EP1261716A2 - Procede d'identification rapide et precise de micro-organismes - Google Patents

Procede d'identification rapide et precise de micro-organismes

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Publication number
EP1261716A2
EP1261716A2 EP00980462A EP00980462A EP1261716A2 EP 1261716 A2 EP1261716 A2 EP 1261716A2 EP 00980462 A EP00980462 A EP 00980462A EP 00980462 A EP00980462 A EP 00980462A EP 1261716 A2 EP1261716 A2 EP 1261716A2
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EP
European Patent Office
Prior art keywords
primer
target sequence
sequence
oligonucleotide
organism
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.)
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EP00980462A
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German (de)
English (en)
Inventor
Zhiping Liu
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Apollo Biotechnology Inc
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Apollo Biotechnology Inc
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Publication date
Application filed by Apollo Biotechnology Inc filed Critical Apollo Biotechnology Inc
Publication of EP1261716A2 publication Critical patent/EP1261716A2/fr
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • Infectious diseases represent an increasingly serious public health concern. Since multiple infectious agents can cause the same or similar symptoms, the identification of the pathogen is crucial for the correct diagnosis and proper treatment of the illness.
  • the etiologic agents for pneumonia and meningitis to name just two serious diseases, include more than a dozen different bacteria and several viruses and fungi.
  • Most of the current diagnostic procedures involve culturing the bacteria for identification, a process that usually requires several days and often gives negative results. Culturing is not only a lengthy process, but certain pathogens (i.e., mycoplasma, mycobacteria, and viruses) are notoriously difficult to grow outside the host.
  • etiologic agents for pneumonia which includes: the classic pathogens Streptococcus pneumoniae, Enter obacteriaceae, Staphylococcus aureus, Chlamydia pneumoniae, Escherichia coli, Legionella pneumophila, and Pseudomonas aeruginosa; the atypical agents Mycoplasma pneumonia, Mycobacteria, and Pneumocystis carinii (predominantly in immuno-compromised patients); and a variety of viruses and fungi (Kayser 1992; Tan, 1999).
  • bacterial meningitis major etiologic agents include: Neisseris meningitidis, Haemophilus influenza, and Streptococcus pneumoniae (Tunkel and Scheld, 1993). Since the proper medical treatment for these infections varies substantially depending on the agent, it is important to rapidly and accurately identify the pathogen.
  • DNA hybridization probes and PCR offer considerable promise for the development of microbial diagnostics (Abele-Horn et al, 1998; Ramirez et al, 1996).
  • the present invention takes advantage of the fact that certain coding sequences are highly conserved in a number of organisms (e.g., eubacteria). By properly choosing PCR primers from among these conserved sequences, one set of PCR primers (or a set of degenerate primers) can be used for the amplification of an unknown DNA sample (with several possible and different genomic origins) for the purpose of revealing its identity.
  • an additional set of primers can be designed based on the same principle for nested-PCR (i.e., a second set of primers within the bounds of the first set of primers).
  • hybridization probes will be chosen from the less conserved sequences (horizontally in evolution) flanked by the PCR primers.
  • the same principles can be applied for identifying any number of microorganisms including, for example, viruses and eukaryotic cells, such as fungi.
  • the invention provides a method of identifying an organism among a population of organisms in a biological sample, the method comprising obtaining genetic material from the sample; contacting the genetic material with at least a first primer and at least a related second primer corresponding to a pair of conserved regions in the genome of the population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences and is characteristic of the organism; amplifying the target sequence; contacting a solid support comprising a probe substantially complementary to the target sequence with the amplified target sequence; and detecting hybridization of the target sequence to the probe, wherein hybridization is indicative of the presence of the organism in the sample.
  • the invention provides a method of diagnosing a disease or disorder associated with an organism, comprising obtaining genetic material from a sample; contacting the genetic material with at least a first primer and at least a related second primer corresponding to a pair of conserved regions in the genome of a population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences and is characteristic of the organism; amplifying the target sequence; contacting a solid support comprising a probe substantially complementary to the target sequence with the amplified target sequence; and detecting hybridization of the target sequence to the probe, wherein hybridization is indicative of the presence of the organism in the sample and correlating the organism to the disease or disorder.
  • the invention provides an array of oligonucleotide probes immobilized on a solid support, the array comprising a plurality of probes having a sequence corresponding to a species specific polynucleotide target sequence wherein the species specific target sequence is flanked by oligonucleotide sequence that are conserved across a population of organisms.
  • the population of organisms can be of the same family or genus or cause the same disease or disorder.
  • the invention provides a kit comprising, at least one container having therein an at least one oligonucleotide primer complementary to a conserved region of genetic material in a population of organisms; and a solid support having attached thereto a species-specific probe capable of hybridizing to a target sequence, the target sequence flanked by the at least one primer.
  • the invention provides a method of identifying at least two organisms from a population of organisms in a biological sample, comprising obtaining genetic material from the biological sample; contacting the genetic material with at least a first primer and at least a related second primer corresponding to a pair of conserved regions in the genome of the population of organisms, wherein the first primer hybridizes " upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences and each target sequence is characteristic of one of the at least two organisms; amplifying the target sequence; providing a solid support comprising at least two probes selected from the at least two different organisms, wherein the at least two probes comprise sequences that are substantially complementary to the target sequence in the organism from which the probe sequences were selected; contacting the solid support with amplification products of the amplified target sequence; and detecting hybridization of the target sequence to the probe, wherein hybridization to a probe is indicative of the presence of the cones
  • the invention provides a method of distinguishing a presence of at least two organisms from a population of organisms in a biological sample, comprising obtaining genetic material from the biological sample; contacting the genetic material with at least a first primer and at least a related second primer conesponding to a pair of conserved regions in the genome of the population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences and each target sequence is characteristic of one of the at least two organisms; amplifying the target sequence; providing a solid support comprising at least two probes selected from the at least two different organisms, wherein the at least two probes comprise sequences that are substantially complementary to the target sequence and differentially hybridize to the target sequence depending on a hybridization condition; contacting the solid support with amplification products of the amplified target sequence under a hybridization condition wherein hybridization to a probe corresponding to any one of the
  • the at least two different organisms may be selected from two different organisms comprise bacteria, yeast, paramecia, trypanosoma, unicellular eukaryotes, and viruses.
  • the invention provides a method of identifying a target sequence in a biological sample, comprising obtaining genetic material from the biological sample; contacting the genetic material with at least a first primer and at least a related second primer conesponding to a pair of conserved regions in the genome of a population of organisms, wherein the first primer hybridizes upstream and the second primer hybridizes downstream of a target sequence in the genetic material in the sample, and further wherein the target sequence is less conserved than the primer binding sequences; amplifying the target sequence; and determining the sequence of amplification products of the amplified target sequence.
  • the invention provides a method for identifying an organism associated with the sequenced target sequence by comparing the sequence of the amplified target with a known sequence of the corresponding target in the organism.
  • a method for increasing the efficiency of coupling of an oligonucleotide to a solid substrate comprising applying a positive electrostatic potential to a surface of the solid substrate, whereby the positive electrostatic potential increases a concentration of oligonucleotides and negatively charged molecules to the surface of the solid substrate.
  • a method for increasing the efficiency of coupling of an oligonucleotide to a glass substrate by forming an Epoxy derivative of a surface of the glass substrate, the method comprising applying an Epoxy derivative to the surface of the glass substrate.
