WO2020096782A1 - Dosages universels ou à large plage et procédé de diagnostic spécifique d'échantillons à marqueurs multiples à l'aide d'un séquençage non optique - Google Patents

Dosages universels ou à large plage et procédé de diagnostic spécifique d'échantillons à marqueurs multiples à l'aide d'un séquençage non optique Download PDF

Info

Publication number
WO2020096782A1
WO2020096782A1 PCT/US2019/057866 US2019057866W WO2020096782A1 WO 2020096782 A1 WO2020096782 A1 WO 2020096782A1 US 2019057866 W US2019057866 W US 2019057866W WO 2020096782 A1 WO2020096782 A1 WO 2020096782A1
Authority
WO
WIPO (PCT)
Prior art keywords
primers
microorganisms
dna
specific
sample
Prior art date
Application number
PCT/US2019/057866
Other languages
English (en)
Inventor
Scot E. Dowd
Original Assignee
Dowd Scot E
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/182,140 external-priority patent/US20190062815A1/en
Application filed by Dowd Scot E filed Critical Dowd Scot E
Publication of WO2020096782A1 publication Critical patent/WO2020096782A1/fr

Links

Classifications

    • 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
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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

Definitions

  • the present invention relates in general to the field of diagnosing and monitoring industrial and environmental microbial processes, medical and veterinary diagnosis and medical and veterinary treatment, and more particularly, to universal or broad range assays and multi-tag sample specific diagnostic process using non-optical sequencing.
  • Microorganisms are traditionally identified by their ability to utilize different substrates as a source of carbon and nitrogen through the use of biochemical tests such as the API20ETM system (bioMerieux).
  • biochemical tests such as the API20ETM system (bioMerieux).
  • API20ETM system bioMerieux
  • clinical microbiology laboratories use methods including disk diffusion, agar dilution and broth microdilution.
  • the detection and identification of biological agents are important in determining the best course of treatment and / or eradication of the biological agent in natural infections, and other cases; such as, but not limited to, biological warfare.
  • identifications based on biochemical tests and antibacterial susceptibility tests are cost-effective, generally two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to identify the bacteria from clinical specimens as well as to determine their susceptibility to antimicrobial agents.
  • Clinical Specimens Tested in Clinical Microbiology Laboratories Most clinical specimens received in clinical microbiology laboratories are urine and blood samples. The remaining percentage of clinical specimens comprise various biological fluids including sputum, pus, cerebrospinal fluid, synovial fluid, respiratory tract aspirate, deep pus, ear aspirate, pleural and pericardial fluid, peritoneal fluid, and others. Infections of the urinary tract, the respiratory tract and the bloodstream are usually of bacterial etiology and require antimicrobial therapy. Typically all clinical samples received in a clinical microbiology laboratory are tested routinely for the identification of bacteria and antibiotic susceptibility.
  • the bactecTM system (from Becton Dickinson) and the bactalertTM system (from Organon Teklika corporation) are the two most widely used automated blood culture systems. These systems incubate blood culture bottles under optimal conditions for growth of most bacteria. Bacterial growth is monitored continuously to detect early positives by using highly sensitive bacterial growth detectors. Once growth is detected, a gram stain is performed directly from the blood culture and then used to inoculate nutrient agar plates. Subsequently, bacterial identification and susceptibility testing are carried out from isolated bacterial colonies with automated systems as described previously. Blood culture bottles are normally reported as negative if no growth is detected after an incubation of 6 to 7 days. Normally, the vast majority of blood cultures are reported negative.
  • DNA-Based Assays with any specimen. There is an obvious need for rapid and accurate diagnostic tests for the detection and identification of algae, archaea, bacteria, fungi and parasites directly from clinical specimens.
  • Common diagnostic methods involving DNA sequencing use florescence detected by a camera or laser and/or other optical method for signal detection and measurement.
  • the process of DNA sequencing specifically refers to the determination of the nucleotide order of a particular DNA fragment.
  • DNA-based technologies are rapid and accurate and offer a great potential to improve the diagnosis of infectious diseases [6-8]).
  • the Universal DNA probes and amplification primers which are objects of the present invention for DNA sequencing applications using non-optical base detection methods are applicable for the detection and identification of algae, archaea, bacteria, fungi, and parasites directly from any clinical specimen such as blood, urine, sputum, cerebrospinal fluid, pus, genital and gastro-intestinal tracts, skin or any other type of specimens.
  • These assays are also applicable for the detection and identification, or confirmation of organism identification from microbial cultures (e.g. blood cultures, bacterial or fungal colonies on nutrient agar, or liquid cell cultures in nutrient broth).
  • the DNA based tests proposed in this invention are superior in terms of both speed and accuracy to standard biochemical methods currently used for routine diagnosis from any clinical specimens in microbiology laboratories. Since these tests can be performed in less than 48 hours, they provide the clinician with new diagnostic tools which should contribute to a better management of patients with infectious diseases. Specimens from sources other than humans (e.g. other primates, birds, plants, mammals, farm animals, livestock, food products, environment such as water or soil, and others) may also be tested with these assays.
  • sources other than humans e.g. other primates, birds, plants, mammals, farm animals, livestock, food products, environment such as water or soil, and others
  • Specimens negative for bacteria would not be amplified by this assay.
  • those that are positive for any bacterium would give a positive amplification signal, and could be moved forward in the processing pipeline into sequencing analysis for organism identification.
  • hyper variable regions of conserved genes of fungi and parasites could serve to map the organisms to its most closely related taxonomic level, and establish the presence of that specific organism in the specimen known to be pathogenic or opportunistic pathogens.
  • DNA amplification and sequencing technologies offer several advantages over conventional methods for the identification of pathogens and antimicrobial agents resistance genes from clinical samples [6, 9]). There is no need for culture of the pathogens, hence the organisms can be detected directly from clinical samples, thereby reducing the time associated with the isolation and identification of pathogens, and reducing the amount of hazardous biological material that need be disposed. Furthermore, DNA-based sequencing assays are more accurate for microbial identification than currently used phenotypic identification systems which are based on biochemical tests and/or microscopic examination.
  • DNA-based sequencing technologies are currently used in clinical microbiology laboratories, mainly for the detection and identification of fastidious bacterial pathogens such as Mycobacterium tuberculosis , Chlamydia trachomatis , Neisseria gonorrhoeae as well as for the detection of a variety of viruses [10]
  • fastidious bacterial pathogens such as Mycobacterium tuberculosis , Chlamydia trachomatis , Neisseria gonorrhoeae
  • DNA sequencing based tests for the detection and identification of bacterial pathogens which are detectable by the present invention, for example: Staphylococcus sp. (U.S. Pat. No. 5,437,978), Neisseria sp. (U.S. Pat. No. 5,162,199 and European patent serial no. 0,337,896,131) and Listeria monocytogenes (U.S. Pat. Nos. 5,389,513 and 5,089,386).
  • the diagnostic tests described in these patents are based either on rRNA genes or on genetic targets detected by optical detection based sequencing techniques, different from those described in the present invention. To our knowledge there are no other patents published by others describing the use of non-optical based sequencing technology described in the present invention for microbiological diagnostic purposes.
  • Bacteria, fungi and parasites encompass numerous well-known microbial pathogens. Other microorganisms could also be pathogens or associated with human diseases. For example, achlorophylious algae of the Prototheca genus can infect humans. Archaea, especially methanogens, are present in the gut flora of humans [11, 12] Methanogens have been associated to pathologic manifestations in the colon, vagina, and mouth [11, 13, 14]
  • sequences for DNA-based assays from genomes available from public databases for (i) the species-specific detection and identification of commonly encountered bacterial, fungal and parasitical pathogens, (ii) the genus- specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (iii) the family-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (iv) the group-specific detection and identification of commonly encountered bacterial, fungal or parasitical pathogens, (v) the universal detection of algal, archaeal, bacterial, fungal or parasitical pathogens, and/or (vi) the specific detection and identification of antimicrobial agents resistance genes, and/or (vii) the specific detection and identification of bacterial toxin genes.
  • Patent publication WO98/20157 describes proprietary tuf DNA sequences as well as tuf sequences selected from public databases (in both cases, fragments of at least 100 base pairs), as well as oligonucleotide probes and amplification primers derived from these sequences. All the nucleic acid sequences described in that patent publication can be used in: (a) detecting the presence of bacteria and fungi; and (b) detecting specifically at the species, genus, family or group levels, the presence of bacteria and fungi and antimicrobial agents resistance genes associated with these pathogens.
  • kits need to be improved, since the ideal kit and method should be capable of diagnosing close to 100% of microbial pathogens and associated antimicrobial agents resistance genes and toxins genes.
  • infections caused by Enterococcus faecium have become a clinical problem because of its resistance to many antibiotics. Both the detection of these bacteria and the evaluation of their resistance profiles are desirable.
  • Non-optical genomic sequencing methods developed for the detection of pathogens in humans and animals fulfill this need by utilizing a non-optical sequencing platform, different than what was originally patented.
  • rRNA ribosomal RNA genes
  • SSU small subunit
  • 18S rRNA genes in eukaryotes
  • the rRNA genes are also the most commonly used targets for universal detection of bacteria [17, 18]and fungi [19]
  • HSP heat shock proteins
  • DNA sequencing technology is opening many new fields, and is finding novel applications in biology and medicine that go far beyond the initial goal of elucidating the order of nucleotide bases in a molecule of DNA.
  • Nucleic acid sequencing refers to the process of determining the primary structure of an unbranched biopolymer, which results in a symbolic linear depiction know as a ‘sequence’ that summarizes much of the atomic level structure of the sequenced molecule.
  • the process of DNA sequencing specifically refers to the determination of nucleotide order of a particular DNA fragment. It is now possible to analyze entire genomes of bacteria, fungi, viruses, animals, and plants.
  • the major limitations to current sequencing methods are the accuracy of the sequence, the length of an individual fragment (template) that can be sequenced, the cost of the sequence analysis, and the length of time it takes to determine the sequence.
  • Some recent efforts have made significant progress towards the development of methods that improve the ability to prepare genomes for sequencing, and to successfully sequence large numbers of templates simultaneously.
  • the DNA sequencing technologies can be reviewed and considered in a variety of ways. However, for the purposes of this patent, we can separate the technologies fundamentally based on the type of detection method used in the technique to determine nucleotide order. These detection platforms can be separated into Optical and Non-Optical based methods of genome sequencing. Since the inception of genome sequencing in the 1970’s until now, Optical genome sequencing techniques have predominated sequencing technology, and are denoted by the requirement for imaging technology, electromagnetic intermediates either in the form of X-rays.
  • Maxam-Gilbert Sequencing The first two sequencing methods were described in 1977. Maxam and Gilbert described a chemical degradation method [26], and Sanger described an enzymatic dideoxy method (also called the chain-terminator method) [22], which became the method of choice since it was perceived to be more efficient and use fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert. Maxam and Gilbert’s method requires radioactive labeling at one 5' end of the DNA, typically by a kinase reaction using gamma-32P ATP, and purification of the DNA fragment to be sequenced. The fragments are visualized by exposing the gel with the separated fragments to X-ray film, presenting a series of bands that each correspond to a labeled DNA fragment. From these fragments, the DNA sequence could be inferred.
