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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
  • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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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
  • 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
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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
  • C12Q1/6876—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
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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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
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

• This invention is in the field of molecular diagnostics. Specifically, the invention relates to the simultaneous detection of a plurality of distinct biological entities in a synchronous amplification reaction(s).
• the compositions and methods of the present invention facilitate large-scale and high throughput detection of biological entities suspected to be present in a variety of biological, industrial, or environmental samples.
• the methods of the present invention are also useful in monitoring the treatment of a disease caused by one or more pathogenic biological entities.
• PCR polymerase chain reaction
• the multiplex PCR technique again exhibits several intrinsic drawbacks.
• developing a multiplex PCR protocol that yields • equivalent amounts of each PCR product can be difficult and laborious. This is due to variations in annealing rates and temperature of the primers in the reaction as well as varying polymerase extension rates for each sequence at a given Mg 2+ concentration.
• primer, Mg 2+ , and salt concentrations, along with annealing temperatures are adjusted to balance primer annealing rates and polymerase extension rates in the reaction.
• compositions and methods applicable for rapid and accurate detection of multiple biological entities, especially for those that are present in extremely small quantities.
• development of these compositions and methods should greatly facilitate screening for pathogenic biological entities in a diversity of biological or environmental samples.
• the present invention satisfies these needs and provides related advantages as well.
• SUMMARY OF THE INVENTION A principal aspect of the present invention is the design of a rapid and sensitive genetic analysis that is amenable for large-scale detection of multiple biological entities. Accordingly, the present invention provides a method of simultaneously detecting a plurality of distinct biological entities that are suspected to be present in a test sample.
• the method involves the steps of a) placing aliquots of nucleic acids present in the test sample into at least two sites of a multi-site test device, each of said sites containing a nucleic acid primer pair that yields a distinct population of amplified nucleic acid fragments representative of one or a subset of biological entities; b) simultaneously carrying out a nucleic acid amplification reaction at each of said sites; and c) detecting amplified nucleic acid fragments in each test site, wherein the presence of the amplified nucleic acid fragments in more than one test site is indicative of the presence of a plurality of distinct biological entities present in the sample.
• the nucleic acid amplification reaction comprises one or more reactions selected from the group consisting of polymerase chain reaction
• PCR reverse transcription polymerase chain reaction
• RT-PCR reverse transcription polymerase chain reaction
• LCR-PCR ligase chain polymerase chain reaction
• TMA transcription mediated amplification
• NASBA nucleic acid sequence-based amplification
• the nucleic acid primer pairs employed for the amplification reaction may be conjugated to a detectable label such as a radioisotope label, a luminescent label, a colorimetric label, or an enzyme-based label.
• the multi-site test device employed in the synchronous detection method may optionally contain at least one test site having a reagent that specifically binds to one or a subset of biological entities or reacts with a by-product of the one or the subset of biological entities suspected to be present in the sample.
• the reagent is an antibody or a functional fragment thereof that is specific for the biological entity under investigation.
• the method further comprises the steps of a) contacting a target biological entity suspected to be present in the test sample with the antibody contained in selected test sites under conditions suitable for target-antibody complex formation; and b) detecting the formation of target-antibody complex, wherein the formation of such complex is indicative of the presence of the biological entity to which the antibody binds.
• the present invention provides a method of detecting differential representation of a multiplicity of biological entities suspected to be present in at least two test samples.
• the method comprises the following steps: (a) amplifying nucleic acids present in a first test sample using a multi-site test device, wherein the test device has at least two test sites each containing a nucleic acid primer pair that yields a distinct population of amplified nucleic acid fragments representative of one or a subset of biological entities; (b) detecting the amplified nucleic acid fragments in each site that form a first amplification pattern representative of the multiple biological entities present in the first sample; (c) amplifying nucleic acids present in a second test sample using said multi- site test device; (d) detecting the amplified nucleic acid fragments in each site that form a second amplification pattern representative of the multiple biological entities present in the second sample; and (e) comparing the amplification patterns, thereby detecting the . differential representation of a multiplicity of biological entities present in the test samples.
• the amplification patterns are generated by the same multi-site test device. In another aspect, the amplification patterns are
• the present invention also provides a kit for simultaneous detection of a plurality of distinct biological entities that are suspected to be present in a test sample.
• the invention kit comprises (a) reactants necessary for an amplification reaction; and (b) a multi-site test device that is adaptable for a synchronous nucleic acid amplification reaction, wherein the test device has at least two test sites each containing a nucleic acid primer pair that yields a distinct population of amplified nucleic acid fragments representative of one or a subset of biological entities; and wherein the multi-site test device optionally has one or more sites serving as positive or negative control(s).
• the kit may further comprise a test site containing a reagent that specifically binds to one or a subset of biological entities or reacts with a by-product of the one or the subset of biological entities suspected to be present in the sample.
• the kit may be employed for detecting blood-born pathogens, those that are associated with a variety of infectious diseases including but not limited to respiratory and sexually transmitted diseases.
• This invention further provides a computer-based system for detecting the presence or absence of a plurality of distinct biological entities in a test sample, said presence is indicated by similarities in amplification patterns that are generated by amplifying nucleic acids derived from a test and a control sample using the invention kit.
• the computer-based system generally includes a) a data storage device comprising a reference amplification pattern and a test amplification pattern, wherein the reference amplification pattern is generated by amplifying nucleic acids of a control with the primer pairs contained in the kit of this invention; and wherein the test amplification pattern is generated by amplifying nucleic acids of the test sample with the same primer pairs; b) a search device for comparing the test amplification pattern to the reference amplification pattern of the data storage device of (a) to detect the similarities in amplification patterns; and c) a retrieval device for obtaining said similarity in amplification patterns of (b).
