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WO1994025572A1 - Mycobacteriophages rapporteurs specifiques d'especes mycobacteriennes - Google Patents

Mycobacteriophages rapporteurs specifiques d'especes mycobacteriennes Download PDF

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Publication number
WO1994025572A1
WO1994025572A1 PCT/US1994/004788 US9404788W WO9425572A1 WO 1994025572 A1 WO1994025572 A1 WO 1994025572A1 US 9404788 W US9404788 W US 9404788W WO 9425572 A1 WO9425572 A1 WO 9425572A1
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Prior art keywords
reporter
mycobacterial
mycobacteriophages
mycobacteriophage
genes
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PCT/US1994/004788
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English (en)
Inventor
William R. Jacobs, Jr.
Barry R. Bloom
Graham F. Hatfull
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Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University
University Of Pittsburgh
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Application filed by Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University, University Of Pittsburgh filed Critical Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University
Priority to AU69052/94A priority Critical patent/AU6905294A/en
Publication of WO1994025572A1 publication Critical patent/WO1994025572A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to mycobacterial species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of making such reporter mycobacteriophages, and the use of such reporter mycobacteriophages, for example, to rapidly diagnose mycobacterial infection and to assess drug susceptibilities of mycobacterial strains in clinical samples.
  • this invention relates to the use of mycobacterial species-specific luciferase reporter mycobacteriophages to diagnose tuberculosis and to assess the drug susceptibilities of the various strains of Mycobacterium tuberculosis (M. tuberculosis).
  • transcriptional promoters and reporter genes are introduced into the genomes of mycobacterial species-specific mycobacteriophages.
  • reporter genes may be the genes for luciferase or the ⁇ -galactosidase gene, and provide the DNA which encodes the production of a gene product.
  • the reporter mycobacteriophages may be used for diagnosing mycobacterial infections by incubating same with samples which may contain the specific mycobacteria of interest. If the mycobacteria of interest is present, then the reporter mycobacteriophages introduce the recombinant nucleic acids which encode expression of the gene product into the mycobacteria of interest, and the mycobacteria then express the gene product.
  • the expressed reporter gene product may be detected by a suitable assay, for example, through the detection of photons or the conversion of an easily assayable chemical reaction. The presence of such gene product indicates that the sample contains the mycobacteria of interest, and hence the mycobacterial species-specific reporter mycobacteriophages may be used to detect and thereby diagnose the specific mycobacterial infection.
  • the mycobacteria species-specific reporter mycobacteriophages of this invention may be used to assess the drug susceptibilities of various strains of mycobacteria. If antibiotic drugs are added to the sample containing the reporter mycobacteriophages and the gene product is detected, the mycobacteria is metabolically active and hence resistant to the antibiotic drug.
  • tuberculosis (and other mycobacterial infections) which is rapid, sensitive and specific, which method is also capable of assessing the drug susceptibilities of the various strains of M. tuberculosis and other mycobacterial strains. It is critical that a mycobacterial strain be assessed for drug resistance rapidly because a patient infected with a strain of M. tuberculosis or another mycobacteria must be treated immediately with the particular antibiotic drug(s) to which the strain is not resistant, and not with antibiotic drug(s) to which the strain is resistant, or the patient may die. Currently, the most rapid test available for the diagnosis of M.
  • tuberculosis is the staining of sputum samples for acid-fast bacilli, which is a tedious procedure, and which procedure has low sensitivity.
  • Alternative methods for diagnosis require cultivation of the bacilli for approximately two to six weeks followed by classification of the cultured organism.
  • Typical diagnostic tools include biochemical tests, analysis of mycolic acids and serotyping. All of these tests are time-consuming.
  • More recently, the use of oligonucleotide probes and Polymerase Chain Reaction have been suggested for the identification of M. tuberculosis species. Although these methods may be useful approaches, their uses in a clinical setting have not yet been determined. Further, these methods do not distinguish between live and dead organisms, and are therefore of limited use in the determination of drug sensitivities of clinical isolates.