  • the Sanger method uses dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators to generate a set of nucleic acid fragments which are different in length by one nucleotide.
  • ddNTPs dideoxynucleotide triphosphates
  • Each one of these chain terminating dideoxynucleotides e.g. ddATP, ddGTP, ddCTP, and ddTTP
  • the labeled DNA fragments are size separated by gel electrophoresis with single nucleotide resolution.
  • Variations in the electrophoretic process include applications of slab gels, capillaries, or microfluidic devices using denaturing polyacrylamide-urea gels, or other gradient poor-size polymer matrices.
  • the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray fdm or gel image.
  • Different variations of chain- termination sequencing have included tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled with a fluorescent dyes.
  • Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation.
  • these fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
  • Dye-terminator sequencing is differentiated by labeling the chain terminator ddNTPs each with a different and unique fluorescent dye that emits light at a unique wavelength. This permits sequencing in a single reaction, rather than four reactions as in the labeled- primer method. Even though the Sanger sequencing was the only method utilized in the parallel consortia that determined the complete human genome, many limitations of the Sanger processed were realized; such as, the need for gels or polymers used as sieving separation media for the fluorescently labeled DNA fragments, the low number of samples which could be analyzed in parallel, and the difficulty of total automation of the sample preparation methods. These limitations shifted focus to develop techniques without gels allowing sequence determination on very large numbers of samples in parallel.
  • each bead When PCR amplification cycles are completed and after denaturation, an individual bead with a single amplified fragment is placed at the top end of an etched fiber in an optical fiber chip, created from a glass fiber bundle.
  • Each glass fiber serves as optical waveguide, which transfers light to its other end attached to a CCD camera, enabling positional detection of emitted light. Therefore, each bead has an addressable position in the light guide chip, containing hundreds of thousands of available positions.
  • polymerase enzyme and primer are added to each of the beads, along with one unlabeled nucleotide per bead, thus starting the synthesis of the complementary strand.
  • Illumina (Solexa) Genome Analyzer The Solexa sequencing platform was first commercialized in 2006, and was acquired by Illumina in 2007. The functioning principles of this instrument are based on the same sequencing-by-synthesis chemistry. DNA fragments are ligated at both ends to adapters and, after denaturation, immobilized at one end on a solid support. The surface of the support is coated densely with the adapters and the complementary adapters. Each single -stranded fragment, immobilized at one end on the surface, creates a ‘bridge’ structure by hybridizing with its free end to the complementary adapter on the surface of the support. In the mixture containing the PCR amplification reagents, the adapters on the surface act as primers for the PCR amplification.
  • PCR amplification is needed as a step in this system as well to ensure sufficient light signal intensity for reliable detection of added bases.
  • the PCR step creates clusters of single-stranded DNA fragments on the surface of the support called‘polonies’.
  • the novelty of this system occurs in the next step following amplification, where the reaction mixture for the sequencing reactions and DNA synthesis is supplied onto the surface and contains primers, four reversible terminator nucleotides each labeled with a different fluorescent dye and the DNA polymerase. After incorporation into the DNA strand, the terminator nucleotide, as well as its position on the support surface, is detected and identified by its fluorescent dye at the CCD camera.
  • Applied Biosystems ABI SOLiD system ABI introduced the SOLiD system in 2007 uses ligation chemistry as its primary platform. In this technique, DNA fragments are ligated to adapters then bound to beads. A water droplet in oil emulsion contains the amplification reagents and only one fragment bound per bead; DNA fragments on the beads are amplified by emulsion PCR. Once amplified, the DNA are denatured After DNA, and the beads are deposited onto a glass support surface. In the next steps, a primer is hybridized to the adapter, followed by the hybridization of a mixture of oligonucleotide octamers followed by the addition of the ligation mixture.
  • the DNA sequence is determined by interrogating every 1st and 2nd base in each ligation reaction. Multiple cycles of ligation, detection and cleavage are performed with the number of cycles determining the eventual read length of the DNA strand.
  • the sequencing process may be continued in the same way with another primer shorter by one base than the previous one, and in fact is done so five times. Through this primer‘reset’ process, theoretically every base is interrogated in two independent ligation reactions by two different primers. Thus, the sequence read length is shorter, respectively speaking, at about 35 bases. However, the method has proven to be very accurate as a result of this dual interrogation type format.
  • Non-Optical Methods of Genome Sequencing The previously outlined optical based methods are still hindered by relatively large reaction volume size needed to prepare templates that are detectable by theses systems, the need for special nucleotide analogues as reagents, and complicated enzymatic and/or chemiluminescence reactions to generate detectable optical signals. As a result of these limitations, a major shift towards non-optical based sequencing methods occurred, resulting in the development of sequencing techniques with two other major categories of detection schemes; sequencing based mass spectrometry, and sequencing based on integrated circuits.
  • Nucleic Acid Sequencing based on Mass Spectrometry In U.S. Patent No. 7,501,251, methods are described for detecting a target nucleic acid in a biological sample using RNA amplification using a primer comprising a sequence that is complementary to a polynucleotide sequence in the target nucleic acid, and a sequence that encodes an RNA polymerase promoter. The RNA polymerase that recognizes the promoter is used to synthesize RNA. The newly synthesized RNA is detected by mass spectrometry, which establishes the presence or absence of that target RNA in the biological sample.
  • the detection systems of mass spectrometers provide a means of determining the individual mass and charge of volatilized molecules in a vacuum as the trajectory of the‘flying’ molecule is influenced by combinations of electric and magnetic fields.
  • This technique is an example of MS-based proteomics; a discipline made possible by the availability of gene and genome sequence databases and technical and conceptual advances primarily in the area of protein ionization methods.
  • the Ion Torrent was developed by Life Technologies using the ion-sensitive field-effect transistor (ISFET) due to its sensitivity to hydrogen ions and compatibility with CMOS processes [35]
  • ISFET ion-sensitive field-effect transistor
  • the Ion Torrent was not the first effort to detect both single-nucleotide polymorphisms [36], monitor DNA synthesis [37], or electronically sequence DNA[38] None of these earlier attempts were able to produce de novo DNA sequence, address issues of delivering template NDA to the sensors, or scale the entire system to large arrays [35]
  • ISFETs Prior to the Ion Torrent, ISFETs were limited in the number of sensors per array, the yield of working independent sensors and readout speed [39], and had issues protecting the electronic circuitry from fluid once the sensors were exposed [40] With this new technology, 25 million bases can be generated from chips containing 1.2 million sensors. This capability was demonstrated in by Rothberg et. al [35]
  • U.S. Patent No. 7,948,015 focuses on the development of the ion sensor chips, supporting instrumentation, and software to enable de novo DNA sequencing for applications requiring millions to billions of bases.
  • the method described here will utilize universal or broad range primers and individual sample specific barcodes or tags as have been well described in the literature, in order to analyzed batches or multiple specific specimens or subjects or sample detecting many specific targets that are grouped together to create a single assay.
  • This assay will be analyzed using a cost effective semi conductor technology or other non-optical method for determining the sequence of molecular material such as proteins or nucleic acids (RNA or DNA).
  • One example is to sequence each of the multiplexed analytes based upon pH generation detected using a semi-conductor or other chip-based technology. This allows many analytes to be screened all at once using broad range (e.g. kingdom specific, genus specific, family or class or sub-groups of organisms or targets) to be screened all at once and allows many different samples to be analyzed all together.
  • broad range e.g. kingdom specific, genus specific, family or class or sub-groups of organisms or targets
  • the present invention includes provide methods, compositions, and workflows, or components thereof, devices and methods based upon non-optical sequencing processes that improve and reduce the cost of genetic evaluation of microbial populations and ecologies in any environment, and further provide the ability to perform comprehensive microbial population characterization in a system that directs treatments or remedies or enhancements or remediations, thereby these embodiments will make such treatments, remedies or enhancements or remediations specific to the subject or the environment and the needed therapeutic trajectory to enhance the health and efficiency of a given animal, human or environmental system. To target and enhance the specific delivery of the treatment more convenient, targeted, and effective methods based upon lower cost diagnostic and microorganism evaluation.
  • the present invention provides a universal, sensitive and ubiquitous method that uses non-optical nucleic acid sequencing methods and universal gene targets (targets that are universal among all microorganisms such as the 16s gene for archaea and bacteria and the 18s gene for fungi, and the ITS gene for fungi) for determining the presence and/or amount of nucleic acids, thus detecting and determining the identity of microorganisms from any algal, archaeal, bacterial, fungal or parasitical species in any sample suspected of containing said nucleic acids.
  • the present invention can be used to investigate, define, or discover from an antimicrobial agents resistance gene, and optionally, from antimicrobial agents toxin gene the detected and identified organisms chemical sensitivities and drug susceptibilities.
  • the present invention includes a method for determining the identify of an organism or virus in a sample comprising the steps of: isolating DNA or RNA from the sample; combining the DNA or RNA directly or with one or more universal or target specific amplification primers, wherein the one or more primers are specific for one or more group of target microorganisms or virus; amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase; and contacting the amplification product with one or more species-, organism- or virus-specific detectable marker.
  • the species-, organism- or viral particle-specific detectable marker is selected from a tag, label, or barcode.
  • the amplification product is further sequenced with a non-optical nucleic acid sequencer.
  • the organism is defined further as a bacteria and the universal primers are specific for 16S ribosomal nucleic acids.
  • the organism is defined further as a fungi and the universal primers are specific for 18S ribosomal nucleic acids.
  • the organism is defined further as a fungi and the universal primers are specific for ITS nucleic acids.
  • the universal primers are specific for at least one of 23s ribosomal nucleic acids, nirS, rpoB, COX1, rbcL, LSU, 28S, fusA, ileS, lepA, leuS, pyrG, recA, recG, rplB, or SSU.
  • the step of amplification comprises PCR or linear amplification followed by non-optical sequencing of amplicons or direct non-optical sequencing or DNA and RNA to identify microorganisms.
  • primers are universal primers selected for a single specific species, wherein amplification and detection of a product is species specific.
  • the method further comprises the step of enriching the DNA or RNA using at least one of magnetic bead hybridization, precipitation, PCR, multiplex PCR, or RT-PCR.
  • the primers for the step of amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase are selected from SEQ ID NOS.: 1 to 283.
  • the primers are universal primers and the method further includes the steps of detecting and identifying unknown, novel or previously unidentified microorganisms using non-optical sequencing.
  • the primers are universal primers or organism specific primers and the method further includes the steps of detecting and identifying known or suspected microorganisms using non-optical sequencing.
  • the method further comprises the step of using non-optical sequencing to identify and quantitate microorganisms fn another aspect, the method further comprises the step of using non-optical sequencing to diagnose an environmental, industrial, veterinary, or medical sample for microorganisms that are either known, suspected, unknown, novel, or previously unidentified. In another aspect, the method further comprises the step of using non-optical sequencing to characterize the microbiological composition of an environmental, industrial, veterinary, or medical sample.