  • FIG. 1 shows an alignment of conserved sequence used as primers in the methods and compositions of the invention.
  • FIG. 2 schematically illustrates a method of using a microorganism identification chip involving hybridization of PCR amplification products of an unknown sample using primers according to the present invention to specific probes immobilized on a solid substrate.
  • FIG. 3 shows the effect of primer concentration on amplification by individual set of PCR primers and mixed PCR primers for a RecA gene fragment.
  • FIG. 4 illustrates a comparison between specific (FIG. 4A) and mixed (FIG. 4B) primers.
  • FIG. 5 shows the results from a mutation that disrupts 3'-end hair-pin formation in a primer for S. aureus FtsY gene.
  • the inventor has determined that there are important sets of protein/DNA sequences that are highly conserved among different pathogens.
  • the genes in question code for proteins involved in essential cellular processes, such as for example, chromosome partition, cell division, genes associated with pathogenicity, ' cell wall proteins, and other functions easily identifiable by those skilled in the art.
  • One conserved sequence for example, is the Fts 7Spolili_ gene, which codes for a product that has proved to be essential for bacterial chromosome partition.
  • Figure 1 shows a partial sequence alignment of FtsK proteins from various bacteria. Pair-wise comparison shows that these bacteria have about 50-70% sequence homology. Moreover, further analysis reveals that these coding sequences are conserved only in eubacteria; they are absent in archaebacteria and eukaryote genomes, reflecting the fact that chromosome partition/segregation in archaebacteria and eukaryotic organisms is mediated through different mechanisms. Thus, the FtsK coding sequence would be useful as a signature probe for bacterial pathogens.
  • coding sequences such as FtsZ, FtsQ, topoisomerases, tRNA synthetases, etc.
  • conserved non-coding sequences such as, for example rDNA
  • degenerate PCR primers can be designed to amplify these sequences.
  • conserved sequences are provided by way of example only, other conserved sequence can be readily identified and are applicable to the methodology and compositions described herein, as discussed below.
  • the rationale for choosing highly conserved coding sequence to design the PCR primers is to simplify, for example, the diagnosis procedure in a clinical setting, where reliability and reproducibility are major concerns. For a given infectious disease and a particular patient, symptoms are often caused by one, out of many possible, etiologic agents.
  • the challenge is to design a single PCR reaction that can reliably amplify a nucleic acid (i.e., DNA or RNA) sample from anyone of these possible pathogens for further analysis, such as, for example, by reverse dot blot hybridization. Selecting PCR primers for highly conserved coding sequences make this possible, although a mixture of degenerate primers may be used in place of a single primer as the number of pathogens to be surveyed increases.
  • PCR amplification with degenerate primers is widely used in academia to clone conserved genes from a new organism based on a known protein sequence (Rose et al,
  • degenerate primer means, for example, introducing mixed nucleotides at one or more positions into the primer to account for possible coding sequence variations as a result of the degeneracy of the genetic code.
  • the coding sequences chosen to be analyzed are typically known or have been determined first. Consequently, a much better design of the degenerate primer pair is to use an equimolar mixture (or, the two degenerate primers of the pair in a defined ratio) of the actual coding sequences from the pathogen(s) to be surveyed that correspond to the same conserved peptide sequence.
  • One advantage of this system is a significant reduction in primer degeneracy, compared to the design of introducing mixed nucleotides at multiple positions and thus, less complication for the PCR reaction.
  • the latter design is viable when the number of pathogens to be covered by the assay is low.
  • Another advantage of this system is to enable one to normalize the rates of individual PCRs in the course of a multiplex reaction.
  • nucleic acid sequence-specific hybridization pioneered by Southern (Southern, 1975) allows highly specific detection of a particular polynucleotide sequence in an extracted DNA sample.
  • the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions.
  • An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
  • An example of progressively higher stringency conditions is as follows: 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC and 0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed.
  • PCR polymerase chain reaction technique
  • the PCR reaction amplifies the target sequence from a clinical sample, although the primer hybridization and the subsequent amplification provide specificity to some extent (i.e., only amplifying genetic material from the pathogens).
  • the identification of the pathogens with high specificity derives from sequence specific hybridization, by choosing hybridization probes from the sequences flanked by the PCR primers. Each probe has an exact match in a particular pathogen. Due to codon bias, the nucleotide sequence corresponding to a conserved protein sequence varies among pathogens. This fact allows one skilled in the art to easily design probes that are sufficiently different from each other in such a way that only one probe hybridizes, under stringent conditions, to the PCR product amplified from a particular pathogen. Recent advances in microarray technology make hybridization to multiple probes a relatively easier task.
  • the hybridization probe(s) is spotted in discrete areas on a biochip, to streamline the hybridization process. This approach is very useful in a clinical setting if the biochip has a built-in sensor array, with each probe corresponding to a sensor.
  • the sensor anay will record and store the hybridization signals, which can be retrieved later, or in real-time, with other conventional devices, such as a desktop computer.
  • Non-natural analogs of nucleic acids may also be used as the probes.
  • PNA peptide nucleic acid
  • PNAs are nucleic acid analogs with an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units replacing the phosphodiester linkages.
  • the purine or pyrimidine bases are linked to each unit via a methylene carbonyl linker.
  • PNAs are resistant to enzymatic degradation and hybridize to complementary nucleic acid sequences with higher affinity than analogous DNA oligomers. The hybridization follows Watson-Crick base-pairing rules (Soomets et al, 1999).
  • PNA probes can be used in place of the DNA probes described above.
  • PNAs have been exploited as an alternative for making biochips in an array format (Weiler et al, 1997).
  • other possible nucleic acid analogs may also be used as probes so long as they hybridize to the target nucleic acids in a sequence specific manner.
  • PCR polymerase chain reaction
  • PCR is a method for amplifying a polynucleotide sequence using a polymerase and two oligonucleotide primers, one complementary to one of two polynucleotide strands at one end of the sequence to be amplified and the other complementary to the other of two polynucleotide strands at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample.
  • the primers used for PCR amplification according to the method of the invention embrace oligonucleotides of sufficient length and appropriate sequence that provides initiation of polymerization of a significant number of nucleic acid molecules containing the target nucleic acid under the conditions of stringency for the reaction utilizing the primers. In this manner, it is possible to selectively amplify polynucleotides for further analysis.
  • the term "primer” as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least eight, which sequence is capable of initiating synthesis of a primer extension product that is capable of hybridizing to a target nucleic acid strand in order to initiate polymerase activity.
  • the oligonucleotide primer typically contains 15-22 or more nucleotides, although it may contain fewer nucleotides so long as the primer is of sufficient specificity to allow essentially only the amplification of the desired target nucleotide sequences (e.g., the primer is substantially complementary).
  • Experimental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH.
  • the DNA polymerase is preferably a thermostable DNA polymerase, such as Taq polymerase, Tthl polymerase, VENT polymerase or Pflx polymerase.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded.
  • the primer is first treated to separate the strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide compound.
  • Primers used according to the method of the invention are designed to be "substantially" complementary to each strand of a target nucleotide sequence to be amplified.
  • Substantially complementary means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to function. In other words, the primers should have sufficient complementarity with the flanking sequences to hybridize therewith and permit amplification of the nucleotide sequence.
  • the 3' terminus of the primer that is extended has perfectly base paired complementarity with the complementary flanking strand.