  • Mycobacterium avium (M. avium) is a mycobacteria which is often found in immunosuppressed patients. This mycobacteria is typically disseminated throughout the bodies of immunosuppressed patients, such as
  • This invention relates to broad host range and mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages, and the use of such reporter mycobacteriophages to rapidly diagnose mycobacterial infection, such as M. tuberculosis, and to distinguish which strains of the mycobacteria are drug-resistant.
  • reporter mycobacteriophages To produce these reporter mycobacteriophages, reporter genes and transcriptional promoters are introduced into the genomes of mycobacterial species-specific mycobacteriophages.
  • the promoter and reporter gene-containing mycobacteriophages are then incubated with a clinical sample which may contain the mycobacteria of interest, such as M. tuberculosis.
  • the reporter mycobacteriophages are specific for the mycobacteria which is sought to be detected.
  • the reporter mycobacteriophages efficiently introduce the recombinant nucleic acids which encode the expression of the reporter gene's gene product into the mycobacteria of interest, and the mycobacteria then express the gene product.
  • a substrate or other means capable of allowing for the detection of the gene product is then added to the sample. If the gene product or the signal generated by the gene product is detected, the presence of the infectious mycobacteria is known, thereby diagnosing the
  • drugs such as antibiotics may be added to a sample containing the reporter mycobacteriophages of this invention. If the mycobacteria are susceptible to a drug after exposure to the drug, the mycobacteria will be killed. However, drug-resistant mycobacteria will continue to be metabolically active in the presence of the drug, and will continue to express the detectable gene product of the reporter genes.
  • the reporter mycobacteriophages of the invention are temperate, and have increased sensitivity for use in drug screening.
  • the preferred reporter genes of the present invention are the Firefly luciferase lux gene (FFlux), the luciferase lux genes of Vibrio fischeri, the luciferase lux genes of Xenorhabdus luminescens and the E. coli ⁇ -galactosidase gene (lacZ).
  • Some preferred promoters of the present invention are hsp60 and gene 71-70-69 promoters, and the preferred mycobacteriophages are L5, TM4 and DS6A. These reporter mycobacteriophages are preferably used for the rapid diagnosis of tuberculosis and M. avium infection, and the accurate assessment of drug susceptibilities of the various strains of M. tuberculosis and M. avium.
  • FIGURE 1 (which is comprised of Figure 1A and Figure 1B) represents the genome organization of mycobacteriophage L5;
  • FIGURE 2 represents a luciferase shuttle plasmid pYUB180 wherein reporter gene FFlux is fused to the BCG hsp60 promoter;
  • FIGURE 3 represents the amount of luciferase activity of M. smegmatis which contains the pYUB180 shuttle plasmid and the FFlux gene;
  • FIGURE 4 (which is comprised of Figure 4A, Figure 4B and Figure 4C) represents the effect of various antibiotic drugs on the metabolic activity of control mycobacteria and drug resistant mycobacteria in the presence of reporter mycobacteriophages which contain luciferase reporter genes;
  • FIGURE 5 represents shuttle plasmid phAE39 wherein the reported gene is FFlux, the promoter is hsp ⁇ O, the phage is TM4 and the cosmid is pYUB216;
  • FIGURE 6 represents luciferase activity
  • FIGURE 7 represents a flow chart for cloning different promoters into TM4 : : lux shuttle phasmid phAE39;
  • FIGURE 8 represents a schematic diagram of the luciferase reporter mycobacteriophages phAE39 and phAE40;
  • FIGURE 9 represents the production of light (photons) by mycobacteria following infection with the luciferase reporter phage phAE40;
  • FIGURE 10 (which is comprised of Figure 10A, Figure 10B and Figure 10C) represents a comparison of the kinetics of light production following phage infection of drug-sensitive BCG cells to drug-resistant BCG mutant cells;
  • FIGURE 11 (which is comprised of Figure 11A, Figure 11B and Figure 11C) represents a comparison of drug-sensitive M. tuberculosis and drug-resistant M. tuberculosis using the luciferase reporter phage assay;