  • the method further comprises the step of generating a report using non-optical sequencing to determine the relative percentage of microorganisms in an environmental, industrial, veterinary, or medical sample and based on those finding selecting at least one of a treatment, a therapy, an improvement, or a remediation.
  • Another embodiment of the present invention includes a method for determining the identify of an organism or virus in a sample comprising the steps of: isolating a DNA or RNA from the sample; combining the DNA or RNA with one or more universal amplification primers, wherein the one or more primers are specific for one or more target organisms or virus; amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase; and sequencing the amplified products with a non-optical nucleic acid sequencer.
  • the method further comprising the step of contacting the amplification product with a species-, organism- or virus-specific detectable marker is selected from a tag, label, or barcode.
  • the organism is defined further as a bacteria and the universal primers are specific for 16S ribosomal nucleic acids. In another aspect, the organism is defined further as a fungi and the universal primers are specific for 18S ribosomal nucleic acids. In another aspect, the organism is defined further as a fungi and the universal primers are specific for ITS nucleic acids. In another aspect, the step of amplification comprises PCR or linear amplification. In another aspect, the primers are universal primers selected for a single specific species, wherein amplification and detection of any product will be species specific.
  • the method further comprises the step of enriching the DNA or RNA using at least one of magnetic bead hybridization, precipitation, PCR, multiplex PCR, or RT-PCR.
  • the universal primers are specific for at least one of 23 s ribosomal nucleic acids, nirS, rpoB, COX1, rbcL, LSU, 28S, fusA, ileS, lepA, leuS, pyrG, recA, recG, rplB, or SSU.
  • the primers for the step of amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase are selected from SEQ ID NOS.: 1 to 283.
  • the primers are universal primers and the method further includes the steps of detecting and identifying unknown, novel or previously unidentified microorganisms using non-optical sequencing.
  • the primers are universal primers or organism specific primers and the method further includes the steps of detecting and identifying known or suspected microorganisms using non-optical sequencing.
  • the non-optical sequencer is used to identify and quantitate microorganisms fn another aspect, the non-optical sequencer is used to diagnose an environmental, industrial, veterinary, or medical sample for microorganisms that are either known, suspected, unknown, novel, or previously unidentified.
  • the non-optical sequencer is used to characterize the microbiological composition of an environmental, industrial, veterinary, or medical sample.
  • the method further comprised the step of determining the relative percentage of microorganisms in an environmental, industrial, veterinary, or medical sample.
  • the method further comprises the step of generating a report using non-optical sequencing to determine the relative percentage of microorganisms in an environmental, industrial, veterinary, or medical sample and based on those finding selecting at least one of a treatment, a therapy, an improvement, or a remediation.
  • Another embodiment of the present invention includes a method for determining the identify one or more target organisms, microorganisms, or viruses or groups of target organisms, microorganisms, or viruses of in a sample comprising the steps of: isolating DNA or RNA from the sample; combining the DNA or RNA directly or with one or more universal or target specific amplification primers, wherein the one or more primers are specific for the one or more target microorganisms, or viruses or groups of target organisms, microorganisms, or viruses; amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase with the one or more universal or target specific amplification primers; contacting the amplification product with one or more species-, organism- or virus-specific detectable markers; detecting the amplification product with a non-optical detector; and determining the presence or absence of the target organisms, microorganisms, or viruses or groups of target organisms, microorganisms, or viruses in the sample, and a copy
  • Another embodiment of the present invention includes a method for determining the identify of an organism or virus in a sample comprising the steps of: isolating a DNA or RNA from the sample; combining the DNA or RNA with one or more universal amplification primers, wherein the one or more primers are specific for one or more target organisms or virus; amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase, wherein the primers for the step of amplifying the DNA, or the RNA following reverse transcription with a reverse transcriptase selected from SEQ ID NOS: 1-283; and detecting the amplification products with a non-optical nucleic acid sequencer; and determining the identity of the microorganism with a non-optical nucleic acid sequencer.
  • Figure 1 shows the non-optical genetic sequencing method and system for evaluation of microorganisms from a variety of sources of the present invention.
  • the present invention relates generally to an approach that utilize non-optical genetic sequencing methods for diagnosing and evaluating microorganisms in any sample type including human, animal, and environmental samples.
  • diagnostic methods can be done using specific assays for universal and more specific gene targets (targets that are universal among all microorganisms such as the 16s gene for Achaea and bacteria and the 18s gene for fungi, and the ITS gene for fungi or more specific genes such as nuclease genes for virus species) for detecting the presence of and determining the identity and relative abundance of microorganisms, including protozoa, fungi, archaea, bacteria and viruses in any given sample.
  • targets targets that are universal among all microorganisms such as the 16s gene for Achaea and bacteria and the 18s gene for fungi, and the ITS gene for fungi or more specific genes such as nuclease genes for virus species
  • This non-optical method of sequencing DNA is more cost effective than all optical sequencing methods used to date, thereby applying novel technological innovation to microbial diagnostic application whereby we are decreasing the cost of diagnosing and evaluating human, animal and environmental samples of all kinds and thereby increasing the utility and applicability of such diagnostics for a variety of purposes.
  • the present invention is directed to the resolution of the complete microbial communities of etiologic agents and commensal flora present in samples including, but not limited to, animals, humans, environmental, clinical, or other samples, including samples of unknown origin from which knowledge of the complete microbial community is of scientific and/or medical interest. This method is also able to detect and help identify novel pathogens.
  • the invention is further directed to the determination of detailed genetic information about the individual organisms which are detected and identified that make up in the sample’s microbiome including identification of genes that predict or indicate sensitivity or susceptibility of the organism to antimicrobials, antibiotics, or other chemical compounds, ions, or elements.
  • the autoSCAN-Walk-Away system (Dade Behring) identifies both gram-negative and gram-positive bacteria species from standardized inoculums in as little as 2 hours, and gives susceptibility patterns to most antibiotics in 5 to 6 hours.
  • this system has a particularly high percentage (i.e. 3.3 to 40.5%) of non-conclusive identifications with bacterial species other than Enterobacteriaceae
  • Enterobacteriaceae the percentage of non-conclusive identifications was 2.7 to 11.4%.
  • Pathogenic Bacteria examples include: Abiotrophia defective; Achromobacter piechaudii; Achromobacter xylosoxidans; Acinetobacter beijerinckii; Acinetobacter calcoaceticus; Acinetobacter haemolyticus;; Acinetobacter seohaensis; Acinetobacter iwoffii; Acinetobacter johnsonii; Acinetobacter junii; Acinetobacter septicus; Acinetobacter ursingii; Actinomyces odontolyticus; Aerococcus sanguinicola; Aerococcus viridians; Aggregatibacter segnis; Alistipes fmegoldii; Anaplasma phagocytophila; Anaplasma phagocytophilum; Arcobacter cryaerophilus; Atopobium rimae; Babesia divergens; Babesia duncani; Babesia microt
  • Pathogenic Fungi examples include: Examples of Pathogenic Fungi; Aspergillus aculeatus; Aspergillus alliaceus; Aspergillus caesiellus; Aspergillus caespitosus; Aspergillus candidus; Aspergillus carneus; Aspergillus clavatus; Aspergillus clavatus; Aspergillus deflectus; Aspergillus egyptiacus; Aspergillus fischerianus; Aspergillus flavus; Aspergillus flaws; Aspergillus foetidus; Aspergillus fumigatus; Aspergillus fumigatus; Aspergillus glaucus; Aspergillus nidulans; Aspergillus niger; Aspergillus ochraceus; Aspergillus oryzae; Aspergillus parasiticus; Asper
  • the term“prepared or isolated from” when used in reference to polynucleotides “prepared or isolated from” a pathogen refers to both polynucleotides (e.g., DNA or RNA, including cDNA produced therefrom) extracted and/or purified from a microorganism, and to polynucleotides that are copied from the transcriptosome of a microorganism, e.g., by a process of reverse-transcription or DNA polymerization using native DNA or RNA as a template.
  • Polynucleotides of the pathogen may be isolated from a specimen in conjunction with host nucleic acid.
  • pathogen refers to a microorganism, which causes disease in another organism (e.g., animal or plant) by directly infecting the other organism, or by producing agents that causes disease in another organism (e.g., bacteria that produce pathogenic toxins and the like).
  • pathogens include, but are not limited to bacteria, protozoa, fungi (e.g., molds and yeasts), helminths (e.g., cestodes, nematodes and trematodes), viroids and viruses, or any combination thereof, wherein each pathogen is capable, either by itself or in concert with another pathogen, of eliciting disease in vertebrates including but not limited to mammals, and including but not limited to humans.
  • the term“pathogen” also encompasses microorganisms, which may not ordinarily be pathogenic in a non-immunocompromised host. Specific nonlimiting examples of bacterial pathogens include [list species that were discovered to be in wounds by your invention] .
  • viral pathogens include Herpes simplex virus (HSV)1, HSV2, Epstein Barr virus (EBV), cytomegalovirus (CMV), human Herpes virus (HHV) 6, HHV7, HHV8, Varicella zoster virus (VZV), hepatitis C, hepatitis B, adenovirus, Eastern Equine Encephalitis Virus (EEEV), West Nile virus (WNE), JC virus (JCV), and BK virus (BKV).
  • Fungi such as Candida spp, Aspergillus spp., Cryptococcus spp, Histoplasma spp, Pneumocystis spp, and Stachybotrys spp.
  • Helminths such as schistosoma spp, Paragonimus spp, Trichenella spp. Taenia spp., Cchinococcus spp. Hymenolepis spp, Strongyloides spp, Dracunculus spp.
  • Protozoa such as Cryptosporidium spp, Encephalitozoon spp., Giardia spp., Plasmodium spp., Phytophthora spp., and Kneallhazia spp., etc.
  • microorganism includes prokaryotic and eukaryotic microbial species from the Domains of Archaea, Bacteria, and Eucarya, the latter including yeast and filamentous fungi, helminths, protozoa, algae, or higher Protista.
  • microbe is used interchangeably with the term microorganism.
  • bacteria refers to a domain of prokaryotic organisms.
  • Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (i) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (ii) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green
  • Gram -negative bacteria include cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.
  • Gram-positive bacteria include cocci, nonsporulating rods, and sporulating rods.
  • the genera of Gram-positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.
  • the term“detection” refers to the at least qualitative determination of the presence or absence of a microorganism in a specimen.
  • the term“identification” also includes the detection of a microorganism, i.e., determining the genus, species, or strain of a microorganism according to its recognized taxonomy in the art and as described in the present specification.
  • identification further includes the quantification of a microorganism in a specimen, e.g., the copy number of the microorganism in a microliter (or a milliliter or a liter) or a microgram (or a milligram or a gram or a kilogram), or swab or any other type or quantity of any type of specimen containing or suspected of containing microorganisms.
  • the term“immunocompromised subject or individual” refers to an individual who is at risk for developing infectious diseases, because the immune system of the individual is not working at optimum capacity.
  • the individual is immunocompromised due to a treatment regimen designed, for example, to prevent inflammation or to prevent rejection of a transplant.
  • the term“specimen” refers to a biological material or environmental sample from any source containing or suspected of containing 1 or more microorganisms of any type which can be evaluated for the microbiological presence of microorganisms, which is isolated from its natural environment (including the body such as skin, mucosa, internal organs, and fluids or a body cavity collected by lavage, water, soil, feces, etc) and contains a polynucleotide DNA or RNA.
  • a biological fluid includes, but is not limited to, blood, plasma, serum, sputum, urine, pus or other wound exudate, infected tissue sampled by wound debridement or excision, cerebrospinal fluid, lavage, and leucopoiesis specimens, for example.