  • Oligonucleotide primers used according to the invention are employed in any amplification process that produces increased quantities of target nucleic acid.
  • one primer is complementary to the negative (-) strand of the nucleotide sequence and the other is complementary to the positive (+) strand.
  • Polymerase I (Klenow) or Taq DNA polymerase and nucleotides or ligases, results in newly synthesized + and -strands containing the target nucleic acid. Because these newly synthesized nucleic acids are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target mutant nucleotide sequence) defined by the primer.
  • the product of the amplification reaction is a discrete nucleic acid duplex with termini conesponding to the ends of the specific primers employed.
  • the terms "forward” and "reverse" primers are interchangeable and used to define any one of a pair of related primers useful for the amplification of a target segment between the two primers. Those of skill in the art will know of other amplification methodologies that can also be utilized to increase the copy number of target nucleic acid.
  • primers are designed that conespond to highly conserved regions of the genome of a family or a genus of organisms.
  • these primers are selected from regions of the genome that code for conserved proteins.
  • These primers can be degenerate depending upon the sequence homology of the target polynucleotide to be amplified.
  • the primers flank a region of a gene (i.e., the target polynucleotide sequence) that is not highly conserved across species.
  • target polynucleotides will be amplified, for example, by PCR and the resulting PCR product then further analyzed as described more fully below.
  • Genetic material is a material containing any nucleic acid (DNA or RNA) sequence or sequences either purified or in a native state such as a fragment of a chromosome or a whole chromosome, either naturally occurring or synthetically or partially synthetically prepared nucleic acid sequences, nucleic acid sequences which constitute a gene or genes and gene chimeras, e.g., created by ligation of different nucleic acid sequences.
  • DNA sequence is a sequence of a linear or circular DNA molecule comprised of any combination of the four DNA monomers, i.e., nucleotides of adenine, guanine, cytosine and thymine, which codes for genetic information, such as a code for an amino acid, a promoter, a control or a gene product.
  • a specific DNA sequence is one that has a known specific function, e.g., codes for a particular polypeptide, a particular genetic trait, or affects the expression of a particular phenotype.
  • Gene is the smallest, independently functional unit of genetic material that codes for a protein product or controls or affects transcription and comprises at least one DNA sequence.
  • a "coding sequence” is a polynucleotide sequence that is transcribed and/or translated into a polypeptide.
  • Specific hybridization to microarrays In general, Southern techniques and PCR can be used to identify particular genomic sequences.
  • DNA chip i.e., biochip
  • the DNA chip is a streamlined version of dot-blot analysis, a variation of Southern's method. Through miniaturization, a large number of probe sequences are deposited onto the surface of a solid support. The identity of the target sequence is defined by its specific hybridization to a probe or probes on the chip. The main advantage of this method is that it can survey a large number of probes with relative ease.
  • oligonucleotides probes are immobilized to a solid support at defined locations (i.e., known positions). This immobilized anay is sometimes refened to as a "biochip.”
  • the solid support can be, for example, a nylon
  • oligonucleotide probes nucleic acid sequences complementary to a species-specific target sequence.
  • the PCR products are detected and distinguished by use of "biochips.”
  • the chips are designed to contain probes exhibiting complementarity to a particular reference sequence from an organism of interest (e.g., viral, prokaryotic, eukaryotic).
  • the probes present on the chip are sequences flanked by the degenerate PCR primers PI and P2.
  • the chips are used to read a target sequence comprising either the reference sequence itself or variants of that sequence representing the various species specific amplification products or target sequences.
  • the sequence selected as a reference sequence can be from anywhere in the target organism with the proviso that they are flanked by the degenerate PCR primers PI and P2 to the sequences A and B of the particular species or organism as shown in Figure 2.
  • a reference sequence can be from anywhere in the target organism with the proviso that they are flanked by the degenerate PCR primers PI and P2 to the sequences A and B of the particular species or organism as shown in Figure 2.
  • probe sequence is usually about 5, 10, 20, 50, 100, 5000, 1000, 5,000 or 10,000 bases in length, and typically about 20-2000 bases in length.
  • the reference sequence can contain the entire region coding for the target sequence of interest or a fragment thereof.
  • Various densities of the reference sequence may be present on the chip such as, for example, about 2 to more than 10,000 probe sequences/cm 2 or more (e.g., 100,000 probe sequence/ cm 2 ) typically about 10 to less than 1,000 probe sequences/cm 2 .
  • the array of probes is usually laid down in rows and columns, such a physical arrangement of probes on the chip is not essential.
  • the data from the probes can be collected and processed to yield the sequence of a target inespective of the physical arrangement of the probes on a chip.
  • the hybridization signals from the respective probes can be reasserted into any conceptual array desired for subsequent data reduction whatever the physical arrangement of probes on the chip.
  • the length of probe can be important in distinguishing between a perfectly matched probe and probes showing a single-base mismatch with the target sequence.
  • the discrimination is usually greater for short probes.
  • Shorter probes are usually also less susceptible to formation of secondary structures.
  • the absolute amount of target sequence bound, and hence the signal is greater for larger probes.
  • the probe length representing the optimum compromise between these competing considerations may vary depending on inter alia the GC content of a particular region of the target DNA sequence. In some regions of the target, short probes (e.g., 11 mers) may provide information that is inaccessible from longer probes (e.g., 19 mers) and vice versa.
  • Maximum sequence information can be read by including several groups of different sized probes on the chip as noted above.
  • the second reference or target sequence can be a control sequence to determine accuracy of the amplification reaction or a control sequence to measure or quantitate the amount of target sequence in a sample.
  • the process and principal of analysis for this secondary sequence is the same as that for the initial or target sequence.
  • the total number of probes on the chips depends on a number of factors, including the number of potential organisms to be identified, the length of the reference sequence and the options selected with respect to inclusion of multiple probe lengths and secondary groups of probes to provide confirmation of the assay.
  • target polynucleotide or target genetic material whose sequence or identity is to be determined, is usually isolated, in the case of therapeutic diagnostics, from a clinical fluid (e.g., urine, blood, plasma, sputum, cerebrospinal fluid, tracheal aspirate or pleural fluid) or tissue sample in the form of RNA or DNA.
  • a clinical fluid e.g., urine, blood, plasma, sputum, cerebrospinal fluid, tracheal aspirate or pleural fluid
  • tissue sample in the form of RNA or DNA.
  • the RNA can be reverse transcribed to DNA, and the cDNA product then amplified by techmques known to those of skill in the art.
  • target polynucleotides are prepared by PCR amplification in the presence of labeled nucleoside triphosphates.
  • the resulting PCR products are hybridized under appropriate conditions to a probe sequence on a biochip and the unhybridized material washed away with buffer.
  • the chip is subsequently scanned by autoradiography or in real time to determine the presence of hybridized product at particular locations on the biochip.
  • a hybridized product is indicative of the presence of a microorganism conesponding to the probe sequence located on the biochip.
  • Bacterial sepsis and related septic shock are frequently lethal conditions caused by infections which can result from certain types of surgery, abdominal trauma and immune suppression related to cancer, transplantation therapy or other disease states. It is estimated that over 700,000 patients become susceptible to septic shock-causing bacterial infections each year in the United States alone. Of these, 160,000 actually develop septic shock, resulting in 50,000 deaths annually.