  • FIGURE 12 represents a schematic diagram of the extrachromosomal plasmid pYUB180 and the integration plasmid pGS16;
  • FIGURE 13 represents the expression of luciferase by
  • FIGURE 14 represents a DNA fragment of the L5 segment defined by the coordinates 3,150-7,143 after cleaving with Xba I and Bcl I;
  • FIGURE 15 represents the DNA fragment of L5 after insertion into plasmid pMV2611acZ and cleaving with Xba I and BAMHI to produce plasmid pGS11;
  • FIGURE 16 represents plasmid pGS12 which was produced by cleaving pGS11 DNA with Xba I and Hind III, and inserting fragment 4,013bp into plasmid pMD31;
  • FIGURE 17 represents plasmid pGS22, which was produced by cutting plasmid pYUB216 with Hind III and converting the sticky ends to blunt ends by Klenow enzyme and dNTP's;
  • FIGURE 18 represents plasmid pGS24, which was produced by inserting plasmid pGS22 into the NHE I site of pGS12 plasmid;
  • FIGURE 19 (which is comprised of Figure 19A and Figure 19B) represents a double crossover event between plasmid pGS24 and L5;
  • FIGURE 20 (which is comprised of Figure 20A and Figure 20B) represents hybridized bands detected by autoradiography
  • FIGURE 21 (which is comprised of Figure 21A-1, Figure 21A-2, Figure 21B-1 and Figure 21B-2) represents a map of expected DNA fragments resulting from a pair of homologous recombination events in common flanking sequences when FFlux is inserted into the L5 genome in a corresponding location to that in pGS24;
  • FIGURE 22 represents restriction enzyme mapping and Southern blot hybridization for phGS1 and phGS5;
  • FIGURE 23 represents determination of the luciferase activity of pGS24, phGS1 and phGS5;
  • FIGURE 24 represents luciferase activity as determined after liquid infection of M. smegmatis mc 2 155 with phGS1 and phGS5;
  • FIGURE 25 represents a comparison of luciferase activity of phGS5 with clear plaque mutant derivatives that are not competent to form lysogens;
  • FIGURE 26 represents the activity of phAE40 and the L5 : :FFlux phages following infection with M. smegmatis mc 2 155;
  • FIGURE 27 represents the sensitivity of phage phGS5 after infecting M. smegmatis mc 2 155;
  • FIGURE 28A represents the detection of luciferase activity after liquid infection of serial dilutions of M. smegmatis with phGS18;
  • FIGURE 28B represents the light produced (RLU);
  • FIGURE 29 represents the result of liquid infection of nonlysogen and lysogen strains of M. smegmatis with phAE40, phGS18 and phGS26;
  • FIGURE 30 represents a list of L5 reporter mycobacteriophages of the invention which have been developed.
  • FIGURE 31 represents an outline of a method which can be used to diagnose tuberculosis and determine drug susceptibility using reporter mycobacteriophage DS6A.
  • This invention is directed to mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages and the use of such reporter mycobacteriophages for the rapid diagnosis of mycobacterial infections and the accurate assessment of mycobacterial drug susceptibilities.
  • mycobacterial species-specific mycobacteriophage genomes are modified by introducing therein transcriptional promoters and reporter genes whose gene product can be sensitively detected.
  • the reporter mycobacteriophages may then be incubated with clinical samples suspected of containing the mycobacteria of interest, either directly of after culture, and the samples tested for the presence of the reporter gene product, thereby diagnosing mycobacterial infection.
  • the method of this invention allows for rapid diagnosis because only the amount of time necessary for the reporter mycobacteriophages to infect their host cells and the amount of time necessary for the host cells to synthesize the reporter gene product are required to allow for diagnosis.
  • the amount of time required for the reporter mycobacteriophages to infect their host cells and for the host cells to synthesize the reporter gene product is between ten minutes and sixteen hours.
  • the assessment of drug susceptibilities with the reporter mycobacteriophages of this invention is accurate because the reporter mycobacteriophages only allow for the detection of metabolically active mycobacterial organisms, the presence of which metabolic activity indicates that a drug has not killed the mycobacteria and that the mycobacteria is resistant to the drug.
  • the L5 reporter mycobacteriophages of this invention are temperate, i.e., they are able to exist in bacterial cells as prophages integrated into mycobacterial genomes without causing cell lysis. Because the L5 reporter mycobacteriophages do not cause cell lysis, they replicate as part of the bacterial genomes in bacterial cells.
  • the integrated reporter phages express high levels of luciferase activity, since the luciferase reporter phages can be stably maintained. This growth causes amplification of photon signal. Because temperate phages possess the ability to site specifically integrate into mycobacterial genomes, they are replicated as part of the mycobacterial chromosome. In addition, the integrated luciferase reporter phages confer to the infected cell the ability to produce amounts of luciferase activity comparable to plasmid transformed cells and 100 to 1000 times more luciferase activity than phage-infected cells.
  • the luciferase lysogens can be readily used to screen for drug activity by simply observing the inhibition of growth measured by proportional luciferase activity.
  • temperate L5 reporter mycobacteriophages results in a more sensitive assay for drug screening, as compared to the use of lytic reporter mycobacteriophages.
  • Mycobacteriophage L5 a temperate virus with a broad host-range among mycobacteria, is the most thoroughly characterized of the mycobacteriophages.
  • L5 particles are morphologically simi lar to the f ami ly of phages that includes phage ⁇ and contain a linear dsDNA genome with cohes ive ends .
  • the inventors have determined the DNA sequence of the entire genome of L5 , as well as several gene functions .
  • DNA sequence of the L5 mycobacteriophage is as follows :
  • ATCTACGCCA CTCCTGACGG GTGGCTGTCA AGGATACTCA CCTTCCCTAC TAATGAGGGG 240
  • CTAAGAGCCC CTCTCTATAG AGCGCCGCAC AGGCGGCGCG ATAAGAGCGC CACCAGGCGC 300
  • GATCCTCAAC TACGAGGGTC CAGGGACCGT CGAGGTCTCC GACGAGAAGC TCGCCGAAGC 2280
  • GTCTCCCCCT CACATCATCG GCCCGTCCTG GCAGAAGACG GTCGATGGTG AGTGGTATCT 6720
  • CTGGCTCACG TCGGATACCC CAGGCTCTAC GTCGACTCAG TCGCCGAGCG CCAGGCCGTC 8640
  • CTGTAGGTGA GTGGCTGCGA CTGCTGCAGG TGCTGTTCCC CGAAATCCAA CGGCGGTATG 10020
  • ACTTCGCCTA CAGCTCAGCG GAAGCCGCAG GGCTCAACCT CGATGACGAG ACCGTGATCG 10440
  • TCGAGGCCAT CAAGGCCGCT GTCGGGATCA AGAAGTAACC CACCCAACAG ATCTCAAGGA 11340
  • CAAGGTCGCG CAGACCGGCG ACTCGATGTT CGAGGGCTAC CTCGAGCCCG AGCAGGCCCA 11460
  • GGCCCAGTCC CAGATCGAGT GGCGAGACCA GCGGTGGGCG CTCTTCGGAG ACGCCACCGT 13440
  • TCGTCATCCT CAACGACGCG AAGCAGGGGC CGCGCTCCTG GCTGTCGCGA GACTCCGAGT 18600
  • CAAGAGGTAA ACCCCTTGAG GTCACGGTGA TGTCGTGCAT CGCGTACGAC CCGTTCTGGT 18780
  • CTACTCCCCC TGGTTCATGG GCACCAGCCC GATAGCACCC CACGTCGTGT TCGAAGAAGG 20400
  • CTGCGCCCGA CCGGGACAGA AATACATAGA GAACCTATGG ATGTAGGAGG CACAAAAAAA 26280
  • CTCCTCGACC GACTCGCGCT CCACGCGGAT CAGCCGGGGA CCGAGCCGAA CGGCCTTGAG 26640
  • GGCACCAGCG ACCGACTCAC GTCGGGCACC AACGGTGTCA GGCTCTTGCA GACCCTCGCG 29880
  • GAGCCTCTTC CTCTTCGACT ACCTCGTCTA CCCGGCGGAA TAACTCCGCT AGTTCTGCGG 30840
  • TAGTAGTAGT CAACGACCTT GTCCCAGTTG AAGGTTCGGG ACGTGCCGTC ATCGAACGCG 31500
  • CTCCATACAT CAGATCCTTT CCAGCAGAGC AGCTTTGCCC TGCGATGTGA CTAGTGAGTT 37860
  • CTACCGCGTC CTCGATGTTC TGCTCGCGAA CAGCCCGCGT AGCTCGTTCG AGCGACCATC 41340
  • GCGCGGCCCA GACCGGGGGT AGCGGATCCC CCGCCGACCC TCGGATGTAG AGCGATTGTC 41580 TAGGTGTGTA CACCTTCCTC CTCGTGGATG TGATTGACCA GGTCATAGAT CTCGTCGCGA 41640