  • a specimen may also be an environmental specimen such as soil, water, or animal or human waste to detect the presence of a pathogen in an area where an outbreak of disease related to a specific pathogen has occurred.
  • a specimen may also be obtained from a tissue bank or other source for the analysis of archival samples or to test samples prior to transplantation.
  • a specimen useful in the method described herein may be any plant, animal, bacterial or viral material containing a polynucleotide, or any material derived there from.
  • a specimen is suspected of containing at least one of a plurality of known or unknown or potential or opportunistic pathogens or commensal organisms for any of a number of reasons.
  • a soil specimen may be suspected of containing a pathogen if humans or animals living close to the location where the soil specimen was collected show symptoms of a condition or diseases associated with a soil pathogen few environments and therefore few specimens are sterile and do not contain some type of microorganism.
  • a specimen is any collection of source material sampled from any environment.
  • Specimens taken from such a subject may be suspected of containing at least one of a plurality of known unknown, suspected, opportunistic or potential pathogens or commensal organisms, even in the absence of infection.
  • the term“reverse transcript” refers to a DNA complement of an RNA strand generated by an RNA-dependent DNA polymerase activity.
  • primer pair“capable of mediating amplification” is understood as a primer pair that is specific to a target polynucleotide, has an appropriate melting temperature, and does not include excessive secondary structure. Guidelines for designing primer pairs capable of mediating amplification are well documented in the literature. The present invention can also take advantage of a single specific primer for amplification.
  • condition that promote amplification are the conditions for target amplification provided by the manufacturer for the enzyme used for amplification of template. It is understood that an enzyme may work under a range of conditions (e.g., buffer pH, ion concentrations, temperatures, concentrations of enzyme or target). It is also understood that several temperatures may be required for amplification (e.g., three in PCR for annealing primer to template, extending primer as the complement of template, and denaturing extended primer from template). Conditions that promote amplification need not be identical for all primers and targets in a reaction, and reactions may be carried out under suboptimal conditions where amplification is still possible.
  • conditions that promote amplification need not be identical for all primers and targets in a reaction, and reactions may be carried out under suboptimal conditions where amplification is still possible.
  • the term“aliquot” refers to a sample volume taken from an amplification reaction mixture.
  • the volume of an aliquot can vary, but will generally be constant within a given experimental run. An aliquot will be less than the volume of the entire reaction mixture. Where there are X aliquots to be withdrawn during an amplification regimen, the volume of an aliquot will be less than or equal to 1/X times the reaction volume.
  • the term“dispense” means to dispense, transfer, withdraw, extrude or remove.
  • the phrase“dispensing an aliquot from the reaction mixture at plural stages” refers to the withdrawal of an aliquot at least twice, and preferably at least about 3, 4, 5, 10, 15, 20, 30 or more times dining an amplification reaction.
  • A“stage” will refer to a point at or after a given number of cycles, or, where the amplification regimen is non-cyclic, will refer to a selected time at or after the initiation of the reaction.
  • the term“separating” nucleic acids in a sample refers to a process whereby they are separated by size (i.e., length).
  • the method of separation should be capable of resolving nucleic acid fragments that differ in size by ten nucleotides or less (or, alternatively, by ten base pairs or less, e.g., where non-denaturing conditions are employed).
  • Preferred resolution for separation techniques employed in the methods described herein includes resolution of nucleic acids differing by five nucleotides or less (alternatively, five base pairs or less), up to and including resolution of nucleic acids differing by only one nucleotide (or one base pair).
  • the term“size distinguishable by capillary electrophoresis” refers to a difference of at least one nucleotide (or base pair), but preferably at least 5 nucleotides (or base pairs) or more, up to and including 10 nucleotides (or base pairs) or more.
  • the term“distinct from” when used in reference to the length of a polynucleotide means that the length of the polynucleotide is distinguishable from the length of another by capillary electrophoresis.
  • the term“amplified product” refers to polynucleotides that are copies of a particular polynucleotide, produced in an amplification reaction.
  • An“amplified product” according to the one embodiment, may be DNA or RNA, and it may be double-stranded or single-stranded.
  • An amplified product is also referred to herein as an“amplicon.”
  • the term“amplification” or“amplification reaction” refers to a reaction for generating a copy of a particular polynucleotide sequence or increasing the copy number or amount of a particular polynucleotide sequence.
  • polynucleotide amplification may be a process using a polymerase and a pair of oligonucleotide primers for producing any particular polynucleotide sequence, i.e., the whole or a portion of a target polynucleotide sequence, in an amount that is greater than that initially present.
  • Amplification may be accomplished by the in vitro methods of the polymerase chain reaction (PCR).
  • amplification methods include, but are not limited to: (a) ligase chain reaction (LCR) (see Wu & Wallace, 1989, Genomics 4: 560-569 and Landegren et al., Science, 1988, 241: 1077-1080); (b) transcription amplification (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177); (c) self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci.
  • LCR ligase chain reaction
  • NABSA nucleic acid based sequence amplification
  • target polynucleotide refers a polynucleotide to be analyzed.
  • a target polynucleotide may be isolated or amplified before being analyzed.
  • the target polynucleotide may be comprised of a sequence that lies between the hybridization regions of two members of a pair of oligonucleotide primers that are used to amplify the target.
  • a target polynucleotide may be RNA or DNA (including, e.g., cDNA).
  • microbe-specific target polynucleotide refers to a target polynucleotide as defined above, wherein the target polynucleotide is prepared or isolated from a specimen suspected of containing a pathogen, and which is present in only one member of the group of different pathogens that are being analyzed (i.e., the target polynucleotide has a unique sequence and is specific for detection of the pathogen’s genera or species).
  • oligonucleotide primer refers to a polynucleotide molecule (i.e., DNA or RNA) capable of annealing to a polynucleotide template and providing a 3' end to produce an extension product that is complementary to the polynucleotide template.
  • the conditions for initiation and extension usually include the presence of four different deoxyribonucleoside triphosphates (dNTPs) and a polymerization-inducing agent such as a DNA polymerase or reverse transcriptase activity, in a suitable buffer (“buffer” includes substituents which are cofactors, or which affect pH, ionic strength, etc.) and at a suitable temperature.
  • dNTPs deoxyribonucleoside triphosphates
  • buffer includes substituents which are cofactors, or which affect pH, ionic strength, etc.
  • the primer as described herein may be single- or double-stranded.
  • the primer is preferably single-stranded for maximum efficiency in amplification.
  • “Primers” useful in the methods described herein are less than or equal to 100 nucleotides in length, e.g., less than or equal to 90, or 80, or 70, or 60, or 50, or 40, or 30, or 20, or 15, but preferably longer than 10 nucleotides in length.
  • label or“detectable label” refer to any moiety or molecule that can be used to provide a detectable (preferably quantifiable) signal.
  • A“labeled nucleotide” e.g., a dNTP
  • label polynucleotide
  • the term“linked” encompasses covalently and non-covalently bonded, e.g., by hydrogen, ionic, or Van der Waals bonds. Such bonds may be formed between at least two of the same or different atoms or ions as a result of redistribution of electron densities of those atoms or ions.
  • Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass spectrometry, binding affinity, hybridization radiofrequency, nanocrystals, and the like.
  • a nucleotide useful in the methods described herein can be labeled so that the amplified product may incorporate the labeled nucleotide and becomes detectable.
  • a fluorescent dye is a preferred label according to the one embodiment.
  • Suitable fluorescent dyes include fluorochromes such as Cy5, Cy3, rhodamine and derivatives (such as Texas Red), fluorescein and derivatives (such as 5-bromomethyl fluorescein), Lucifer Yellow, IAEDANS, 7-Me.sub.2N-coumarin-4-acetate, 7-OH-4-CH.sub.3-coumarin-3-acetate, 7- NH 2 -4-CH 3 -coumarin-3 -acetate (AMCA), monobromobimane, pyrene trisulfonates, such as Cascade Blue, and monobromorimethyl-ammoniobimane (see, for example, DeLuca, 1982, Immunofluorescence Analysis, in Antibody As a Tool, Marchalonis, et al., eds., Wiley, which is incorporated herein by reference).
  • fluorochromes such as Cy5, Cy3, rhodamine and derivatives (such as Texas Red), fluorescein and derivatives (such as 5-bromo
  • labeled nucleotide refers to a synthetic or biochemically derived nucleotide analog that is intrinsically fluorescent, e.g., as described in U.S. Patents 6,268,132 and 5,763,167, Hawkins et al. (1995, Nucleic Acids Res., 23: 2872-2880), Seela et al. (2000, Helvetica Chimica Acta, 83 : 910-927), Wierzchowski et al. (1996, Biochimica et Biophysica Acta, 1290: 9-17), Virta et al.
  • intrinsically fluorescent it is meant that the nucleotide analog is spectrally unique and distinct from the commonly occurring conventional nucleosides in their capacities for selective excitation and emission under physiological conditions.
  • the fluorescence typically occurs at wavelengths in the near ultraviolet through the visible wavelengths.
  • fluorescence will occur at wavelengths between 250 nm and 700 nm and most preferably in the visible wavelengths between 250 nm and 500 nm.
  • the terms“detectable label” or“label” refer to a molecule or moiety capable of generating a detectable signal, either by itself or through the interaction with another label.
  • The“label” may be a member of a signal generating system, and thus can generate a detectable signal in context with other members of the signal generating system, e.g., a biotin-avidin signal generation system, or a donor- acceptor pair for fluorescent resonance energy transfer (FRET) (Stryer et al., 1978, Ann. Rev.
  • nucleotide refers to a phosphate ester of a nucleoside, e.g., mono, di, tri, and tetraphosphate esters, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose (or equivalent position of a non-pentose“sugar moiety”).
  • nucleoside e.g., mono, di, tri, and tetraphosphate esters
  • the term“nucleotide” includes both a conventional nucleotide and a non-conventional nucleotide which includes, but is not limited to, phosphorothioate, phosphite, ring atom modified derivatives, and the like, e.g., an intrinsically fluorescent nucleotide.
  • the term“conventional nucleotide” refers to one of the“naturally occurring” deoxynucleotides (dNTPs), including dATP, dTTP, dCTP, dGTP, dUTP, and dITP.
  • dNTPs deoxynucleotides
  • non-conventional nucleotide refers to a nucleotide, which is not a naturally occurring nucleotide.
  • naturally occurring refers to a nucleotide that exists in nature without human intervention.
  • non-conventional nucleotide refers to a nucleotide that exists only with human intervention.
  • A“non-conventional nucleotide” may include a nucleotide in which the pentose sugar and/or one or more of the phosphate esters is replaced with a respective analog. Exemplary pentose sugar analogs are those previously described in conjunction with nucleoside analogs.
  • Exemplary phosphate ester analogs include, but are not limited to, alkylphosphonates, methylphosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoroamidates, boronophosphates, etc., including any associated counterions, if present.
  • a non-conventional nucleotide may show a preference of base pairing with another artificial nucleotide over a conventional nucleotide (see Ohtsuki et al., 2001, Proc. Natl. Acad.
  • the base pairing ability may be measured by the T7 transcription assay as described in Ohtsuki et al. (2001).
  • Other non-limiting examples of“artificial nucleotides” may be found in Lutz et al. (1998, Bioorg. Med. Chem. Lett., 8: 1149-1152); Voegel & Benner (1996, Helv. Chim. Acta 76, 1863-1880); Horlacher et al. (1995, Proc. Natl. Acad. Sci., 92: 6329-6333); Switzer et al. (1993, Biochemistry 32: 10489-10496); Tor & Dervan (1993, J. Am. Chem. Soc.
  • A“non-conventional nucleotide” may also be a degenerate nucleotide or an intrinsically fluorescent nucleotide.
  • Degenerate nucleotide refers to a nucleotide that may be able to basepair with at least two bases of dA, dG, dC, and dT.
  • a non-limiting list of degenerate nucleotides that basepairs with at least two bases of dA, dG, dC, and dT include: inosine, 5-nitropyrole, 5-nitroindole, hypoxanthine, 6H,8H,4-dihydropyrimido[4,5c][l,2]oxacin-7-one (P), 2-amino-6-methoxyaminopurine, dPTP, and 8-oxo-dGTP.