  • Gram-negative bacterial infections comprise the most serious infectious disease problem seen in modern hospitals. Two decades ago, most sepsis contracted in hospitals was attributable to more acute gram positive bacterial pathogens such as Staphylococcus and Streptococcus. By contrast, the recent incidence of infection due to gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, has increased.
  • Gram-negative bacteria now account for some 200,000 cases of hospital-acquired infections yearly in the United States, with an overall mortality rate in the range of 20% to
  • Gram-negative sepsis is a disease syndrome resulting from the systemic invasion of gram negative rods and subsequent endotoxemia.
  • the severity of the disease ranges from a transient, self-limiting episode of bacteremia to a fulminant, life threatening illness often complicated by organ failure and shock.
  • the disease is often the result of invasion from a localized infection site, or may result from trauma, wounds, ulcerations or gastrointestinal obstructions.
  • the symptoms of gram-negative sepsis include fever, chills, pulmonary failure and septic shock (severe hypotension).
  • Gram-negative infections are particularly common among patients receiving anticancer chemotherapy and immunosuppressive treatment. Infections in such immuno- compromised hosts characteristically exhibit resistance to many antibiotics, or develop resistance over the long course of the infection, making conventional treatment difficult.
  • the ever-increasing use of cytotoxic and immunosuppressive therapy and the natural selection for drug resistant bacteria by the extensive use of antibiotics have contributed to gram-negative bacteria evolving into pathogens of major clinical significance.
  • the Gram-negative bacteria are a diverse group of organisms and include Spirochetes such as Treponema and Borrelia, Gram-negative bacilli including the Pseudomonadaceae, Legionellaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellaceae, Gram-negative cocci such as Neisseriaceae, anaerobic Bacteroides, and other Gram- negative bacteria including Rickettsia, Chlamydia, and Mycoplasma.
  • Spirochetes such as Treponema and Borrelia
  • Gram-negative bacilli including the Pseudomonadaceae, Legionellaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellaceae
  • Gram-negative cocci such as Neisseriaceae, anaerobic Bacteroides
  • other Gram- negative bacteria including Rickettsia, Chlamydia, and Mycoplasma.
  • Gram-negative bacilli are important in clinical medicine. They include (1) the Enterobacteriaceae, a family that comprises many important pathogenic genera, (2) Vibrio, Campylobacter and Helicobacter genera, (3) opportunistic organisms (e.g.,
  • Gram-negative bacilli are the principal organisms found in infections of the abdominal viscera, peritoneum, and urinary tract, as well secondary invaders of the respiratory tracts, burned or traumatized skin, and sites of decreased host resistance. Cunently, they are the most frequent cause of life threatening bacteremia. Examples of pathogenic Gram-negative bacilli are E.
  • coli (diarrhea, urinary tract infection, meningitis in the newborn), Shigella species (dysentery), Salmonella typhi (typhoid fever), Salmonella typhimurium (gastroenteritis), Yersinia enterocolitica (enterocolitis), Yersinia pestis (black plague), Vibrio cholerae (cholera), Campylobacter jejuni (enterocolitis), Helicobacter jejuni (gastritis, peptic ulcer), Pseudomonas aeruginosa (opportunistic infections including burns, urinary tract, respiratory tract, wound infections, and primary infections of the skin, eye and ear), Haemophilus influenzae (meningitis in children, epiglottitis, otitis media, sinusitis, and bronchitis), and Bordetella pertussis (whooping cough).
  • Vibrio is a genus of motile, Gram-negative rod shaped bacteria (family Vibrionaceae). Vibrio cholerae causes cholera in humans; other species of Vibrio cause animal diseases. E. coli colonize the intestines of humans and warm blooded animals, where they are part of the commensal flora, but there are types of E. coli that cause human and animal intestinal diseases. They include the enteroaggregative E. coli (EaggEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E.coli (EIEC), enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). Uropathogenic E.
  • EaggEC enteroaggregative E. coli
  • EHEC enterohaemorrhagic E. coli
  • EIEC enteroinvasive E.coli
  • EPEC enteropathogenic E. coli
  • ETEC enterotoxigenic E. coli
  • UPEC urinary tract infections.
  • NMEC neonatal meningitis E. coli
  • calf septicaemia bovine mastitis
  • porcine oedema disease and air sac disease in poultry.
  • the pathogenic bacteria in the Gram-negative aerobic cocci group include Neisseria, Moraxella (Branhamella), and the Acinetobacter.
  • the genus Neisseria includes two important human pathogens, Neisseria gonorrheae (urethritis, cervicitis, salpingitis, proctitis, pharyngitis, conjunctivitis, pharyngitis, pelvic inflammatory disease, arthritis, disseminated disease) and Neisseria meningitides (meningitis, septicemia, pneumonia, arthritis, urethritis).
  • Gram-negative aerobic cocci that were previously considered harmless include Moraxella (Branhamella) catarrhalis (bronchitis and bronchopneumonia in patients with chronic pulmonary disease, sinusitis, otitis media) has recently been shown to be an common cause of human infections.
  • the Neisseria species include N. cinerea, N. gonorrhoeae, N. gonorrhoeae subspecies kochii, N. lactamica, N. meningitidis, N. polysaccharea, N. mucosa, N. sicca, N. subflava, the asaccharolytic species N.flavescens, N. caviae, N cuniculi and N. ovis.
  • the strains o ⁇ Moraxella (Branhamella) catarrhalis are also considered by some taxonomists to be Neisseria.
  • Other related species include Kingella, Eikenella, Simonsiella, Alysiella, CDC group EF-4, and CDC group M-5.
  • Veillonella are Gram-negative cocci that are the anaerobic counte ⁇ art of Neisseria. These non-motile diplococci are part of the normal flora of the mouth. Specific E. coli phenotypes have been associated with intestinal diseases, notably dianhoea, and extraintestinal conditions including urinary tract infections and meningitis in the newborn. Like many pathogens, E. coli strains produce adhesins structures that mediate attachment to eukaryotic cells and which can be distinguished by their specificity for receptors on the target cell. Adhesins can represent the filamentous, hair-like structures known as fimbriae or pili, or they may be nonfilamentous components of the cell surface.
  • FI A type 1 fimbrial adhesins recognize the sugar a-mannose in glycoproteins, whereas mannose-resistant (MR) adhesins bind to eukaryotic receptors other than mannose.
  • MR mannose-resistant
  • a wide range of filamentous adhesins are produced by different E. coli strains with specificities for various receptors on human and animal tissues. Pathogenic strains may contain sets of genes encoding one or more types of fimbriae, sometimes in combination with nonfimbrial adhesins.
  • this invention can also be used to test food-borne bacteria, such as E. coli and Salmonella etc. Such safety measures will reduce the actual number of infections caused by food-borne pathogens. Selection of probes
  • PCR and Southern blot e.g., a dot blot version or "biochip” technology
  • sensitivity and specificity or accuracy
  • primers that would work on genomic DNA from many different microbial pathogens.
  • the subsequent Southern blot analysis would be less informative due to cross-species hybridization.
  • the ideal genomic regions are highly conserved coding sequences (for designing the PCR primers) flanking a less conserved coding sequence (for designing the hybridization probe).
  • conserved non-coding regions such as 16S rDNA, can also be used for this kind of analysis, except that greater efforts are required to eliminate possible artifacts.