• a computer readable medium having recorded thereon an array of amplification patterns generated by amplifying nucleic acids derived from a plurality of test samples using a kit of this invention.
• the computer-implemented method comprises the steps of (a) providing a database comprising amplification patterns generated by amplifying nucleic acids derived from at least two test samples using an invention kit; (b) receiving at least two amplification patterns for comparison; (c) determining the differences, if any, in the selected amplification patterns; and (d) displaying the results of said determination.
• All methods, kits, computer-systems and computer readable medium embodied in the present invention may be applied to detecting a plurality of distinct biological entities present in a biological, industrial, or environmental sample.
• Members of the plurality of distinct biological entities to be tested may differ in one or more of the characteristics selected from the group consisting of phylum, family, genus, species and strain of origin.
• the one or the subset of the biological entities is a microorganism selected from the group consisting of bacterium, virus, fungus, and protozoa. More preferably, the virus is a hepatitis-causing virus such as HAN, HBN, HCN, HDN, HEN, HGN, TTN or any subtype thereof.
• polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
• polynucleotide and “nucleotide” as used herein are used interchangeably.
• Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
• polynucleotides a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
• sequence of nucleotides may be interrupted by non-nucleotide components.
• a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
• a "fragment” or “segment” of a nucleic acid is a small piece of that nucleic acid.
• a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
• primer refers to a short polyonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a polymerase and at a suitable temperature and pH.
• the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method.
• PCR polymerase chain reaction
• Luminescence is the term commonly used to refer to the emission of light from a substance for any reason other than a rise in its temperature.
• atoms or molecules emit photons of electromagnetic energy (e.g., light) when then move from an "excited state” to a lower energy state (usually the ground state); this process is often referred to as "radiative decay”.
• electromagnetic energy e.g., light
• photoluminescence the luminescence process is referred to as "photoluminescence”.
• the exciting cause is an electron, the luminescence process is referred to as "electroluminescence”.
• electroluminescence results from the direct injection and removal of electrons to form an electron-hole pair, and subsequent recombination of the electron-hole pair to emit a photon.
• Luminescence which results from a chemical reaction is usually referred to as “chemiliiminescence”.
• Luminescence produced by a living organism is usually referred to as “bioluminescence”.
• photoluminescence is the result of a spin-allowed transition (e.g., a single-singlet transition, triplet-triplet transition)
• the photoluminescence process is usually referred to as "fluorescence”.
• fluorescence emissions do not persist after the exciting cause is removed as a result of short-lived excited states which may rapidly relax through such spin-allowed transitions.
• photoluminescence is the result of a spin-forbidden transition (e.g. , a triplet-singlet transition)
• the photoluminescence process is usually referred to as "phosphorescence”.
• phosphorescence emissions persist long after the exciting cause is removed as a result of long-lived excited states which may relax only through such spin-forbidden transitions.
• a "luminescent label" of the present invention may have any one of the above- described properties.
• a “linear sequence” or a “sequence” is an order of nucleotides in a polynucleotide in a 5' to 3' direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polynucleotide.
• a “partial sequence” is a linear sequence of part of a polynucleotide which is known to comprise additional residues in one or both directions.
• a linear sequence of nucleotides is "identical" to another linear sequence, if the order of nucleotides in each sequence is the same, and occurs without substitution, deletion, or material substitution. It is understood that purine and pyrimidine nitrogenous bases with similar structures can be functionally equivalent in terms of Watson-Crick base-pairing; and the inter-substitution of like nitrogenous bases, particularly uracil and thymine, or the modification of nitrogenous bases, such as by methylation, does not constitute a material substitution.
• RNA and a DNA polynucleotide have identical sequences when the sequence for the RNA reflects the order of nitrogenous bases in the polyribonucleotides, the sequence for the DNA reflects the order of nitrogenous bases in the polydeoxyribonucleotides, and the two sequences satisfy the other requirements of this definition. Where one or both of the polynucleotides being compared is double-stranded, the sequences are identical if one strand of the first polynucleotide is identical with one strand of the second polynucleotide.
• a linear sequence of nucleotides is "essentially identical" to another linear sequence, if both sequences are capable of hybridizing to form a duplex with the same complementary polynucleotide.
• hybridize as applied to a primer refers to the ability of the primer to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues in a hybridization reaction. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
• the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
• the hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. Sequences that hybridize under conditions of greater stringency are more preferred. It is understood that hybridization reactions can accommodate insertions, deletions, and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align.
• essentially identical sequences of about 40 nucleotides in length will hybridize at about 30°C in 10 x SSC; preferably, they will hybridize at about 40 °C in 6 x SSC; more preferably, they will hybridize at about 50 °C in 6 x SSC; even more preferably, they will hybridize at about 60 °C in 6 x SSC, or at about 40 °C in 0.5 x SSC, or at about 30 °C in 6 x SSC containing 50% formamide; still more preferably, they will hybridize at 40 °C or higher in 2 x SSC or lower in the presence of 50% or more formamide.
• sequences that correspond or align more closely to the invention disclosed herein are comparably more preferred.
• essentially identical sequences are at least about 80% identical with each other, after alignment of the homologous regions.
• the sequences are at least about 85% identical; more preferably, they are at least about 90% identical; more preferably, they are at least about 95% identical; still more preferably, the sequences are 100% identical.