  • the term“Opposite orientation” refers to one nucleotide sequence complementary to the sense strand of a target polynucleotide template and another nucleotide sequence complementary to the antisense strand of the same target polynucleotide template. Primers with opposite orientation may generate a PCR-amplified product from matched polynucleotide template to which they complement. Two primers having opposite orientation may be referred to as a“reverse” primer and a“forward” primer.
  • the term“Same orientation” refers to primers that comprise nucleotide sequences complementary to the same strand of a target polynucleotide template. Primers with same orientation will not generate a PCR-amplified product from matched polynucleotide template to which they complement.
  • polynucleotide or“nucleic acid” refer to a polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include without limitation single- and double-stranded polynucleotides, and embrace chemically, enzymatically, or metabolically modified forms of polymerized nucleotides, as well as chemical forms of DNA and RNA characteristic of particles and cells.
  • a polynucleotide may be an isolated or purified polynucleotide or it may be an amplified polynucleotide in an amplification reaction.
  • a“set” of oligonucleotide primers comprises at least two oligonucleotide primers.
  • a“set” of oligonucleotide primers refers to a group of primers sufficient to specifically amplify a nucleic acid amplicon from each member of a plurality of target pathogens— generally, there will be a pair of oligonucleotide primers for each member of said plurality, (it is noted that these primer pairs will, in some aspects, also be used to amplify one or more competitor or internal standard templates).
  • a“pair” of oligonucleotide primers are two oligonucleotide primers.
  • a“pair” of oligonucleotide primers are used to produce an extended product from a double-stranded template (e.g., genomic DNA or cDNA)
  • a“pair” of oligonucleotide primers are used to produce an extended product from a double-stranded template (e.g., genomic DNA or cDNA)
  • it is preferred that the pair of oligonucleotide primers hybridize to different stand of the double-stranded template, i.e., they have opposite orientations.
  • the term“Isolated” or“purified” refers to a naturally -occurring substance has been removed from its normal cellular environment or is synthesized in a non-natural environment (e.g., artificially synthesized).
  • an“isolated” or“purified” substance may be in a cell-free solution or placed in a different cellular environment.
  • “purified” does not necessarily imply that a sequence is the only nucleotide present, but that it is essentially free (at least about 90% or 95%, up to 99- 100% pure) of non-nucleotide or polynucleotide material naturally associated with it.
  • cDNA refers to complementary or copy polynucleotide produced from an RNA template by the action of an RNA-dependent DNA polymerase activity (e.g., reverse transcriptase).
  • the term “Complementary” refers to the ability of a single strand of a polynucleotide (or portion thereof) to hybridize to an anti-parallel polynucleotide strand (or portion thereof) by contiguous base-pairing between the nucleotides (that is not interrupted by any unpaired nucleotides) of the anti-parallel polynucleotide single strands, thereby forming a double-stranded polynucleotide between the complementary strands.
  • a first polynucleotide is said to be“completely complementary” to a second polynucleotide strand if each and every nucleotide of the first polynucleotide forms base-paring with nucleotides within the complementary region of the second polynucleotide.
  • a first polynucleotide is not completely complementary (i.e., partially complementary) to the second polynucleotide if one nucleotide in the first polynucleotide does not base pair with the corresponding nucleotide in the second polynucleotide.
  • the degree of complementarity between polynucleotide strands has significant effects on the efficiency and strength of annealing or hybridization between polynucleotide strands. This is of particular importance in amplification reactions, which depend upon binding between polynucleotide strands.
  • an oligonucleotide primer is“complementary” to a target polynucleotide is if at least 50% (60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more) nucleotides of the primer form base pairs with nucleotides on the target polynucleotide.
  • the term“analyzing” when used in the context of an amplification reaction refers to a qualitative (i.e., presence or absence, size detection, or identity etc.) or quantitative (i.e., amount) determination of a target polynucleotide, which may be visual or automated assessments based upon the magnitude (strength) or number of signals generated by the label.
  • The“amount” (e.g., measured in pg, pmol, or copy number) of a polynucleotide may be measured by methods well known in the art (e.g., by UV absorption or fluorescence intensity, by comparing band intensity on a gel with a reference of known length and amount), for example, as described in Basic Methods in Molecular Biology (1986, Davis et al., Elsevier) and Current Protocols in Molecular Biology (1997, Ausubel et al., John Wiley).
  • One way of measuring the amount of a polynucleotide in one embodiment is to measure the fluorescence intensity emitted by such polynucleotide, and compare it with the fluorescence intensity emitted by a reference polynucleotide, i.e., a polynucleotide with a known amount.
  • a reference polynucleotide i.e., a polynucleotide with a known amount.
  • Capillary tubes are available with inner diameters from about 10 pm to 300 pm, and can range from about 0.2 cm to about 3 m in length, but are preferably in the range of 0.5 cm to 20 cm, more preferably in the range of 0.5 cm to 10 cm.
  • a microfluidic microcapillary e.g., available from Caliper or Agilent
  • the use of a microfluidic microcapillary is specifically encompassed within the meaning of capillary electrophoresis.
  • the term“aymptomatic” refers to an individual who does not exhibit physical symptoms characteristic of being infected with a given pathogen, or a given combinations of pathogens.
  • the term“Plurality” refers to more than two, for example, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more 10 or more etc.
  • the methods described here utilize a non-optical genetic sequencing approach that can identify, provide relative quantification, relative abundance or absolute identification/resolution of these quantitative factors of all known, unknown, suspected, commensal, opportunistic, pathogens and microorganisms within any given type of sample known or suspected to contain 1 or more microorganisms.
  • pathogens may be suspected pathogens, unknown or previously undescribed pathogens, opportunistic pathogens, commensal organisms that provide synergistic contribution to pathogenicity and polymicrobial communities that act together to create infection or subclinical disease including organisms in biofilm or any other phenotype or compilation within a sample hereafter referred to as pathogens) contained within a sample.
  • pathogens may be suspected pathogens, unknown or previously undescribed pathogens, opportunistic pathogens, commensal organisms that provide synergistic contribution to pathogenicity and polymicrobial communities that act together to create infection or subclinical disease including organisms in biofilm or any other phenotype or compilation within a sample hereafter referred to as pathogens
  • the pathogen is selected from the group consisting of: bacteria, fungi (e.g., molds and yeasts), helminths, protozoan, viruses, and combinations thereof.
  • the pathogen is selected from the group consisting of: bacteria, fungi, viruses, and combinations thereof.
  • the pathogen is selected from the group consisting of: bacteria, viruses, and combinations thereof.
  • the pathogens may be microbes belonging to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different genera (especially bacterial and/or viral genera); the pathogens may be bacteria belonging to at least five, at least ten, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 different species (especially bacterial and/or viral species).
  • An infection may be a suspected infection, subclinical infection, a potential infection, a future infection, or a past infection hereafter referred to as infection.
  • a specimen may be from any environment including bodily fluids, feces, tissue, debrided materials, swabbed surfaces, biopsies, aqueous materials, fluids collected from any source, surfaces of any type, soil, food, etc., including any environment that contains microorganisms.
  • a specimen is any form of content removed in whole or in part from an environment intended for analysis of microorganisms using non-optical genetic sequencing methods and apparatus for determining the genetic sequence of RNA or DNA either directly with shotgun sequencing or targeted using a set of primers in pairs or in multiplex where pairs of primers are 2 or more together.
  • This non-optical genetic DNA and RNA sequencing is used to evaluate any type of sample suspected to contain or known to contain microorganisms. Such sample is hereafter referred to as a specimen.
  • Diagnostic, screening, monitoring, or testing for microorganisms or microbial pathogens causing an infection or microorganisms hindering or enhancing an ecological or industrial process is typically conducted for a subjects or environments or industrial processes who present symptoms characteristic of clinical infection presumably by one or more pathogenic microorganisms, or in a subject who has been in contact with another having one or more pathogenic infections, or in a subject who are otherwise suspected to have developed an infectious disease resulting from one or more pathogens or a process that has a microorganism causing contamination or fouling or improvement or enhancement or remediation of an infection, industrial process, or improvement of ecological processes or environmental improvements that can be monitored for microorganisms.
  • Figure 1 shows the non-optical genetic sequencing evaluation detection method and system for the identification of microorganisms from a variety of sources.
  • Figure 1 shows the methods and use of the present invention in the field of diagnosing and monitoring industrial and environmental microbial processes, medical and veterinary diagnosis and medical and veterinary treatment, and more particularly, to universal or broad range assays and multi-tag sample specific diagnostic process using non-optical sequencing.
  • the skilled artisan will recognize that the present invention can be used to detect and/or identify a wide variety of nucleic acid containing samples.
  • Figure 1 begins with the isolation of a sample that contains a biological material that includes any type of nucleic acid at step 20.
  • samples include but are not limited to industrial processing, bioreactors, waste and wastewater 10, samples from human samples 12, veterinary samples 14, airborne samples 16 (e.g., filters containing biological samples such as pollen or viruses), or other environmental samples 18 that may include molds, soil samples, etc.
  • Samples containing or suspected of containing microorganisms undergo DNA or RNA extraction at step 22, after which DNA or RNA is sequenced directly or as part of a process that uses barcoding of universal or targeted amplicons derived from PCR.
  • the sample obtained from step 22 is further processed on sequencing beads at step 24, which the skilled artisan would understand to be an optional step.
  • the DNA is sequencing using a non- optical genetic sequencing method at step 26.
  • the detection of pH changes as hydrogen atoms are released and detected by semi-conductor based pH meters is measured at the signal and the nucleic acid sequence is determined at step 28. As nucleotides are flowed in any order of A, T, G, C when an A is added to the sequencing strand it releases a H+ ion which creates a pH change.
  • the present invention allows the user to use very small samples to determine the nature of the nucleic acids in the sample using non-optical sequencing and provides for the first time the determination of relative amounts of virus, bacteria, fungi, etc., in a sample, the determination of which organisms are present (either as a species in general or with great detail as to the nature of the organism), can be used to quantitatively and/or qualitatively to determine the presence or absence of a wide, medium or narrow spectrum of organisms.
  • RNA and DNA are extracted from a given human, animal or other environmental sample. This DNA is prepared using method to generate a sequencing library that is then sequenced using non-optical genetic sequencing apparatus and method to evaluate the metagenome or consortium of genetic information from the given sample. This provides information on the identity of microorganisms, their antimicrobial resistance potential as well as their virulence factors.
  • primers Universal, specific, semi-universal, targeting kingdoms, super-kingdoms, targeting phylums, targeting all classes, families, orders, genus or species of microorganisms.
  • the present inveniton incudes the use of 1, 2, 3, 4, 5, 6, 7, 8, 910, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250 or more primers.
  • Barcodes Types of barcodes are selected oligonucleotides that may be from 2 nucleotides to 200 nucleotides in length (preferably from 4 nucleotides to 10 nucleotides in length) and are used to identify, barcode, or define which sequences are derived from which specimen so that many specimens can be combined together in a non-optical genetic sequencing reaction and afterwards deconvoluted to resolve which genetic information resolves or relates to which sample.