  • the following advantages of using conserved protein coding sequences for diagnostic assay in a microarray format are significant in the selection of signature probes for a microorganism. Firstly, use of conserved protein coding sequences results in a different type of diagnostic test than comparable ribosomal DNA based approach.
  • Another important criteria for a good diagnostic assay is its accuracy.
  • the built-in redundancy generated by using two or more independent loci for identification enables one to achieve better accuracy.
  • the present invention allows the selection of multiple target sequences, from hundreds of conserved protein coding sequences in a microorganism, to be used in a single diagnostic test.
  • conserved coding sequences are selected such that they are highly conserved at both ends of an operationally defined gene fragment and more divergent in the intervening coding sequence. For example, a preliminary analysis of FtsZ gene suggests that it has a high degree of conservation throughout.
  • Type I and Type II topoisomerases are also examples of highly conserved genes in prokaryotes and eukaryotes. For a given organism, these functions are often encoded by multiple genes that share sequence similarity. Whereas these properties make them less preferred for application in the present invention, segments of these genes may still be suitable for the pu ⁇ oses of this application.
  • an oligonucleotide comprising any 5 uninterrupted nucleotides in a disclosed probe sequence is suitable for the application of this invention.
  • probe as used herein is thus intended to encompass any 5 uninterrupted nucleotides of a specific claimed or disclosed probe sequence.
  • an "universal primer” is used to amplify the target sequence, followed by sequencing of the amplified target. Comparison of this sequence with known sequence data enables the identification of the microorganism. In fact, the bacterial rDNA locus has been utilized in this fashion (e.g., in ribotyping). A variation of this scheme is to determine the sequence of the amplified sequence by on-chip hybridization to a high-density oligonucleotide microarray (as described in U.S. Pat. Nos. 5,202,231 and 5,002,867, inco ⁇ orated herein by reference).
  • the present invention encompasses creation of an "universal primer” by mixing together related primers. It differs from conventional multiplex PCR primers in that all the primer pairs amplify the same genetic locus, albeit from different organisms. It also differs the conventional degenerate PCR primers which inco ⁇ orate mixed base(s) at certain position(s) on the primer during its chemical synthesis.
  • the advantage of mixing a number of primers of specific sequences over a single degenerate primer is two fold. One is to significantly reduce degeneracy of the primer. The other is to allow normalization of the individual reaction rates by adjusting the conesponding primer concentrations. The point is illustrated in the following example. Sequences of primers for the RecA gene of 11 different microorganisms and a degenerate consensus sequences are shown in the following table: Table 1. Aligned sequences of RecA primers and a consensus sequence.
  • the RecA primers have different lengths, varying at the 5'-end. This normalizes the melting temperature (Tm) of the primers, such that each conesponding PCR is performed at the same annealing temperature.
  • Tm melting temperature
  • the 3'-end of a group of primers is determined and the 5'-end is extended according to the sequence until the primer reaches the desired Tm. Since proper annealing at the 3' end of the primer is essential for the PCR, a prefened mode of the invention has four out of five bases matched at the 3' end of the primers.
  • the present invention allows further normalization of the reaction rate of each individual PCR by adjusting the concentration of the corresponding primer pair in the primer mixture. Since all primers are related, especially at the 3'-end, by design, a primer running low at the later cycles can be compensated by the others, achieving the effect of a single pair of "universal primers".
  • Figure 3 shows the effect of primer concentration on amplification by individual set of PCR primers and mixed PCR primers for a RecA gene fragment.
  • Standard 50 ⁇ l PCR reactions were carried out at various primer concentration, using genomic DNA from Legionella pneumonia (Lp), Staphylococcus aureus (Sa), and Streptococcus pneumonia (Sp) as the template. After a 27 cycle reaction, 10 ⁇ l aliquots were taken from each tube, resolved on a 2% agarose gel and then stained with ethidium bromide (EtBr).
  • the DNA templates added to the PCR reaction were as follows: Legionella pneumonia, lane 2, 5, 8, and 11; Staphylococcus aureus, lane 3, 6, 9, and 12; Streptococcus pneumonia, lane 4,7, 10, and 13.
  • Primer concentrations for the reactions were: lane 2-4, 1 ⁇ M of specific primer; lane 5-7, 0.33 ⁇ M specific primer; lane 8-10, 0.11 ⁇ M of specific primer; lane 11-13, 1.0 ⁇ M of mixed primers (an equimolar mixture of nine different pairs of primers, the effective concentration for a specific pair of primer being the same as in lanes 8-10).
  • Lane 1 includes a 100 bp DNA size marker.
  • the primers used were: Legionella pneumonia, SEQ 36 and 49; Staphylococcus aureus, SEQ 33 and 46; Streptococcus pneumonia, SEQ 32 and 45.
  • Other primers that comprised the equimolar primer mixture were: SEQ ID NOS 35 and 48, 30 and 43, 34 and 47, 29 and 42, 31 and 44, 28 and 41.
  • the reaction rates are not the same, as shown in Figure 3. However, for each PCR, the reaction rate can be controlled by adjusting the primer concentration as shown in Figure 3.
  • the primers have similar Tm, the reaction for S. aureus and L. pneumonia RecA are slower than that for S. pneumonia RecA.
  • reaction rates of a multiplex PCR can be normalized by mixing primer pairs at unequal molar ratios.
  • Figure 4A shows RecA PCR for eight different bacteria, using specific primers. It is evident that the reaction rates are different, even though the primers were normalized to a similar Tm (68 to 70 °C). When primers are mixed at the appropriate ratios (see Table 2), the reaction rates are normalized, as shown in Figure 4B.
  • the primers used for panel A were: lane 1, SEQ ID NOS 30 and 43; lane 2, SEQ ID NOS 29 and 42; lane 3, SEQ ID NOS 33 and 46; lane 4, SEQ ID NOS 35 and 48; lane 5, SEQ ID NOS 28 and 41; lane 6, SEQ ID NOS 36 and 49; lane 7, SEQ ID NOS 34 and 47; lane 8, SEQ ID NOS 32 and 45.
  • the primers used for panel B were the same in all reactions, "universal primers" mixed according to the ratios shown in Table 2.
  • One method of achieving the proper primer mixing ratio is to titrate each specific primer pair, by dilution, in a linear reaction (e.g. 25 cycles PCR) and then, select the primer concentration for each specific primer pair that gives a comparable reaction rate to the others.
  • a linear reaction e.g. 25 cycles PCR
  • the original primer pair designed for S aureus FtsY were 5'- TGTGAATGGTGtTGGTAAAACAAC-3' (derived from wild type S. aureus FtsY gene sequence; SEQ ID NO 10 is a mutated version of this primer in which the "t” is changed to "A") and 5'-TTTGTAAACGTCCAGCGGTATC-3' (SEQ ID NO 23 is wild type sequence).
  • SEQ ID NO 10 is a mutated version of this primer in which the "t” is changed to "A”
  • SEQ ID NO 23 is wild type sequence
  • PCR experiments using a mutation that disrupts 3 '-end hair-pin formation in a primer for S. aureus FtsY gene is shown in Figure 5.
  • Standard PCRs were carried out for S. aureus FtsY, using different primer pairs.
  • the PCR products were resolved on a 2% agarose gel, stained with EtBr.
  • the same backward primer (SEQ ID NO 23) was used for both reactions, but the forward primer was: lane 2, primer derived from wild type sequence; lane 3, mutated primer (i.e., SEQ ID NO 10).