• nucleic acid sequences are essentially identical, a sequence that preserves the functionality of the nucleic acid with which it is being compared is particularly preferred. Functionality may be established by different criteria, such as ability to hybridize with a target polynucleotide, ability to effectively amplify a target sequence to eld a substantially homogenous multiplicity of products, and the ability to extend the 3' end sequence complementary to a target sequence in a nucleotide sequencing reaction.
• a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
• “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
• T m (°C) 2 x (A+T) + 4 x (C+G)
• A, T, G and C represent the number of adenosine, thymidine, guanosine and cytosine residues in the primer.
• Primer pairs employed in the detection system of the present invention generally have similar melting temperatures.
• the difference between the highest and the lowermost melting temperatures of the primer pairs contained in the plurality of test sites is less than 15 °C, more preferably less than 10 °C, and even more preferably less than 5 °C.
• a amplification pattern "database” denotes a set of stored data which represent a collection of amplification patterns generated by amplifying nucleic acids derived from a plurality of test samples using the multi-site test device of the present invention.
• a "subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
• a “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive” or “negative”. For example, where the purpose of the experiment is to detect a plurality of distinct biological entities, it is generally preferable to use a positive control (a subject or a sample from a subject containing the biological entity suspected to be present in the test sample), and a negative control (a subject or a sample from a subject lacking the biological entity).
• the term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one antibody combining site.
• the term includes both polyclonal and monoclonal antibodies.
• An “antibody combining site” or “binding domain” is formed from the folding of variable domains of an antibody molecule(s) to form three- dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows an immunological reaction with the antigen.
• An antibody combining site may be formed from a heavy and/or a light chain domain (VH and NL, respectively), which form hypervariable loops which contribute to antigen binding.
• the term “antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, univalent antibodies, the Fab proteins, and single domain antibodies.
• An antibody “specifically binds" to a polypeptide if it binds with greater affinity or avidity than it binds to other reference polypeptides or substances.
• Antigen as used herein means a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor.
• Antigens can include peptides, proteins, glycoproteins, polysaccharides and lipids; portions thereof and combinations thereof.
• the antigens can be those found in nature or can be synthetic.
• Phathogen refers to any biological substance(s) that causes directly or indirectly a disease state in a subject.
• a central aspect of the present invention is the design of a rapid, sensitive genetic analysis that is amenable for high-throughput detection of multiple biological entities. Distinguished from the previously described genetic analyses, the present invention incorporates a multiple test format that permits simultaneous screening of various biological and environmental constituents.
• the present invention provides a method of simultaneously detecting the presence or absence of a plurality of distinct biological entities that are suspected to be present in a biological sample.
• the method comprises the following steps: a) placing aliquots of nucleic acids present in the test sample into at least two sites of a multi-site test device, each of said sites containing a nucleic acid primer pair that yields a distinct population of amplified nucleic acid fragments representative of one or a subset of biological entities; b) simultaneously carrying out a nucleic acid amplification reaction at each of said sites; and c) detecting amplified nucleic acid fragments in each test site, wherein the presence of the amplified nucleic acid fragments in more than one test site is indicative of the presence of a plurality of distinct biological entities present in the sample.
• Multi-site testing device
• the multi-site test device embodied in the present invention comprises a plurality of compartments separated from each other by a physical barrier resistant to the passage of liquids and forming an area or space referred to as "test site". At least two of the test sites of the device contain a primer pair that yields a distinct population of amplified nucleic acid fragments representative of one or a subset of biological entities suspected to be present in a biological sample. Optionally, one or more test sites of the device may also contain a reagent that specifically binds to one or a subset of biological entities or reacts with a by-product of the one or the subset of biological entities present in the test sample.
• the test sites contained within the device can be arrayed in a variety of ways.
• the test sites are arrayed on a multi-well plate. It typically has the size and shape of a microtiter plate having 96 wells arranged in an 8x12 format. ' One advantage of this format is that instrumentation already exists for handling and reading assays on microtiter plates; extensive re-engineering of commercially available fluid handling devices is thus not required.
• the test device may vary in size and configuration. It is contemplated that various formats of the test device may be used which include, but are not limited to thermocycler, lightcycler, flow or etched channel PCR, multi-well plates, tube strips, microcards, petri plates, which may contain internal dividers used to separate different media placed within the device, and the like.
• test device employed in the present invention.
• material with which the device is fabricated does not interfere with amplification reaction and/or immunoassays.
• a preferred multi-site testing device is made from one or more of the following types of materials: (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polypropylene, and polystyrene.
• test Samples encompasses any samples suspected to contain biological entities. It is not intended to be limited as regards to the source of the sample or the manner in which it is made. Generally, the test sample can be biological and/or environmental samples. Biological samples may be derived from human or other animals, body fluid, solid tissue samples, tissue cultures or cells derived therefrom and the progeny thereof, sections or smears prepared from any of these sources, or any other samples that contain nucleic acids. Preferred biological samples are body fluids including but not limited to urine, blood, cerebrospinal fluid, spinal fluid, sinovial fluid, semen, ammoniac fluid, cerebrospinal fluid (CSF), and saliva.
• body fluids including but not limited to urine, blood, cerebrospinal fluid, spinal fluid, sinovial fluid, semen, ammoniac fluid, cerebrospinal fluid (CSF), and saliva.
• biological sample may include food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste.
• Environmental samples are derived from environmental material including but not limited to soil, water, sewage, cosmetic, agricultural and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable, and non-disposable items.