  • Database formation a nucleotide or protein database containing genetic information from all known microorganisms, formatted or raw to promote comparison of sequencing data to known or existing data for use in identifying microorganisms, characterizing microbial populations.
  • Methods are provided for performing DNA extraction from a wide range of specimen, then performing of microbial non-optical barcoded amplicon genetic sequencing that can detect and identify, through computational or bioinformatics methods, the profde of microorganisms within the specimen, the genes associated with antimicrobial resistance, the genes associated with virulence or pathogenicity.
  • the method further, utilizes a database of known sequence information to compare against sequence information derived from the specimen to identify which microorganisms are present in the specimen. This in turn is followed by subsequent computational or bioinformatics algorithms which draws from a database containing information on antibiotic susceptibility, resistance information and previous treatment outcomes to obtain a profde of those antibiotics or therapeutics, which may be utilized to treat or positively impact the microbial profile identified.
  • the computational system then generates interpretive diagnostic, virulence, antimicrobial resistance, and microbial ecology reports that elucidate the microbial assemblage and characteristics contained within the specimen and provide the associated therapeutic, treatment, remediation, or optimization options.
  • a preferred embodiment of the diagnostic, microorganism community assessment or microbial ecology method is the employment of non-optical sequencing processes (all previous patents dealing with genetic evaluation or sequence information have all been based upon optical sequencing methods by contrast), to identify microorganisms that may or may not be specifically targeted by assay thereby providing novel universal diagnostic methods based upon novel NON-optical genetic sequencing processes, this genetic sequencing using non-optical machines and processes for determining the microbial composition of any given sample is then followed by a computational system to characterize the microbial and genetic resistance profiles, virulence factors, and genetic information relevant to the health of a given sample source including animals, humans, and environmental sources, then computation methods are utilized to evaluate the genetic non-optical sequencing data and provide reports and interpretations.
  • An advantage of some embodiments is that it provides a cost effective molecular diagnostic method and microbial ecology characterization method. This improves the ability of clinicians, bioremediation specialists to evaluate, remediate, treat, monitor infections including polymicrobial and biofilm phenotype infections, microbial populations involved in industrial processes, microbial populations causing improvement or decay of industrial processes (such as oil well or water well fouling), veterinary infections, animal health enhancement such as feedlot productivity related to improving the microbial populations in an animals gastrointestinal tract thereby improving the utilization of feed (feed efficiency and conversion in animals), the use of probiotics and prebiotics to improve health and the associated monitoring of the microbial populations, bacterial infections not conducive to diagnosis by traditional culture based methodology.
  • Another advantage is the ability to utilize the microbial profiles to determine which antibiotics may be utilized to most efficiently and effectively control or treat an infection, remediate an industrial process, enhance animal health, improve the efficiency of an industrial process related to microbial processes and assemblages and to do this evaluation diagnostic and monitoring of the microorganism populations using non-optical sequencing methods which are lower in cost than optical sequencing methods and to provide microbial ecology and infection monitoring in a comprehensive, less expensive and more rapid manner.
  • Another advantage is that non-optical sequencing by being less expensive can generate more data and provide a more comprehensive evaluation of microbial assemblages allowing computational methods to provide a diagnostic, monitoring and therapeutic indication and interpretive report that can be utilized by a clinicians, industrial scientists, environmental microbiologist, and other individuals needing to optimize, diagnose treat, remediation, improve microbial processes, to easily evaluate and personalize approaches for improving or optimizing or changing or medially treating their subject, bioreactor, environment, animal in a personalized specific and targeted manner.
  • Another advantage is that this method does not rely on the ability of a microorganism to be grown in the laboratory.
  • Another advantage is that hard to culture, fastidious organism, organisms in biofilm phenotype and viable but non-culturable organism can be identified and all organisms can be quantified or relatively quantified. Another advantage is that patient-specific therapeutic regimes can be identified for clinicians to address the complex nature of polymicrobial or poor culturing microbial infections. Another advantage is that an algorithm for identifying such therapeutics, which can best target a specific microbial polymicrobial infection, can be determined.
  • the methods disclosed herein permit identifying the presence and/or the relative or the specific quantity of two or more microorganisms, particularly bacterial, fungal, helminthal, protozoan or viral pathogens that may be present in any given environmental or biological specimen.
  • the methods perform such utility through the using of non-optical genetic (DNA or RNA) sequencing methods of directly extracted RNA or DNA from the environmental or biological specimen.
  • the methods permit the detection and quantification of pathogens or microorganism via non- optical genetic sequencing of all available or specific polynucleotides, e.g., DNAs or RNAs isolated from an environmental, biological, or clinical specimen, both within a panel of reactions, in a multiplex format and in a highly parallelized sequencing pyrosequencing or future non-optical sequencing format, that can further permit the determination of levels (e.g., ratios, percentages, and quantities) for two or more target polynucleotides in a single reaction.
  • levels e.g., ratios, percentages, and quantities
  • Identification and quantification of pathogen specific targets in a specimen has a myriad clinical and microbial ecology utilities specifically to identification of differences between environments, to identify microorganisms and guide treatment, remediation, therapy, enhancement, optimization or other method of changing or monitoring the microorganisms from any given sample.
  • the methods described herein use shotgun approaches to generate sequence data for all aspects of the genetic makeup of the microoganisms in any sample or by using non-optical sequencing approaches to evaluate generated Polymerase chain reaction amplification products of known sizes that both differ from each other at the sequence level in specific regions of the polynucleotide and are the same or similar or conserved (same) in specific regions of the polynucleotide.
  • a set of oligonucleotide primers that are specific and target a DNA or RNA molecule isolated from the specimen that can be used to identify a given species, strain, genus, family, class, phylum, or order of microorganism by targeting non-conserved or conserved regions of a gene or part of the genetic material of the organism or a combination of the two.
  • the methods described herein relate to methods of estimating or determining the identification and/or quantification of microorganisms in a specimen following isolation (e.g., extraction or purification) of polynucleotides from the specimen, the method comprising: for a given pathogen specific target polynucleotide, selecting a pair of amplification primers that will generate a target amplicon of known length upon amplification of the target, e.g., by PCR or RT-PCR. The method will provide a relative or absolute quantification of the amount of the target, e.g., by spiking or applying known concentrations of a given template or the use of quantitative universal PCR approaches or other format of polymerase chain reaction.
  • methods described herein relate to the detection of selected pathogens in pre- symptomatic immunocompromized subjects. Since development of clinical symptoms can be subclinical in many infections and in immunocompromized subjects, particularly transplant recipients undergoing immunosuppressant therapy, quantitative rapid and or comprehensive detection of viral, bacterial and protozoan pathogens provides a means to guide therapy during the early stages of infection.
  • the methods analyze a specimen suspected of containing any of a polymicrobial community of predetermined or unknown pathogens by screening a specimen for a known and unknown pathogens specific, universal, semi-universal or conserved targets to be used in a nucleic acid amplification reaction to produce an amplicon from each pathogen specific target.
  • the methods include selecting a series of pathogen-specific or kingdom based universal or semi-universal primer pairs wherein each primer pair corresponds to and is targeted to polynucleotide sequences specific to a corresponding pathogen or conserved or universal for all known or unknown microorganisms.
  • the series of pathogen- specific primers or universal or semi-universal domain, kingdom, phylum, class, family, genus or species specific primers when used together produce amplicons of distinct sizes such that the presence of a specific or group of known or unknown pathogen in the specimen. Amplicons are detected by resolving a portion of the amplification mixture to determine if amplicons are present, and is so, their size and then amount of amplicon. Portions of the specimen may be sampled at intermediate points during amplification to determine when amplicons are first detectable, or at the end of amplification. Portions of the specimen may be sampled for downstream non-optical genetic sequencing.
  • the methods for quantifying a plurality of predetermined pathogens in a specimen suspected of containing at least one pathogen using non-optical genetic sequencing methods include obtaining a specimen suspected of containing at least one of the predetermined pathogens.
  • the specimen may be obtained from the environment (e.g., soil, water, animal or human waste), from a plant, animal, frozen tissue banks, or human source (e.g., a pathogen carrier or host).
  • Polynucleotides are isolated from the specimen for use as target in an amplification reaction to produce template.
  • Pathogen-specific or universal or semi-universal primers are selected to correspond to each or all of the plurality of pathogens that could be present in the specimen.
  • Control polynucleotides preferably competitor polynucleotides, may also be included in the amplification reaction.
  • the competitor polynucleotides can be templates for amplification by pathogen-specific primers, but produce amplicons of a distinct size from the products amplified from the specifically targeted or universal or semi-universal oligonucleotide primers using the same or any other pathogen-specific universal or semi- universal oligonucleotide primers with specimen-derived or control templates.
  • Competitor polynucleotides are added at multiple specific but differing concentrations (i.e., copy numbers) to allow for determination or estimation of the quantity (i.e., copy number) of a pathogen-specific, universal or semi-universal nucleic acid amplifications generated from the specimen.
  • the methods include monitoring of a series of specimens from the same source for any of a predetermined plurality or multiplicity of pathogens.
  • the methods include obtaining a specimen from a source at regular intervals (e.g., about continually, hourly, daily, weekly, about monthly, about quarterly or yearly) and quantifying the amount or relative amount of the composition of pathogen or multiple pathogens or specific or unknown organisms in the specimen using any amplification method and also followed by non-optical genetic sequencing methods.
  • a source may be any specimen suspected clinically of containing microorganisms.
  • pathogens may be detected in the asymptomatic individual and appropriate measures can be taken, such as modification of administration of compositions that result in immunosuppression of the individual or administration of a therapy to ameliorate and/or treat the pathogen infection.
  • the present invention relates generally to an approach that utilize non-optical methods and universal gene targets (targets that are universal among all microorganisms such as the 16s gene for Achaea and bacteria and the 18s gene for fungi, and the ITS gene for fungi) for detecting the presence of and determining the identity of microorganisms thereby decreasing the cost of the technology and thereby increasing its utility for a variety of purposes.
  • the present invention is directed to the resolution of the complete microbial community of etiologic agents and commensal flora present in samples including, but not limited to, animals, humans, environmental, clinical, or other samples, including samples of unknown origin from which knowledge of the complete microbial community is of scientific and/or medical interest.
  • the invention is further directed to the determination of detailed genetic information about the individual organisms which are detected and identified that make up in the sample’s microbiome including identification of genes that predict or indicate sensitivity or susceptibility of the organism to antimicrobials, antibiotics, or other chemical compounds, ions, or elements.