  • SEQ ID NO 10 mutated primer
  • the hair-pin structure may self-prime at room temperature or the annealing temperature (i.e. 53 °C), extending the primer at the 3' end.
  • the annealing temperature i.e. 53 °C
  • this product can anneal to the template at the original site, it cannot prime the intended PCR reaction due to lack of proper base-pairing at the 3' end, thus becoming a competitive inhibitor.
  • the hair-pin structure is disrupted at 92 °C, yet a certain percentage refold at the annealing temperature, reducing the effective concentration of the forward primer. Both scenarios are consistent with the result shown in Figure 5 that permanent disruption of hair-pin formation via mutations of the primer improves the PCR reaction.
  • base modification of the primers to reduce secondary structures is performed. Because the general location of the primers on the target sequences is fixed for all the pathogens to be identified, such modification enables one to improve the weaker reactions without having to drastically change the primer sequences (e.g., generate primers from a different location on the target sequence).
  • Genomic DNA samples from Mycobacterium tuberculosis and Mycobacterium leprae can be distinguished by nested PCR, followed by sequence specific hybridization.
  • the same sets of primers can be used to amplify the FtsK gene fragment from either genomic DNA, because of the high degree of nucleotide sequence conservation at the chosen FtsK coding regions (a single nucleotide difference in some of the primers is indicated by a capital letter, below).
  • the unknown DNA prepared from a clinic sample will be used as the template for the first PCR reaction, with the primer set of:
  • reaction product After a standard 30-cycle reaction, an aliquot of the reaction product will be used as the template for the second PCR reaction with the primer set of: 5'- ccgcatCtgatcacgccgatcatc-3' (SEQ ID NO:3) and 5'-acgtcGtccgacgggcgtag-3' (SEQ ID NO:4) (both fall within the sequence amplified by SEQ ID NOS: 1 and 2, i.e., internal ⁇ 3' to SEQ ID NO: 1 and 5' to SEQ ID NO:2 - set forth by the first set of primers, and one of the two will have a biotin label at the 5' end).
  • the PCR product will be used directly in a hybridization reaction, probing a Nylon membrane.
  • the Nylon membrane is prepared in such a way that it has two discrete spots with different oligonucleotides attached to the membrane at the two spots, respectively.
  • One oligonucleotide is 5'-atcgacgacttcaacgacaag-3' (SEQ ID NO:5), derived from M. tuberculosis FtsK coding sequence (from Box D shown in Figure 1).
  • the other is from M. leprae FtsK, having the sequence of 5'-atcgacgTGttcaaCgagaag-3' (SEQ ID NO:6).
  • This sequence differs from the first oligonucleotide (i.e., SEQ ID NO:5) at three nucleotide positions (indicated in upper case).
  • SEQ ID NO:5 the first oligonucleotide
  • the specific hybridization pattern can be revealed in a number of way, such as streptavidin conjugated alkaline phosphates, radionucleotide labeling followed by autoradiography or by chemiluminescence (Bronstein et al. 1990). Based on the 5 hybridization result, one can determine the bacterial origin of the unknown DNA sample.
  • Meningitis can be viral or bacterial in origin, with the latter causing the more severe illness.
  • Etiologic agents for bacterial meningitis are usually Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. Cunently, the identification of 10 precisely which bacterium is the culprit requires lengthy laboratory tests.
  • the present invention provides an alternative for rapid and accurate identification. Based on the FtsK coding sequence from these three bacteria, PCR primers and hybridization probes are designed as follows:
  • oligonucleotide sequences are derived from the conserved coding region, Box A ( Figure 1). For doing actual PCR, an equal molar ratio mixture of these three 10 oligonucleotides will be used, which is equivalent to a single primer with a three-fold degeneracy.
  • oligonucleotides are derived from the conserved Box B ( Figure 1). An equimolar mixture is used for the PCR reaction.
  • Hybridization probes :
  • Each oligonucleotide is derivatized with Acrydite (Mosaic Technologies), which will allow them to be immobilized directly in a discrete spot on the surface of a glass slide (pretreated with acrylic silane).
  • the unknown D ⁇ A sample prepared from a clinic sample will be used as the template for the PCR reaction.
  • a single PCR reaction will be performed using the degenerate Forward and Backward primer set (each primer is derivatized with a fluorescent dye during its chemical synthesis).
  • the products are hybridized with the probe panel in situ, followed by a brief wash.
  • the hybridization pattern is then observed under a fluorescent microscope or a confocal microscope.
  • the bacterial origin of the D ⁇ A sample is indicated by the probe that hybridized to the PCR product.
  • Identification of different eukaryotic cells can accomplished based on the same principle.
  • Fungi are known etiologic agents that caused pneumonia, such as Aspergillus parasiticus and Candida albicans.
  • these two different organisms share a number of highly conserved proteins that are involved in various essential cellular processes.
  • beta-tubulin protein is highly conserved both in sequence and function, i.e. mediating chromosome segregation during eukaryotic cell division.
  • the human CDC2 protein is also highly conserved, with an essential function of regulating eukaryotic cell cycle progression.
  • a test based on beta-tubulin gene is described here, although other conserved protein coding sequences can also be used.
  • beta-tubulin from Aspergillus parasiticus and Candida albicans are available from GenBank (Accession number L49386 and M19398 respectively). Different from the bacteria cases describe above, eukaryotic protein coding sequences are usually Interrupted by non-coding sequences, i.e., introns. For a given conserved gene, the number of introns as well as the location of the introns within the gene are not necessarily conserved.
  • the beta-tubulin gene from C. albicans two introns, with the exon 3 encoding amino acids 17 to 449. That from A. parasiticus contains seven introns, with the exon 6 and exon 7 encoding amino acids 54 to 436.
  • PCR primers are chosen from the conserved coding regions in the exon 3 for C. albicans, or from exons 6 and 7 for A. parasiticus.
  • the nucleotide sequences are list below.
  • the DNA extracted from a clinic sample is used as the template for the first PCR reaction.
  • An equal molar mixture of the forward primers as well as that of the backward primers are added to the standard amplification.
  • an aliquot of the product is taken out and used as the template for the second round of amplification (i.e. nested PCR).
  • the primers used for the second PCR reaction are flanked by the first pair of PCR primers, respectively.
  • the second PCR reaction will further amplify the desired product, and offer an additional specificity check.
  • an equal molar mixture of the primers for these two organisms are used for the reaction to avoid possible bias for a particular pathogen.
  • the actual sequences of the second pair of primers are listed below.
  • Second backward primers (each primer is biotinylated at the 5' end):
  • the products are extracted once with phenol once, and hybridized to nylon membrane with a panel of immobilized probes.
  • Two of the probes are from A. parasiticus and one from for C. albicans. All of them are flanked by the second set of PCR primers. Each probe is located within a restricted area of the membrane.
  • the nucleotide sequences of the probes are:
  • the hybridization pattern is display by alkaline phosphatase and chemiluminescent, followed by autoradiography.
  • the specific hybridization to a particular probe indicates the genomic origin of PCR product, hence the identity of the pathogen in the clinic sample.
  • hybridization probe A. para-2
  • Intron 6 sequence of A. parasiticus beta-tubulin gene This intron (thus the hybridization probe sequence) is completely absent from PCR product amplified from for
  • C. albicans genomic DNA It may work better in terms of discriminating between the PCR products.