• Primer pairs employed in the synchronous amplification reaction of the present invention generally exhibit the following characteristics: a) each individual primer of a pair is non-self hybridizing, containing at least about 10 nucleotides, having a melting temperature within the range of 35°C to 85°C; and b) the selected pair is non-cross hybridizing, and c) the selected pair produces a substantially homogenous population of amplified fragments that is representative of the biological entity to be tested in a primer- dependent amplification reaction.
• non-self hybridizing as applied to a primer, means that the primer is incapable of forming intra-molecular duplex mediated by hydrogen-bonding between the bases of the nucleotide residues within the primer in an amplification reaction as defined herein.
• Non-cross hybridizing as applied to primer pairs means that the two individual primers do not hybridize and form a duplex stabilized by hydrogen-bonding between complementary bases in the two primers. In determining whether a primer is non-self hybridizing or a primer pair is non-cross hybridizing, sequence complementarity within the primer and between the primers should be examined. A non-self hybridizing primer typically lacks internal sequence homology necessary for duplex formation. Likewise, a pair of non-cross hybridizing primers does not share a sufficient amount of sequence homology to form a stable double-stranded structure.
• a "substantially homogenous population" of amplified products refers to a mixture of DNA fragments in which the undesired DNA fragments constitute less than about 25% of the total amount of products. Preferably, the undesired fragments constitute less then 20%, more preferably 15% and even more preferably 10%.
• Product homogeneity may be indicated by a number of means, such as agarose gel electrophoresis of the amplified products, followed by visualizing a single DNA band upon staining the gel. Product homogeneity may also be determined by sequencing or quantitative Southern blot analysis using the same primer pair employed in the amplification as probe.
• a proportion of 75% or above between the intensities of the band of predicted size and the bands of unexpected sizes indicates that the amplified products are substantially homogenous.
• the proportion is above 80%, more preferably it is above 85%, even more preferably it is above 90%, still more preferably it is above 95%.
• primer pairs having the above-mentioned characteristics.
• a selected primer pair is specific to the target biological entity to be tested.
• Such unique primer pair lacks substantial sequence homology with any other non-target polynucleotide sequences when optimally aligned, and thus having a low probability of cross-hybridizing with sequences found in any other organisms.
• Sequence alignment and homology searches are often determined with the aid of computer methods.
• a variety of software programs are available in the art. Non-limiting examples of these programs are Blast 1 , Fasta 2 , DNA Star, MegAlign, and GeneJocky. Any sequence databases that contains DNA sequences corresponding to a gene or a segment thereof can be used for sequence analysis. Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS. Sequence similarity can be discerned by aligning the probe sequence against a DNA sequence database. Common parameters for determining the extent of homology set forth by one or more of the aforementioned alignment programs include p value and percent sequence identity.
• P value is the probability that the alignment is produced by chance.
• the p value can be calculated according to Karlin et al. (1990) Proc.Natl. Acad. Sci 87: 2246.
• the p value can be calculated using a heuristic approach such as the one programmed in Blast.
• Percent sequence identity is defined by the ratio of the number of nucleotide matches between the query sequence and the known sequence when the two are optimally aligned.
• a probe sequence is considered to have no substantial homology when the region of alignment exhibits less than 20% of sequence identity, more preferably less than 10% identity, even more preferably less than
• a second consideration of designing the subject primer pair is to select primers which have minimal secondary structures and internal sequence homology. Extensive homology within the probe due to e.g., inverted repeats, promotes self-hybridization, and thus interferes with the binding of the primers to the target sequences.
• primer pairs having similar thermal profiles and internal stability are chosen. This can be achieved by selecting primers with comparable length and G/C content.
• primers Preferably, primers have 50 to 60% G+C composition.
• primers to be employed have a minimal length of about 10 nucleotides, more preferably about 15 nucleotide, and even more preferably about
• the primer pairs contained in at least two of the test sites test device must be capable of yielding substantially homogenous populations of nucleotide fragments
• types of primers added to the test device employed by the present invention may differ in the nature of the target polynucleotides that the primers are capable of amplifying, and specifically the types of genes of a particular biological entities to which the primers correspond.
• the types of genes may be characterized based on one or more of the following features: species origin, sub-species origin, primary structural similarity, involvement in a particular biological process, and their association with a particular disease or disease stage.
• the primers useful for the present invention comprise sequences corresponding to genes representative of viruses, bacteria, fungi, protozoa, or other families of microorganisms. In another aspect, the primers yield amplified products specific for a type or a sub-type of virus, bacterium, fungus and protozoa.
• Viruses generally can be classified into two groups consisting of DNA viruses and RNA viruses.
• the main pathogenic viruses include but are not limited to Smallpox, Adenovirus, Influenza, Herpes simplex, Varicella-zoster Epstein-Barr, Hepatitis A, Hepatitis B, and other hepatitis- causing viruses, HIV, Papillomavirus, Polio, Coxsackie, other enterovirus, Mumps,
• Measles, Rubella, Rhinovirus, Arbovirus encephalitis, Respiratory syncytial virus, Parvovirus, and Rotavirus include Staphylococcus aureus, Staphylococcus epidermidis, Group A streptococci, ⁇ -Hemolytic streptococci, a-Hemolytic streptococci, Pneumococcus (S. pneumoniae), Meningococcus (Neisseria meningitidis), Gonococcus (N. gonorrhoeae), Haemophilus influenzae,
• the selected primers are capable of amplifying genes implicated in a particular disease. Such genes include but are not limited to those associated with sexually transmitted diseases, respiratory diseases, autoimmune diseases, obesity, hypertension,
• the primers employed in the synchronous amplification reaction comprise sequences complementary to blood-born pathogens.