  • DNA or RNA is extracted from specimen(s).
  • a universal primer such as the universal 16s universal ribosomal primers that pick up a broad range of analytes (e.g., bacteria or archaea) or targets and sample or specimen specific barcodes are used to detect any and all a universal tag are utilized to amplify and or label a set of analytes from a specimen(s) to provide a diagnostic or analysis of monitoring method.
  • analytes e.g., bacteria or archaea
  • sample or specimen specific barcodes are used to detect any and all a universal tag are utilized to amplify and or label a set of analytes from a specimen(s) to provide a diagnostic or analysis of monitoring method.
  • a secondary labeling step or direct labeling will incorporate a sample specific tag/label/barcode that are specific to a sample. This allows many samples to be analyzed through non-optical molecular genetic sequencing approaches.
  • the sample can then be analyzed using a biochemical method (such as generation of hydrogen ions) to generate a signal that is then detected using a semi-conductor chip that can act as a highly multiplex pH meter.
  • a biochemical method such as generation of hydrogen ions
  • the method described herein uses universal or broad range primers and individual sample specific barcodes or tags (as have been well described in the literature), in order to analyzed batches or multiple specific specimens or subjects or sample detecting many specific targets that are grouped together to create a single assay.
  • This assay will be analyzed using a cost effective pH sensor, wave guide, semi-conductor technology or other non-optical method for determining the sequence of molecular material such as nucleic acids (RNA or DNA).
  • RNA or DNA nucleic acids
  • One example is to sequence each of the multiplexed analytes based upon pH generation detected using technology exemplified by a semi-conductor or other silicon or wave guide chip-based technology.
  • the present invention allows many analytes to be screened all at once using broad range (e.g. kingdom specific, genus specific, family or class or sub-groups of organisms or targets) to be screened all at once and allows many different samples to be analyzed all together.
  • broad range e.g. kingdom specific, genus specific, family or class or sub-groups of organisms or targets
  • this invention does not utilize optical methods (such as laser or CCD camera, etc.) and instead measures a chemical change or property (e.g. pH change or increase in molecular weight) then the cost of the technology is reduced dramatically thereby improving the feasibility of the diagnostic applications.
  • optical methods such as laser or CCD camera, etc.
  • a chemical change or property e.g. pH change or increase in molecular weight
  • DNA or RNA is extracted using a method to generate purified RNA or DNA, which can be mechanical or enzymatic. Purification generally includes removing some, any or all non-analyte material through selective enrichment of analytes/target or depleting inhibitors.
  • a set of universal or highly comprehensive e.g. for bacteria the 16s gene can be universal for all bacteria, for fungi the ITS1-4 region is highly comprehensive
  • amplifying primers are used.
  • a 16s and ITS assay can be combined.
  • One or more very broad range or universal or comprehensive primers (non-specific primers) targeting a variety of similarly related analytes e.g.
  • RNA and or DNA is extracted (e.g., step 1) from receipt of sample but may be extracted at any time up to the analysis (step #3) from a sample using a chemical or physical method that generates a template.
  • This template contains zero or up to 1 million or more analytes that will be detected directly or after processing through steps 2-3. These analytes may be directly measured without steps 2-3, or steps 2 and 3 may occur together.
  • Step 4 can be integrated with step 3. Together the steps may be used separately or together and in any order within the process.
  • a number of universal or broad range primers that is between 1 and 1 million broad range primers (universal primers e.g. two primers that can amplify by means of the PCR 20% of all bacteria or 80% of all bacteria or 100% of all bacteria or between 20% and 100% of all bacteria).
  • Bacteria in this instance represent one of many targets that may be analytes for diagnostic, monitoring, or research purposes. Samples can then be batched together or may be mixed together with the sample during DNA and or RNA extraction.
  • Each of the specific primers will be targeted for a group of analytes (e.g.
  • primers A universal for target group A primers B universal for target group B, ....Primers N (where N is any number between 3 and 1 million or more) specific for target group N, are utilized to mix with the DNA or RNA from the specimen(s), and then create a Step 3 ligation or an amplification with the purpose of generating directly or indirectly each detectable group specific analyte between 0 and 1 million or more for each specimen.
  • Step 3 can be performed within an enzymatic step or process such as the polymerase chain reaction or directly through a linear amplification process or through other general mechanisms of amplification.
  • DNA and RNA are extracted using one of many common methods from physical and chemical disruption. Many DNA and RNA extraction kits are sold commercially and methods for DNA extraction are well known.
  • Primers are designed to detect groups of targets of known analytes at the kingdom or phylum level (highly universal or comprehensive). These analytes can be, but are not limited to, microorganisms such as the phylum spirochetes or the kingdom bacteria, or the kingdom fungi, e.g., 1 primer set rather than 3000 primer sets. Many samples can be analyzed in multiplex as each individual sample will have a unique barcode/tag or primer. These primers are then mixed in equimolar ratios and utilized to amplify or enrich the targets from the DNA and RNA extracted in step 1. On each primer we have a 4-25bp or larger sample specific barcode. This same barcode is utilized for all samples.
  • BarcodeB-XXXXXXXXX-PrimerNF- Analyte N's SPECIFIC REGION - PrimerNR- YYYYYYYYY-BarcodeB .. which would be specific for sampleB and analyte N groups within Sample B; where barcodes A, B, C, D, etc., are examples of unique barcodes that delineate a specific sample that is then utilized by software downstream to deconvolute information and data and diagnosis when run in a highly multiplexed assay format and where Primers are broad range designed to cover large populations of analytes. Examples include but are not limited to different classes of genes e.g. 16s ribosomal gene, 23s ribosomal, functional genes such as nirS, etc.
  • the present invention can also be used with semi-conductor technology.
  • semi-conductor technology One example of which is the ion torrent technology or the IBM semi-conductor genetic sequencing technology.
  • the key feature and difference in this is the use of non-optical sequencing technologies.
  • This is in contrast to optical sequencing methods.
  • Non-optical sequencing uses a sensor that measures changes in the molecular state of the molecule (in this example DNA) such that as bases are added there is a change in the pH of the environment which can be measured, e.g., through use of a semi-conductor chip.
  • optical methods that produce fluorescence when sequencing or light, which are measured by laser or CCD camera respectively (i.e. measured using optical methods).
  • the highly multiplexed sample is then prepared to create an appropriate library that can be placed within the semi-conductor technology and analytes without fluorescence or other optical technologies.
  • bioinformatics and software tools are utilized to analyze the data and provide reports as the presence of the analytes, concentration, mutations within the analytes, species, genus of the analytes, thereby providing a diagnostic tool that can be used by scientists and physicians.
  • DNA and RNA are extracted using one of many common methods from physical and chemical disruption. Many DNA and RNA extraction kits are sold commercially and methods for DNA extraction are well known.
  • Primers are designed to specifically target broad ranges of analytes. These analytes can be pathogens such as all bacteria or all fungi or all protozoa or comprehensive groups of microorganisms such as all spirochetes.
  • pathogens such as all bacteria or all fungi or all protozoa
  • comprehensive groups of microorganisms such as all spirochetes.
  • primers are designed (1 primer pair for instance) that will detect all spirochetes.
  • the user could label each of these samples with a unique barcode but use the sample primer pair and many samples at the same time could be evaluated for spirochetes (in this example).
  • primers from each sample are mixed in equimolar ratios and utilized to amplify or enrich the targets from the DNA and RNA extracted in step 1.
  • 100 samples 100 unique barcodes.
  • the design can be: 5'XXXXXXXX-PrimerAF and 5' YYYYYYYY-primerAR, where XXXXXXXXX represents the unique barcode label and YYYYYYYYY represents the reverse unique barcode label.
  • the forward label and the reverse label can be the same or different.
  • the primerAF represents the forward primer targeting a group of analytes represented by A and primer AR represents the reverse primer targeting analyte group A.
  • primer AR represents the reverse primer targeting analyte group A.
  • Non-optical sequencing technology such as semi-conductor sensor technology, can then be used to sequence the analyte that have been amplified using very small sample sizes.
  • One example of a non- optical method for sequencing is the Life Technologies“ion torrent” technology or the IBM semi conductor genetic sequencing technology.
  • the highly multiplexed sample is then prepared to create an appropriate library that can be placed within the semi-conductor technology and analyzed without fluorescence or other labeling technologies that rely on optical technologies such as cameras or lasers.
  • bioinformatics and software tools are used to analyze the data and provide reports as the presence of the analytes, concentration, mutations within the analytes, species, genus of the analytes, thereby providing a diagnostic tool that can be used by scientists and physicians.
  • the invention can be used for research purposes.
  • non-optical sequencing technologies such as sequencing using semi-conductor chips, which can measure pH changes and many methods for incorporating both the analyte specific primers with their common code and the sample specific codes.
  • Many sequencing methods that do not rely on optical methods may be utilized.
  • Many different types of computational algorithms can be utilized to take the data from the sequencing device and analyze it in a variety of ways to generate a report to give to the scientist or physician.
  • Many computer languages can be used to design software to analyze and generate reports.
  • Data form analysis may identify new types of pathogens that cause disease and may help to identify new treatments for existing pathogens.
  • the technology can be used as a monitoring technology in addition to a diagnostic technology.
  • the technology and process can be used to identify mutations among hundreds of different genes as part of genetic screening in humans and animals.
  • the technology can be used for parentage testing, for studies on phylogeny and systematics, and the technology and process can be used to identify polymicrobial disease contributors, e.g., may be used to target the top 1000 bacteria associated with skin infections.
  • the user would utilize 1 primer set targeting a broad range of bacteria known to reside in skin infections.
  • This technology differs from prior art due to the use of a combination of primers and non-optical sequencing technologies as a process for diagnostics and research use.
  • the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as“have” and“has”), “including” (and any form of including, such as“includes” and“include”) or“containing” (and any form of containing, such as “contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • “comprising” may be replaced with“consisting essentially of’ or“consisting of’.
  • the phrase“consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention.
  • the term“consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.
  • words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
  • Persing DH PCR protocols for emerging infectious diseases: a supplement to Diagnostic Molecular Microbiology: principles and applications: Amer Society for Microbiology; 1996.
  • Ronaghi M, Uhlen M, Nyren P A sequencing method based on real-time pyrophosphate. Science 1998, 281(5375):363-365.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé pour déterminer l'identité d'un organisme ou d'un virus dans un échantillon. Le procédé consiste à isoler l'ADN ou l'ARN de l'échantillon ; à combiner l'ADN ou l'ARN directement ou avec une ou plusieurs amorces d'amplification universelles ou spécifiques d'une cible, la ou les amorces étant spécifiques pour un ou plusieurs groupes de micro-organismes ou virus cibles ; et à amplifier l'ADN ou l'ARN après une transcription inverse avec une transcriptase inverse ; et à mettre le produit de l'amplification en contact avec un ou plusieurs marqueurs détectables spécifiques d'une espèce, d'un organisme ou d'un virus.
PCT/US2019/057866 2018-11-06 2019-10-24 Dosages universels ou à large plage et procédé de diagnostic spécifique d'échantillons à marqueurs multiples à l'aide d'un séquençage non optique WO2020096782A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16/182,140 US20190062815A1 (en) 2011-12-06 2018-11-06 Universal or Broad Range Assays and Multi-Tag Sample Specific Diagnostic Process Using Non-Optical Sequencing
US16/182,140 2018-11-06