  • This example illustrates that, when the present invention is applied to eukaryotic sample, the hybridization may be chosen from an intron sequence rather than a less conserved protein coding sequence.
  • Virus is another major class of infectious agents.
  • the present invention also provides a mean for the systematic detection of multiple pathogens from this class.
  • viruses certain proteins or functions are highly conserved.
  • the replication of a viral genome is an essential step in the life cycle of the virus. It invariably requires the participation of at least a viral-encoded DNA or RNA polymerase. Within a subclass of viruses, these polymerase are usually conserved, due to evolutionary constrain on the replication function. For example, reverse transcriptase is highly conserved among retroviruses.
  • a method is described that detects and distinguishes a class of single stranded RNA viruses.
  • viral etiologic agents for acute lower respiratory track infection in children include adenovirus (12.7% of the total viral isolates), influenza virus type A (21.1%), -type B (13.9%), parainfluenza virus type 1 (13.5% ), -type 2 (1.3%), -type 3 (16.0%) and respiratory syncytial virus (21.5%).
  • influenza virus type A (21.1%)
  • -type B (13.9%)
  • parainfluenza virus type 1 13.5%
  • -type 2 1.3%)
  • -type 3 (16.0%
  • respiratory syncytial virus 27%
  • the overall viral isolation rate was 22.1%.
  • -type 2 parainfluenza virus type 2
  • PSV-2 -type 3 (PIV-3), and respiratory syncytial virus (RSV) belong to Paramyxoviridae family of enveloped negative-strand RNA viruses. Other members of this family also include Ebola virus, Newcastle disease virus, Sendai virus, Measles virus, and Hendra virus etc.
  • a single PCR reaction can be designed based on the conserved coding sequences within the RNA polymerase gene (L-protein), which will detect all four viruses. Because these are RNA viruses, a reverse transcriptase reaction will be needed to convert the interested genomic RNA into DNA for the PCR reaction.
  • L-protein RNA polymerase gene
  • the coding sequences for amino acids 537 to 542 (IDKAIS) and amino acids 776 to 781 of RSV RNA polymerase (GenBank accession number U39662) are chosen as the PCR primers.
  • the C-terminal primer (A. A. 775 to 781) will also be used as the primer for the reverse transcriptase reaction.
  • Primers for the other viruses will be chosen from the conesponding coding sequences, based on protein sequences alignment.
  • PIV-3 GenBank accession number U51116
  • the primers encode amino acids 497-502 and amino acids 763-768.
  • PIV-1 GenBank accession number AFl 17818
  • PIV-2 (GenBank accession number X57559), the primers conespond to amino acids 475- 480 and 742-747. The actual sequences of these primers are listed below.
  • nucleic acid sample extracted from nasopharyngeal aspirate of a patient will be used as the template.
  • the backward primer pool (an equal molar mixture of the four primers, which is equivalent to a single primer with a four-fold degeneracy) is used to initiate the reverse transcriptase reaction first, according to standard reaction condition.
  • the forward primer pool (each of the primer has a biotin molecule derivatized at the 5' end) is added to start the PCR reaction, following standard protocol.
  • the PCR products are extracted once, and hybridized to a nylon membrane with a panel of immobilized hybridization probes. The exact sequences of the probes conespond to a stretch of non-conserved amino acid sequence of the RNA polymerase, flanked by the PCR primers. They are listed below.
  • Hybridization probes PIV-3 5'-ttgtcttctaatcagaaatca-3'
  • Each probe is spotted onto the membrane in a separate discrete area, and cross- linked to the membrane by UV irradiation. These sequences are sufficiently different to allow the differentiation of specific hybridization to respective PCR products, under stringent conditions. After the hybridization and subsequent washes under stringent condition, the membrane is treated with streptavidin-alkaline phosphatase conjugate. The hybridization signal is reveal by adding chemiluminescent substrate followed by autoradiography. The specific hybridization of the PCR product to a particular viral probe indicates the presence of that virus in the clinic sample. 8. Pneumonia pathogen identification.
  • the pathogens described in examples 2, 3, and 4 can all cause pneumonia.
  • the fastest way, and the economic way, to identify the actual pathogen for a particular patient is to include all these pathogens in a single test, rather than separate tests.
  • the hybridization probes described in above examples (including bacteria, viruses, and fungi) are spotted onto a single chip or a piece of nylon membrane, to obtain a disease-specific probe panel.
  • the PCR reactions described above are carried out in parallel.
  • the amplified products are pooled and hybridized to the disease-specific probe panel in a single step.
  • the specific hybridization of the PCR products to the probe panel is indicated by fluorescence or chemiluminescence as described in previous examples.
  • More pathogens can be included in this single test, based on the principles for the PCR primer and hybridization probe design disclosed herein. Furthermore, for other infectious diseases (such as STD), similar tests can be developed that include all the known etiologic agents in a single assay, based on the same principle.
  • both prokaryotic and eukaryotic cells can be identified simultaneously in a single test, since many genes are highly conserved among them.
  • Candida albicans is a significant respiratory-track pathogen.
  • a database search using E. coli FtsY protein sequence as the query, easily identified its homologue in
  • Candida albicans and the yeast S. cerevisiae. These proteins share about 30% amino acid sequence identity and 50% DNA sequence identity with their prokaryotic counte ⁇ art over a stretch of 300 amino acids.
  • the conesponding coding sequences were retrieved from GenBank and aligned with the prokaryotic FtsY coding sequences, using Clustal W program.
  • PCR primers were designed based on the alignment, such that they were compatible with the bacterial FtsY primers. When standard PCR was performed with these new primer pairs individually, a single product of the expected size was amplified.
  • FtsY PCR products from fungi and bacteria are very similar, they can be easily distinguished by hybridization to specific probes for each organism, derived from the divergent regions flanked by the PCR primers.
  • PCR was performed using a mixture of FtsY primers from S. cerevisiae (SEQ ID NOS 14 and 27), C albicans (SEQ ID NOS 13 and 26), L. pneumophila (SEQ ID NOS 7 and 20), M. pneumoniae (SEQ ID NOS 11 and 24), as well as genomic DNA from these four organisms .
  • the PCR products were labeled with a fluorescent Alexa Fluor 488 dye (from
  • the labeled PCR products were hybridized to a oligonucleotide microarray under stringent conditions.
  • the microarray consisted of two probes each from these four organisms (i.e. SEQ ID NOS 56, 62, 65, 66, 70, 76, 79, and 80). After washing away the unhybridized PCR products, the slide was scanned in a fluorescent scanner and analyzed. All the probes hybridized, suggesting that the FtsY fragment from these four organism were amplified in a single PCR reaction using the "universal primers".
  • labeled PCR from each organism was hybridized to the same oligonucleotide array, under the same conditions. The result were that only the conesponding probe hybridized, suggesting that the hybridization pattern observed in the previous experiment was due to specific hybridization.
  • RNA viruses can also cause pneumonia. It would be beneficial to design a test that can identify these bacteria and viruses in a single assay. Since the evolution of virus is very different from that of bacteria, it is difficult to find a locus that is conserved in both bacteria and viruses. However, certain viral functions are conserved within a subgroup, such as enzyme(s) involved in replicating their genomes.
  • yet another embodiment of present invention is utilized.
  • a locus or loci conserved among the virus subgroup is selected.
  • Several PCR primer pairs based on the sequences from one of the viruses are then designed. PCR is carried out to determine which primer pair(s) is compatible with bacterial PCR and the compatible primer is selected for designing similar primers (i.e. the same amplicon) for other members of the viral group, which would also be compatible with the bacterial PCR.
  • the PCR for a amplicon within the gene encoding human RSV L protein was found to be compatible with the PCR for bacterial RecA. Both viral and bacterial sequences were amplified in a single PCR, using a primer mixture (SEQ ID NOS 34, 47, 36, 49, 105, 106). The PCR product was fluorescently labeled with Alexa Fluor 488 (Molecular Probes) and hybridized to a microanay panel spotted with the relevant probes (SEQ ID NOS 89, 90, 101, 102, 107, 108). After stringent hybridization and wash, the slide was scanned in a fluorescent scanner. All spots showed hybridization. In a separate experiment, labeled individual PCR product from the virus or the bacteria was hybridized to the same panel and only specific hybridizations were observed.
  • FtsY and RecA loci for the identification for twelve different bacteria: Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Haemophilus influenzae, Enter ococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Bacteroides fragilis, Neisseria meningitidis, and E. coli.
  • FtsY and RecA sequences from each of the bacteria was amplified and individually labeled. Each PCR product was then hybridized to a microanay panel containing all the relevant probes under stringent conditions.
  • RNA was extracted from human RSV virus and cDNA was made using a commercial kit (Promega, Cat # A 1260) SEQ ID NO 106.
  • the cDNA was then mixed with extracted genomic DNA from Candida albicans and Mycoplasma pneumonia, and amplified through a 40-cycle PCR.
  • the primers used were SEQ ID NOS 11, 13, 24, 26, 105, and 106.
  • the PCR product was labeled using Alexa Fluor 546 Kit (Molecular Probes), and hybridized to a microanay panel spotted with SEQ NOS 62, 65, 76, 79, 107, and 108.
  • One embodiment of the present invention is to carry out the hybridization in a microanay format, e.g., spotting the hybridization probes as a panel or panels onto glass surface.
  • oligonucleotides does not bind to glass surface easily.
  • Various techniques or methods have been utilized to achieve efficient coupling. In general, these method entail introducing a reactive group into the oligonucleotide and derivatizing the glass surface for coupling (Zammatteo et al, 2000). Generally, the coupling is very inefficient. This is in part due to the fact that surface reaction (where diffusion is the rate- limiting step) is less efficient than the same reaction in solution. Furthermore, part of the reaction used for coupling, i.e. Schiff s base formation, is reversible.
  • the present invention improves the coupling efficiency in two ways.
  • One is to apply an electrostatic potential pe ⁇ endicular to the coupling surface, drawing oligonucleotide or negatively charged molecules to the surface.
  • the other is to use Epoxy derivatized glass surface for the coupling.
  • the Epoxy group is preferably a three member ring, the most active one of this family of compounds. After base-catalyzed ring opening, it can react with a number of functional groups, such as -NH 2 , -OH, and -SH and has been widely used to couple proteins to solid support.
  • an aminated oligonucleotide with a fluorescein label was used.
  • the slide was washed twice with 0.1% SDS, with vigorous shaking for about two minutes each; then washed once with boiling deionized H 2 O for 5 minutes. After a series of fluorescent standards were spotted onto the dried slide, the slide was scanned in a CCD-based fluorescent scanner. The percentage of coupling was defined as the amount of labeled oligonucleotide retained on the slide after the wash divided by the amount of the oligonucleotide originally spotted at the same spot. Table 2 summarizes the improvement on the coupling reaction. Table 2.
  • aldehyde-derivatized glass slides are widely used for coupling aminated oligonucleotides, it is quite inefficient. Applying an electric field increases the coupling efficiency three- fold. The electric field likely moves more negatively charged oligonucleotides to the glass surface, leading to an increase in local concentration and hence more coupling.
  • the Epoxy-derivatized glass surface generates more efficient coupling. In fact, a saturated mono-layer of oligonucleotides on the glass slide can be easily achieved with this method, though it is undesirable for subsequent hybridization reactions. It is conceivable that the coupling efficiency may be further improved by altering other parameter(s), such as increasing the voltage potential across the coupling surface or reducing the ionic strength of the oligonucleotide solution (e.g. lowering the buffer concentration).
  • FtsY gene sequence from B. fragilis has not been published prior to this invention.
  • an equimolar primer mixture was made using FtsY primers from E.coli (SEQ ID NOS 1 and 15), Chlamydia pneumoniae (SEQ ID NOS 1 and 15), Chlamydia pneumoniae (SEQ ID NOS 1 and 15), Chlamydia pneumoniae (SEQ ID NOS 1 and 15), Chlamydia pneumoniae (SEQ ID NOS)
  • nucleotide and protein sequences are refened to and were utilized in various examples mentioned and/or described in the Specification. All of the sequences were obtained through NCBI server (publicly available database), except as otherwise noted.
  • Bacteroides fragilis SEQ ID NO 113 Chlamydia pneumoniae Genbank
  • Contig894 Mycoplasma pneumoniae Genbank AE000040.1 Pseudomonas aeruginosa Genbank AF214677.1 Staphylococcus aureus OUACGT_1280

Abstract

L'invention concerne des procédés et des compositions utilisés pour l'identification rapide de micro-organismes. L'invention concerne également des procédés et des compositions servant à l'identification rapide et simultanée dans un échantillon biologique de multiples micro-organismes, comportant des bactéries, des champignons, des virus et de la levure. L'invention fait également état de procédés et de compositions employant de techniques d'amplification et d'hybridation de séquence spécifique, afin de détecter d'une séquence de polynucléotide spécifique d'une espèce dans un échantillon. L'invention présente aussi de nouveaux procédés associant des sondes d'oligonucléotides à des surfaces en verre à l'efficacité améliorée.
EP00980462A 1999-11-16 2000-11-16 Procede d'identification rapide et precise de micro-organismes Withdrawn EP1261716A2 (fr)

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US47945700A 2000-01-06 2000-01-06
PCT/US2000/031579 WO2001036683A2 (fr) 1999-11-16 2000-11-16 Procede d'identification rapide et precise de micro-organismes

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US20050202414A1 (en) * 2001-11-15 2005-09-15 The Regents Of The University Of California Apparatus and methods for detecting a microbe in a sample
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DE10244456A1 (de) * 2002-09-24 2004-04-15 Hain Lifescience Gmbh Verfahren zum Nachweis und Differenzierung von Bakterien
FI113549B (fi) * 2002-11-19 2004-05-14 Mobidiag Oy Diagnostinen menetelmä hengitystieinfektioita aiheuttavien bakteerien osoittamiseksi ja tunnistamiseksi ja menetelmässä käyttökelpoinen alukeseos
FR2860801B1 (fr) * 2003-10-10 2007-09-21 Bertin Technologies Sa Methode de detection rapide de micro-organismes sur puces a adn
EP2280085A3 (fr) * 2004-11-01 2011-02-23 George Mason University Compositions et méthodes pour le diagnostic de troubles du colon
RU2348695C2 (ru) * 2006-05-23 2009-03-10 Закрытое акционерное общество "Молекулярно-медицинские технологии" Дифференцирующий и специфический олигонуклеотиды для идентификации последовательностей днк инфекционных агентов в биологических материалах, способ видовой идентификации инфекционных агентов, биочип и набор для осуществления этого способа
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