• Non-limiting exemplary pathogens present in a blood sample include Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influnzae, Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.
• MSRA methicillin-resistant Staphylococcus aureus
• Staphylococcus aureus Staphylococcus hominis
• Enterococcus faecalis Pseudomonas aeruginosa
• primers employed in the present invention amplify gene sequences conferring resistance to a variety of antibiotics used in treating a diversity of infectious diseases.
• the primer pairs chosen for the synchronous amplification reaction facilitate the screening for resistance to amikacin, amoxicillin, ampicillin, carbapenem, cephalosporin drug class, chlormaphenocol, clindamycin, ciprofloxacin, erythromycin, gentamicin, methicillin, nitrofurantoin, oxacillin, penicillin G, piperacillin, tetracycline, trimethiprim-sulfidamide, spectinomycin, or vancomycin.
• a variation of this aspect of the invention is the use of these primer pairs to screen for and/or confirm the presence of genetically engineered crops in which one or more marker genes are exogenously introduced.
• marker genes for modifying natural crops include but are not limited to antibiotic resistance genes or herbicide resistance gene.
• an array of primer pairs each being specific for a type or a subtype of hepatitis-causing virus, is employed for a synchronous screening of different types of hepatitis-causing viruses.
• Any oligonucleotide sequences capable of amplifying polynucleotides that are representative of hepatitis-causing virus genomes are candidate primers.
• Exemplary hepatitis-causing viruses include HAV, HBV, HCV, HDV, HGV, TTV and subtypes thereof.
• Non-limiting exemplary primers include oligonucleotide sequences shown in SEQ ID NOS. 1-63. Encompassed by the invention are also primers essentially identical to the aforementioned primer sets.
• primers retain the characteristics and functionalities of the two exemplified primer pairs, even if they may contain mismatched nucleotide sequences. It is known in the art that "perfectly matched" primer is not needed for a specific amplification. Minor changes in primer sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the amplification specificity. For instance, primers may contain 5' extensions or mismatches for incorporating restriction enzyme sites, an ATG start codon, or promoter sequences into the target sequence. Mismatched bases can also be placed internally. In general, as much as 20% base-pair mismatch (when optimally aligned) can be tolerated.
• the primers employed by the present invention comprise control primers, positive or negative, for comparison purposes.
• control primers positive or negative, for comparison purposes.
• selection of an appropriate control primer is dependent on the test sample initially selected and the biological entities which are under investigation.
• the primer pairs used in this invention can be obtained by chemical synthesis, recombinant cloning, e.g. PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired primer pairs by employing a DNA synthesizer or ordering from a commercial service.
• primers may be conjugated to a detectable label.
• Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
• a wide variety of appropriate detectable labels are known in the art, which include luminescent labels, radioactive isotope labels, enzymatic or other ligands.
• the labels may be incorporated by any of a number of means well known to those of skill in the art.
• the label is simultaneously incorporated during the amplification step.
• PCR polymerase chain reaction
• labeled primers or labeled nucleotides can provide a labeled amplification product.
• transcription reaction in which RNA is converted into DNA using a labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) or a labeled primer, incorporates a detectable label into the transcribed nucleic acids.
• a labeled nucleotide e.g. fluorescein-labeled UTP and/or CTP
• a labeled primer incorporates a detectable label into the transcribed nucleic acids.
• amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity.
• Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, Taq polymerase, Tth polymerase, pfu polymerase and/or RNA polymerases such as reverse transcriptase.
• a preferred amplification method is PCR.
• General procedures for PCR are taught in U.S. Patent Nos. 4,683,195 (Mullis et al.) and 4,683,202 (Mullis et al.).
• optimal PCR conditions used for each application reaction are generally empirically determined or estimated with a computer software commonly employed by artisans in the field. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg 2+ ATP concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides.
• the template nucleic acids are denatured by heating to at least about 90°C for 2 to 10 minutes prior to the polymerase reaction.
• PCR reaction contains about 100 ng template nucleic acids, 20 uM of upstream and downstream primers, and 0.05 to 0.5 mm dNTP of each kind, and 0.5 to 5 units of commercially available DNA polymerases.
• RT-PCR reverse transcription PCR reaction
• a reverse transcriptase first coverts RNA molecules to single stranded cDNA molecules, which are then employed as the template for subsequent amplification in the polymerase chain reaction.
• the reverse transcriptase is generally added to the reaction sample after the target nucleic acids are heat denatured.
• the reaction is then maintained at a suitable temperature (e.g. 30-45°C) for a sufficient amount of time (10-60 minutes) to generate the cDNA template before the scheduled cycles of amplification take place.
• Tth DNA polymerase can be employed for RT- PCR.
• Such reaction is particularly useful for detecting the biological entity whose genetic information is stored in RNA molecules.
• Non-limiting examples of this, category of biologieal entities include RNA viruses such as HIV and hepatitis-causing viruses such as HAV and HCV.
• Another important application of RT-PCR embodied by the present invention is the simultaneous quantification of biological entities based on the mRNA level detected in the test sample. An additional application is to assess viability.
• One of skill in the art will appreciate that if a quantitative result is desired, caution must be taken to use a method that maintains or controls for the relative copies of the amplified nucleic acids. Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR can involve simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction.
• One preferred internal standard is a synthetic AW106 cRNA.
• the AW106 cRNA is combined with RNA isolated from the sample according to standard techniques known to those of skill in the art.
• the RNA is then reverse transcribed using a reverse transcriptase to provide cDNA.
• the cDNA sequences are then amplified (e.g., by PCR) using labeled primers.
• the amplification products are separated, typically by electrophoresis, and the amount of radioactivity (proportional to the amount of amplified product) is determined.
• the amount of mRNA in the sample is then calculated by comparison with the signal produced by the known AW106 RNA standard.
• Detailed protocols for quantitative PCR are provided in PCR Protocols, A Guide to Methods and Applications, Innis et al, Academic Press, Inc. N. Y., (1990).
• LCR-PCR ligase chain polymerase chain reaction
• the method involves ligation of a pool of nucleic acids derived from a test sample to a set of primer pairs, each having a target-specific portion and a short anchor sequence unrelated to the target sequences. A second set of primers containing the anchor sequence is then used to amplify the target sequences linked with the first set of primers.
• Procedures for conducting LCR-PCR are well known to artisans in the field, and hence are not detailed herein (see, e.g., WO 9745559, WO 9803673, WO 9731256, and U.S. Patent No. 5,494,810).
• the aforementioned amplification methods are highly sensitive, amenable for large- scale identification of multiple biological entities using extremely small quantities of test sample. Because of the extraordinar sensitivity of these amplification techniques, precautions are required to avoid contamination of reactants and amplified products contained in each test site of a multi-site testing device. A variety of procedures to minimize cross- contamination are known in the art.
• One particularly useful technique is to add and immobilize primers to each test site prior to the addition of test samples and other reactants required for an amplification reaction. Prior immobilization of primer pairs to each test site reduces the chance of non-specific amplification resulting from contaminating primer pairs. Any materials suitable for immobilizing liquid or solid oligonucleotides can be used.
• Such material should not substantially interfere or alter the specificity and efficiency of a PCR- based amplification reaction.
• Non-limiting examples of such materials include geling agent and wax.
• gelling agent refers to a class of compounds obtainable from natural sources or by synthetic means, which is viscous and becomes at least partially solidified at room temperature and atmospheric pressure.
• radiolabels may be detected using photographic film or a phosphoimager (for detecting and quantifying radioactive phosphate incorporation). Fluorescent markers may be detected and quantified using a photodetector to detect emitted light (see U.S. Patent No. 5,143,854 for an exemplary apparatus). Enzymatic labels are typically detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and finally colorimetric labels are detected by simply visualizing the colored label.
• the amplified DNA molecules are visualized by directly staining the amplified products with a DNA-intercalating dye.
• exemplary dyes include but not limited to S YBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold and ethidium bromide.
• the amount of luminescent dyes intercalated into the amplified DNA molecules is directly proportional to the amount of the amplified products, which can be conveniently quantified using a Flurolmager (Molecular Dynamics) or other equivalent devices according to manufacturers' instructions.
• Flurolmager Molecular Dynamics
• a variation of such an approach is gel electrophoresis of amplified products followed by staining and visualization of the selected intercalating dye.
• labeled oligonucleotide hybridization probes e.g. fluorescent probes such as FRET probes and colorimetric probes
• a specific amplification of the genome sequences representative of the biological entity being tested may be verified by sequencing or demonstrating that the amplified products have the predicted size, exhibit the predicted restriction digestion pattern, or hybridize to the correct cloned nucleotide sequences.
• a biological entity can be identified by amplifying its genetic material, by detecting the presence of proteins coded by its genome or the presence of a metabolic by-product of such biological entity.
• the multi-site test device employed by the invention methods includes optionally a reagent that specifically binds to a biological entity or reacts with a by-product produced by such entity.
• a preferred reagent is an antibody specific for an antigen or a class of antigens unique to the biological entity.
• an antibody “specifically binds to” or “specifically recognizes” a biological entity if the antibody can distinguish the biological entity to be tested from other biological entities or substances.
• the antibodies employed by methods of this invention encompass polyclonal and monoclonal antibodies. They include but are not limited to mouse, rat, rabbit and human antibodies. The antibodies may also be functionally equivalents and fragments thereof. A “functional equivalent” refers to an antibody or a fragment thereof, or any molecule having the antigen binding site (or epitope) of the antibody that cross-blocks an exemplified antibody when used in an immunoassay such as immunoblotting or immunoprecipitation.
• Antibody fragments include the Fab, Fab', F(ab') 2 , and Fv regions, or derivatives or combinations thereof. It will further be appreciated that encompassed within the definition of antibody fragment is single chain antibody that can be generated as described in U.S.
• the antibodies useful for the detection methods of this invention can be conjugated to a detectable agent or a hapten.
• the complex is useful to detect the polypeptide(s) (or polypeptide fragments) to which the antibody specifically binds in a sample, using standard immunochemical techniques such as immunohistochemistry as described by Harlow and Lane (1988), supra.
• labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include, but are not limited to, radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.
• the detection of a target biological entity in a test sample using the aforementioned antibodies can be performed before or after the synchronous amplification reaction. It is generally proceeded by contacting the antibodies with the sample under conditions that will allow the formation of antibody-target complexes.
• the reaction may be performed in solution, or on a solid support immobilized with the antibodies or the test sample proteins.
• the formation of the complexes is detected by a number of techniques known in the art.
• the antibodies may be conjugated with a label and unreacted antibodies may be removed from the complexes; the amount of remaining labels thereby indicating the amount of complexes formed.
• the amount of the target biological entities reactive with the antibodies embodied in the present invention can be quantified by standard quantitative immunoassays.
• the antigens representative of the biological entities may be mixed with a pre-determined non-limiting amount of the reagent antibodies specific for the antigens.
• the reagent antibodies may contain a directly attached label, such as an enzyme or a radioisotope, or a second labeled reagent may be added, such as anti-immunoglobulin or protein A.
• a solid-phase assay For a solid-phase assay, unreacted reagents are removed by washing. The amount of label captured in the complex is positively related to the amount of target protein present in the test sample.
• a competitive assay in which the target biological entity is tested for its ability to compete with a labeled analog for binding sites on the specific antibody. In this case, the amount of label captured is negatively related to the amount of target protein present in a test sample. Results obtained using any such assay on a test sample are compared with that of a suitable control sample. The selection of an appropriate control sample is dependent on the test sample initially selected and the biological entities which are under investigation.
• the biological entities to be tested are hepatitis-causing viruses
• one or more counterparts of non-hepatitis-causing viruses can be used as a control.
• a control matches the tissue, and or cell type the tested sample is derived from. It is also preferable to analyze the control and the tested sample in parallel.
• the detection method described herein provides a positional localization of the test site where specific amplification or formation of reagent-target complexes has taken place.
• the position of the test sites where these reactions took place correlates to the specificity of the primer pair or antibody, and hence the identity of the biological entity that is under investigation.
• the detection methods also may yield quantitative measurement of the amount of amplified products or the reagent-complexes formed at each test site and thus a direct measurement of the amount of each biological entity suspected to be present in the sample.
• a collection of the data pattern constitutes an "amplification pattern" that is representative of a multiplicity of biological entities present in a test sample.
• any discrepancies detected in the amplification patterns generated by amplifying target entity sequences derived from different test samples are indicative of the differential presence of a multiplicity of biological entities present in these test samples.
• the amplification patterns to be compared can be generated by the same multi-site test device. In such case, different patterns are distinguished by the distinct positions of the test sites in which specific amplifications are detected.
• the amplification patterns employed for the comparison are generated by different multi- site test devices.
• the test samples employed for the comparative amplification analysis may be biological samples derived from human or other animals. Such biological samples include but are not limited to body fluid, solid tissue samples, tissue cultures or cells derived therefrom and the progeny thereof, sections or smears prepared from any of these sources.
• biological sample may include food products and ingredients such as dairy items, vegetables, meat and meat by-products that are suspected to contain nucleic acids.
• environmental samples may also be used for comparative amplification analysis.
• Non-limiting examples of environmental materials include soil, water, sewage, agricultural and industrial samples, as well as samples obtained from food processing instruments, apparatus, equipment, disposable, and non-disposable items.
• Such comparative analysis may be extended to monitor the progress of a particular therapy by quantifying the relative amounts of a variety of pathogens present in the subject during the course of treatment. For instance, the detection and quantification of a diversity of opportunistic pathogens present in immunocompromised patients (e.g. HIV positive individuals) aid in the evaluation of a particular therapy.
• immunocompromised patients e.g. HIV positive individuals
• a "computer readable medium” refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories, such as magnetic/optical storage media.
• the term "recorded” refers to a process for storing information on computer readable medium.
• a skilled artisan can readily adopt any of the presently know methods for recording information on computer readable medium to generate manufactures comprising the amplification patterns of the present invention.
• a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon an array of amplification patterns of this invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information.
• a variety of data processor programs and formats can be used to store the array information of the present invention on a computer readable medium.
• the array information can be represented in a word processing file including but not limited to doc, txt, wpf, and formatted in commercially-available software such as WordPerfect and Microsoft Word, Excel, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, Informix, SQL or the like.
• the array information can also be represented in comma delimited file, tab delimited file, space delimited file, data interchange format (DIF), quatro pro file, SAS file, SPSS file, flat file, Dbase file, all adobe acrobat files: pdf, document template file, filemaker pro fp3 file, or the like.
• DIF data interchange format
• quatro pro file SAS file
• SPSS file flat file
• Dbase file all adobe acrobat files: pdf, document template file, filemaker pro fp3 file, or the like.
• a skilled artisan can readily adapt any number of data-processor structuring formats (
• the computer readable medium can be incorporated as part of the computer-based system of the present invention, and can be employed for a computer-based analysis as described below.
• the computer-based system of the present invention is designed to detect differential presence of a multiplicity of distinct biological entities by a difference in amplification patterns generated using a kit comprising the multi-site test device of the present invention.
• Such system comprises: a) a data storage device comprising a reference amplification pattern and a test amplification pattern, wherein the reference amplification pattern is generated by amplifying nucleic acids of a control with the primer pairs contained in a kit of claim 51; and wherein the test amplification pattern is generated by amplifying nucleic acids of the test sample with the same primer pairs; b) a search device for comparing the test amplification pattern to the reference amplification pattern of the data storage device of (a) to detect the similarities in amplification patterns; and c) a retrieval device for obtaining said similarity in amplification patterns of (b).
• a computer-based system includes hardware and software.
• the "data storage device” as part of the system refers to memory which can store reference amplification pattern(s) and test amplification pattern(s) that are generated with a kit comprising the multi-site test device of the invention.
• the invention multi-site test device is adaptable to simultaneous amplification of nucleic acids representative of distinct biological entities.
• the data-storage device may also include a memory access device which can access manufactures having recorded thereon the array information of the present invention.
• Non-limiting exemplary data storage devices are media storage, floppy drive, super floppy, tape drive, zip drive, syquest syjet drive, hard drive, CD ROM recordable (R), CD Rom rewritable (RW), M.D. drives, optical media, and punch cards/tape.
• the "search device” as part of the computer-based system encompasses one or more programs which are implemented on the system to compare the test amplification pattern to the reference amplification pattern in order to detect the differences in these amplification patterns.
• a variety of known algorithms are disclosed publicly and a variety of commercially available software useful for pattern recognition can be used in computer- . based systems of the present invention. Examples of array analysis software include Biodiscovery, HP, and any of those applicable for image analyses.
• the retrieval device includes program(s) which are implemented on the system to retrieve the differences in amplification patterns detected by the search device. Hardware necessary for displaying the detected device may also form part of the retrieval device.
• the storage, search, retrieval devices may be assemble as a PC, Mac, Apollo workstation (Cray), SGI machine, Sun machine, UNIX or LINUX based Workstations, Be OS systems, laptop computer, palmtop computer, and palm pilot system, or the like.
• the computer-implemented method comprises the following steps: (a) providing a database comprising amplification patterns generated by amplifying nucleic acids derived from at least two test samples using an invention kit described herein; (b) receiving at least two amplification patterns for comparison; (c) determining the differences, if any, in the selected amplification patterns; and (d) displaying the results of said determination.
• the determining step includes the step of calculating the differences between the amplification intensities of the amplified products localized in predetermined test site on the multi-site test device.
• kits containing a multi-site test device of this invention include those that allow simultaneous detection and or quantification of a plurality of distinct biological entities suspected to be present in a test sample.
• kits embodied by this invention include those that allow simultaneous detection and or quantification of a plurality of distinct biological entities suspected to be present in a test sample.
• Each kit necessarily comprises the reactants and a multi-site test device which render the synchronous amplification procedure possible.
• reagents include, but are not limited to labeled or unlabeled dNTP, polymerase, Mg 2+ , primers, and suitable buffers.
• the multi-site test device is adaptable for a synchronous nucleic acid amplification reaction, wherein the test device has at least two test sites each containing a nucleic acid primer pair that yields a distinct population of amplified nucleic acid fragments representative of one or a subset of biological entities; and wherein the multi-site test device optionally has one or more sites serving as positive or negative control(s).
• the multi-site device contained in the kits may include a reagent that specifically binds to one or a subset of biological entities, or a reagent that reacts with a by-product of the one or the subset of biological entities suspected to be present in the sample.
• the kit may contain reagents useful for extracting nucleic acids from a test sample.
• Each reagent or reactant can be supplied in a solid form or dissolved suspended in a liquid buffer suitable for inventory storage, and later for exchange or addition into the reaction medium when the test is performed.
• Suitable packaging is provided.
• the kit can optionally provide additional components that are useful in the procedure. These optional components include, but are not limited to, reagents for DNA extraction/isolation, buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.
• kits embodied in the invention can be employed to test a variety of biological samples, including body fluid, solid tissue samples, tissue cultures or cells derived therefrom and the progeny thereof, and sections or smears prepared from any of these sources.
• the kits may also be used to test a diversity of environmental samples such as surface matter, soil, water, agricultural and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable, and non- disposable items.
• the kits contain the reactants and device that can be employed for detecting a plurality of microorganisms including but not limited to bacterium, virus, fungus and protozoa.
• kits are designed to aid in the diagnosis of tuberculosis and concurrent detection of micro-plasmids that confer resistance to antibiotics such as ethanbutol, ethionamide, isoniazid, pyrazinamide, rifampin, and streptomycin, amikacin, aminosalicylate acid (teebacin), capreomycin, ciprofloxacin, clofazimine, cycloserine (seromycin), kanamycin, and ofloxaxin.
• antibiotics such as ethanbutol, ethionamide, isoniazid, pyrazinamide, rifampin, and streptomycin, amikacin, aminosalicylate acid (teebacin), capreomycin, ciprofloxacin, clofazimine, cycloserine (seromycin), kanamycin, and ofloxaxin.
• kits enable simultaneous identification of at least two kinds of blood-born pathogens selected from a non-limiting group that consists of Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Enter ococcus faecalis, Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.
• a non-limiting group that consists of Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Enter ococcus faecali
• kits allow simultaneous screening for a variety of sexually transmitted diseases selected from the following: gonorrhea (Neisseria gorrhoeae), syphilis (Treponena pallidum), clamydia (Clamyda tracomitis), nongonococcal urethritis
• hepatitis (Ureaplasm urealyticum), yeast infection (Candida albicans), chancroid (Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis), genital herpes (HSV type I & II), HIV I, HIV II and hepatitis A, B, C, G, as well as hepatitis caused by TTV.
• yeast infection Candida albicans
• chancroid Haemophilus ducreyi
• trichomoniasis Trichomonas vaginalis
• HSV type I & II genital herpes
• HIV I HIV II
• hepatitis A, B, C, G as well as hepatitis caused by TTV.
• kits are capable of synchronously detecting a multiplicity of respiratory pathogens including but not limited to Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus (MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphlococcus aureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae, Escherichia coli, Enterococcus faecalis, Serratia marcescens, Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans, Moraxie ⁇ la catarrhalis, Streptococcus pneumoniae, Citrobacter freundii, Enterococcus faecium, Klebsella oxytoca,
• MSRA methicillin-resistant Staphlococccus aureus
• Pseudomonas fluorscens Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii, Klebsella pneumoniae Legionella pneumophila, Mycoplasma pneumoniae, and Mycobacterium tuberculosis.
• diagnostic or prognostic procedures using the kits of this invention can be performed by clinical laboratories, experimental laboratories, practitioners, or private individuals.

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