Publications (1)

Publication Number Publication Date
WO2020096782A1 true WO2020096782A1 (fr) 2020-05-14

Family

ID=70612184

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/057866 WO2020096782A1 (fr) 2018-11-06 2019-10-24 Dosages universels ou à large plage et procédé de diagnostic spécifique d'échantillons à marqueurs multiples à l'aide d'un séquençage non optique

Country Status (1)

Country Link
WO (1) WO2020096782A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646904A (zh) * 2020-12-14 2021-04-13 深圳市计量质量检测研究院 唐菖蒲伯克霍尔德氏菌及米酵菌酸产毒株的检测方法、检测用荧光pcr引物和探针

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030093830A1 (en) * 1999-06-21 2003-05-15 Alain Ghesquiere Means for identifying the locus of a major resistance gene to the rice yellow mottle virus, and their applications
US20120129794A1 (en) * 2009-07-24 2012-05-24 Dowd Scot E Apparati, methods, and compositions for universal microbial diagnosis, detection, quantification, and specimen-targeted therapy
US20130157874A1 (en) * 2011-12-06 2013-06-20 Scot E. Dowd Universal or broad range assays and multi-tag sample specific diagnostic process using non-optical sequencing
US20170088889A1 (en) * 2015-09-24 2017-03-30 Syracuse University Universal dna profiling
US20170335410A1 (en) * 2016-05-17 2017-11-23 Shoreline Biome, Llc High Throughput Method for Identification and Sequencing of Unknown Microbial and Eukaryotic Genomes from Complex Mixtures
WO2018156664A1 (fr) * 2017-02-21 2018-08-30 Millennium Health, LLC Méthodes et systèmes de test génétique microbien

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030093830A1 (en) * 1999-06-21 2003-05-15 Alain Ghesquiere Means for identifying the locus of a major resistance gene to the rice yellow mottle virus, and their applications
US20120129794A1 (en) * 2009-07-24 2012-05-24 Dowd Scot E Apparati, methods, and compositions for universal microbial diagnosis, detection, quantification, and specimen-targeted therapy
US20130157874A1 (en) * 2011-12-06 2013-06-20 Scot E. Dowd Universal or broad range assays and multi-tag sample specific diagnostic process using non-optical sequencing
US20170088889A1 (en) * 2015-09-24 2017-03-30 Syracuse University Universal dna profiling
US20170335410A1 (en) * 2016-05-17 2017-11-23 Shoreline Biome, Llc High Throughput Method for Identification and Sequencing of Unknown Microbial and Eukaryotic Genomes from Complex Mixtures
WO2018156664A1 (fr) * 2017-02-21 2018-08-30 Millennium Health, LLC Méthodes et systèmes de test génétique microbien

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646904A (zh) * 2020-12-14 2021-04-13 深圳市计量质量检测研究院 唐菖蒲伯克霍尔德氏菌及米酵菌酸产毒株的检测方法、检测用荧光pcr引物和探针

Similar Documents

Publication Publication Date Title
US10167520B2 (en) Universal or broad range assays and multi-tag sample specific diagnostic process using non-optical sequencing
EP3320110B1 (fr) Amorces universelles d'arn ribosomique 16s et leur utilisation pour les analyses microbiologiques et les diagnostics
AU783278B2 (en) Genomic profiling: a rapid method for testing a complex biological sample for the presence of many types of organisms
Ibarz Pavón et al. Multilocus sequence typing
CN108441565B (zh) 人类y染色体37个str基因座的荧光标记复合扩增试剂盒及其应用
WO2000066789A2 (fr) Methode d'identification de micro-organismes a partir d'une matrice polynucleotidique
WO2021099499A1 (fr) Compositions et procédés d'identification rapide et test phénotypique de la sensibilité antimicrobienne de bactéries et de champignons
CA2905410A1 (fr) Systemes et procedes pour la detection de changements de nombre de copie de genome
Diggle et al. Pyrosequencing™: Sequence typing at the speed of light
Thies Molecular approaches to studying the soil biota
Jannes et al. A review of current and future molecular diagnostic tests for use in the microbiology laboratory
CN105349657A (zh) 病原生物的多种分析检测
US20150056626A1 (en) Nucleic acid amplification kits for specific detection of e. coli o157:h7 without co-detection of e. coli o55:h7
Ma Applications and limitations of polymerase chain reaction amplification
US20190062815A1 (en) Universal or Broad Range Assays and Multi-Tag Sample Specific Diagnostic Process Using Non-Optical Sequencing
WO2020096782A1 (fr) Dosages universels ou à large plage et procédé de diagnostic spécifique d'échantillons à marqueurs multiples à l'aide d'un séquençage non optique
Galluzzi et al. Current molecular techniques for the detection of microbial pathogens
WO2021039777A1 (fr) Méthode d'examen de la polyarthrite rhumatoïde
CN108060244A (zh) 一种用于结核分枝杆菌复合群检测的核酸序列及应用
Rao et al. Recent trends in molecular techniques for food pathogen detection
RU2455364C2 (ru) Способ идентификации микобактерий с помощью полимеразной цепной реакции
Kensara et al. Microbiological factors of peri-implantitis: methodologies for biofilm analysis
Eshoo et al. Microbial forensic analysis of trace and unculturable specimens
RU2510856C2 (ru) НАБОР СИНТЕТИЧЕСКИХ ОЛИГОНУКЛЕОТИДОВ ДЛЯ ВЫЯВЛЕНИЯ ДНК ПАРОДОНТОПАТОГЕННОГО МИКРООРГАНИЗМА Candida albicans МЕТОДОМ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ
Figueroa et al. Principles and Applications of Genomic Diagnostic Techniques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19882524

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19882524

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19882524

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC