers that include at least one forward primer having a complementary sequence to the tagging sequence of the fusion probe may also be suitably used so long as the protocol is compatible with the primer-extended templates. Methods for performing these conventional PCR are generally known in the art and can be adapted to work with the PE-PCR strategy of the present invention by those skilled in the art. For example, real-time PCR may also be used to simultaneously amplify the primer-extended templates and quantify the abundance of the microbial DNA.
- the amplified PCR products can be analyzed by any number of convention analytical methods known in the art to characterize and identify their origin.
- the melting curve analysis may be applied to the amplified PCR products to generate a melting curve profile.
- DNA from different microbes will generally have a unique melting curve profile because of their divergent nucleotide content.
- These melting curve profiles can serve as a sort of "fingerprint" for identifying the origin a sample.
- Other analytical methods include, but not limited to sequencing, microarray assay, mass spectroscopy, melting curve including high-resolution melting analysis, denatured HPLC, capillary electrophoresis, agarose and/or polyacrylamide gel electrophoresis, heteroduplex mobility assay (HMA) and NMR spectroscopy.
- kits of the present invention will include a plurality of pre -made fusion probes and degradation/extension reagents (e.g. EK mix) in a convenient package, such as in a vial or a cartridge, but are not limited thereto.
- the kit may further include an instruction manual for directing its use or additional reagents for facilitating PCR amplifications.
- FIG. 6 shows a schematics diagram of an exemplary system of the present invention.
- a system in accordance with embodiments of the present invention has a sample receiving unit 1 for receiving a sample to be analyzed, a processing unit 2 configured to add various reagents to the sample and maintain appropriate reaction conditions.
- microbial DNA may be extracted from sample reservoir 3 into reaction chambers 4 and 5 whereto the fusion probes and EK mix may be added so as to form primer-extended templates.
- the primer-extended templates may then be amplified by PCR in PCR chamber 6.
- the PCR reaction chamber may then be forwarded to an sample analyzing unit 7.
- the processing unit may be configured in a number of different ways. Exemplary means of implementing the processing unit may be a series of microcomputer controlled actuators, or a microfluidic device.
- the analyzing unit may also be implemented in any number of ways depending on the particular mode of analysis to be applied. For example, it may be a mass spectrometer, a DNA sequencing device, a capillary electrophoretic device, or a melting temperature analysis apparatus.
- systems of the present invention are adapted from existing automated PCR systems such as the BD MaxTM system from Becton Dickinson (Franklin Lakes, NJ).
- Non-automated systems may also be assembled using off-shelf components.
- Sample processing may be performed manually or in a separate sample processing/preparation unit.
- PCR reaction may be performed using a standard thermocycler such as the LightCycler ® 480 system by Roche Diagnostics Corporation (Indianapolis, IN).
- Species identification analysis may be performed by high-resolution melting curve analysis using the LightScanner ® 32 system (Idaho Technologies, Salt Lake City, Utah).
- the primer extension product was then subjected to PCR amplification using Ml 3 and the downstream primer TstaG765 corresponding to the Tuf genomic sequences.
- a 391 -bp single PCR product was obtained with 50 fg of bacterial DNA, equivalent to 10 copies of S. aureus genome in the sample.
- no PCR product was observed in the NTC control ( Figure 3A). This result indicates that our PE-PCR is accurate and sensitive down to the range of fentograms.
- the LCGreen I plus reagent set and HR-1 instrument were purchased from Idaho Technology (Salt Lake City, UT).
- the HotStart Taq DNA polymerase was purchased from Protech (Taipei, Taiwan).
- the Fast Hot Start Taq DNA polymerase was purchased from KAPA Biosystems (Woburn, MA).
- the "low- DNA” Taq DNA polymerase was purchased from Takara (Shiga, Japan).
- the ULTRATOOLS Taq DNA polymerase was purchased from Biotools Inc. (Madrid, Spain).
- the LightCycler capillaries were purchased from Roche Applied Science (Indianapolis, IN).
- the DNase I was purchased from Promega (Madison, WI). Bacterial strains were clinical isolates as described previously (ref 11). A complete list of primer and fusion probe sequences is shown in Table 1.
- Isolation of bacterial genomic DNA [0080] The overnight culture bacterial suspension (4 ml) was centrifuged at 10,000 rpm for 10 min and the pellet was resuspended in 4 ml of solution I buffer (25 mM Tris-HCl, pH 7.5, 50 mM glucose, 10 mM EDTA, and 40 ⁇ g/ml lysostaphin). The bacterial suspension was incubated at 37°C for 2 h and the reaction buffer containing 280 ⁇ of 20% SDS and 40 ⁇ of proteinase K (10 mg/ml) and RNase A (10 mg/ml) was added to the bacterial suspension and incubated at 55°C overnight.
- I buffer 25 mM Tris-HCl, pH 7.5, 50 mM glucose, 10 mM EDTA, and 40 ⁇ g/ml lysostaphin.
- the bacterial suspension was incubated at 37°C for 2 h and the reaction buffer containing 280 ⁇ of 20% SDS and 40
- the annealing step consisted of a 20 ⁇ annealing mix containing 8 ⁇ of H 2 0, 5 ⁇ of fusion probe (2 ng/ ⁇ ), 5 ⁇ of bacterial genomic DNA at the indicated concentration, and 2 ⁇ of 10X PCR buffer. The reaction mixture was heated to 95°C for 5 min and was kept at 37°C.
- EK mix consisting of 3 ⁇ of H 2 0, 1 ⁇ of 10X PCR buffer, 5 ⁇ of dNTP (2 mM), 1 ⁇ of Klenow DNA polymerase (5 U/ ⁇ ), and 1 ⁇ of exo I (20 U/ ⁇ ) was added to the annealing mix and incubated at 37°C for 2 h. After heat inactivation at 80°C for 20 min, the reaction mixture was brought up to 50 ⁇ by adding 14 ⁇ of H 2 0, 2 ⁇ of 10X PCR buffer, 1 ⁇ of forward primer Ml 3 (5 ⁇ ), 1 ⁇ of reverse primer (5 ⁇ ), and 1 ⁇ of HotStart Taq DNA polymerase (5 U/ ⁇ ).
- the PCR cycling condition was 1 cycle of 95°C for 10 min, 45 cycles of 95°C for 15 s, and 60°C for 1 min.
- the Ml 3 and the reverse primer was replaced by the primer set of SA-F and SA-R (Table 1) that specifically amplifies S. aureus genomic DNA fragment (ref 43).
- the reaction was proportionally scaled down to 8 ⁇ during binding of fusion probe and primer extension. Then the reaction mixture was brought up to 20 ⁇ by adding 1.5 ⁇ of H 2 0, 2 ⁇ of 10X bovine serum albumin (BSA), 2 ⁇ of 10X LCGreen I plus, 0.8 ⁇ of 10X PCR buffer, 0.4 ⁇ of forward primer M13 (5 ⁇ ), 0.4 ⁇ of reverse primer (5 ⁇ ), and 0.5 ⁇ of HotStart Taq DNA polymerase (5 U/ ⁇ ) and was transferred to the capillary tube.
- Real-time PCR was performed using LightCycler 1.5 instrument and the cycling condition was 1 cycle of 95°C for 10 min, 45 cycles of 95°C for 15 s, and 60°C for 1 min at a transition rate of 20°C/s.
- PE-PCR may be used as the basis for a clinical diagnostic assay to test for bacteremia or sepsis.
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RU2013153505A RU2620953C2 (en) | 2011-05-19 | 2012-05-18 | Methods, systems and compositions for microbial dna detection by pcr |
AU2012255042A AU2012255042B2 (en) | 2011-05-19 | 2012-05-18 | Methods, systems, and compositions for detection of microbial DNA by PCR |
CA2842659A CA2842659A1 (en) | 2011-05-19 | 2012-05-18 | Methods, systems, and compositions for detection of microbial dna by pcr |
BR112013029601A BR112013029601A2 (en) | 2011-05-19 | 2012-05-18 | method for selectively amplifying one or more target bacterial DNAs in a sample, method for detecting a bacterial infection in an individual, fusion probe, kit for generating a DNA model, and system for detecting target bacteria in a sample. |
KR1020137033578A KR20140071968A (en) | 2011-05-19 | 2012-05-18 | Methods, systems, and compositions for detection of microbial dna by pcr |
CN201280031294.3A CN103917660B (en) | 2011-05-19 | 2012-05-18 | Method, system and the composition of microbial DNA are detected by PCR |
EP12786756.2A EP2710155A4 (en) | 2011-05-19 | 2012-05-18 | Methods, systems, and compositions for detection of microbial dna by pcr |
JP2014511592A JP6112623B2 (en) | 2011-05-19 | 2012-05-18 | Methods, systems, and compositions for detection of microbial DNA by PCR |
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RU2728317C1 (en) * | 2019-08-08 | 2020-07-29 | Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) | Method for eliminating false-positive results caused by contamination of dna polymerase with foreign dna fragments, with identification of blatem genes |
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CN105316398A (en) * | 2014-07-30 | 2016-02-10 | 益善生物技术股份有限公司 | Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit |
RU2630673C1 (en) * | 2016-12-19 | 2017-09-11 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) | Method for determination of biomaterials bacterial contamination |
KR102032647B1 (en) | 2018-04-03 | 2019-10-15 | 한양대학교 에리카산학협력단 | Structure for detection of microorganism, manufacturing method thereof, and method for detecting microorganism using the structure |
EP3802867A1 (en) * | 2018-06-01 | 2021-04-14 | Agena Bioscience, Inc. | Products and processes for nucleic acid detection and quantification |
US20210355527A1 (en) * | 2018-09-27 | 2021-11-18 | Cortexyme, Inc. | Methods for detection of microbial nucleic acids in body fluids |
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RU2182176C2 (en) * | 1991-09-24 | 2002-05-10 | Кейгене Н.В. | Method of selective amplification, oligonucleotide and set for selective amplification |
IE20020544A1 (en) * | 2002-06-28 | 2003-12-31 | Univ College Cork Nat Univ Ie | Method for the characterisation of nucleic acid molecules |
JP2008527979A (en) * | 2005-01-12 | 2008-07-31 | アプレラ コーポレイション | Compositions, methods and kits for selective amplification of nucleic acids |
CA2659543C (en) * | 2006-06-06 | 2015-12-29 | Gen-Probe Incorporated | Tagged oligonucleotides and their use in nucleic acid amplification methods |
US7833716B2 (en) * | 2006-06-06 | 2010-11-16 | Gen-Probe Incorporated | Tagged oligonucleotides and their use in nucleic acid amplification methods |
US8603749B2 (en) * | 2006-11-15 | 2013-12-10 | Biospherex, LLC | Multitag sequencing ecogenomics analysis-US |
US20090011417A1 (en) * | 2007-03-07 | 2009-01-08 | George Maltezos | Testing Device |
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US20100323348A1 (en) * | 2009-01-31 | 2010-12-23 | The Regents Of The University Of Colorado, A Body Corporate | Methods and Compositions for Using Error-Detecting and/or Error-Correcting Barcodes in Nucleic Acid Amplification Process |
DE102009012039A1 (en) * | 2009-03-10 | 2010-09-16 | Qiagen Gmbh | Quantification of nucleic acids |
AU2010232439C1 (en) * | 2009-04-02 | 2017-07-13 | Fluidigm Corporation | Multi-primer amplification method for barcoding of target nucleic acids |
WO2011085075A2 (en) * | 2010-01-07 | 2011-07-14 | Gen9, Inc. | Assembly of high fidelity polynucleotides |
US8828688B2 (en) * | 2010-05-27 | 2014-09-09 | Affymetrix, Inc. | Multiplex amplification methods |
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US20120295806A1 (en) | 2012-11-22 |
JP6112623B2 (en) | 2017-04-12 |
KR20140071968A (en) | 2014-06-12 |
AU2012255042A1 (en) | 2014-01-09 |
BR112013029601A2 (en) | 2017-06-13 |
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RU2620953C2 (en) | 2017-05-30 |
RU2013153505A (en) | 2015-06-27 |
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CN103917660A (en) | 2014-07-09 |
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