  • CTCCACTGGG TGTGTCGGTC CTCGCTGGTG TGGTCCCCGA CGTATGGGAA GTGGCTCAGC 41820
  • AAGTCACACC TCCAATTCGT GGGGCTTGAT CTCGTTGGTC ACGTCGTAGT CGTTCAGCAG 45420
  • CAGCGTGGAC CACCTTGCGG CGCTCGCGCC GTACCTTGTC GCGGCCGGCC GGCCGAACCA 48360
  • CTCCCTCCAA GGCTTGCACC GAGTACCACG GCTTGCCCTC GCGGTGCGTG CGGTGCAGGT 48480
  • Integration-proficient plasmid vectors have been constructed which efficiently transform both fast-growing and slow-growing mycobacteria through stable integration of the plasmid sequences into the bacterial chromosomal attachment site (attB).
  • L5 sequence is now known, and because L5 has been previously characterized, the use of transcriptional promoters with this mycobacteriophage may be evaluated efficiently, and host synthesis inhibition may also be evaluated efficiently.
  • Figure 1 represents the genome organization of the entire L5 genome. DNA analysis has indicated that the L5 genome is organized into a right and left arm with the attachment site at the center of the genome. The integration functions have been successfully employed to construct integration-proficient vectors for mycobacteria.
  • L5 genome is not essential for mycobacteriophage growth.
  • gene 71-70-69 may be deleted without affecting the lytic cycle of the L5 phage. Therefore, it may be a suitable region in the L5 mycobacteriophage for the insertion of reporter genes. As a general role, it is critical that reporter genes be inserted into non-essential regions of the mycobacteriophage. Otherwise, the mycobacteriophage will be unable to survive and replicate.
  • the L5 mycobacteriophage may have introduced therein promoter gene 71 fused to reporter gene lacZ, and this reporter mycobacteriophage would be capable of rapid diagnosis of mycobacterial infection and accurate assessment of mycobacterial strain drug susceptibilities.
  • TM4 mycobacteriophage which may be successfully used to produce the reporter mycobacteriophages
  • TM4 has been used to construct a first generation reporter mycobacteriophage, and has the ability to discriminate between M. tuberculosis and BCG.
  • a shuttle plasmid may be employed with TM4, and may be useful in the construction of recombinant and other mycobacteriophages.
  • L5 which is a broad host-range mycobacteriophage
  • TM4 is a species-specific mycobacteriophage.
  • TM4 is not as well characterized as the L5 mycobacteriophage, and therefore it is more difficult to analyze its functions.
  • DS6A is a mycobacteriophage that has been found to be specific for the M. tuberculosis complex of mycobacteria. It has been shown to infect both M. tuberculosis and BCG. It has been demonstrated that DS6A can infect over 3,000 different types of M. tuberculosis strains. Current efforts are under way to develop DS6A shuttle phasmids containing Firefly luciferase genes as the reporter molecule.
  • DS6A mycobacteriophage is specific for only M. tuberculosis strains.
  • L5 and TM4 mycobacteriophages are specific for several mycobacteria, including M. tuberculosis and M. smegmatis.
  • mycobacteriophages which are specific for M. tuberculosis strains only. Because DS6A mycobacteriophage is specific for M.
  • tuberculosis strains only, it can be used to narrow the host specificity of L5 and TM4 mycobacteriophages so that L5 and TM4 mycobacteriophages can be used to accurately diagnose tuberculosis.
  • a clinical sample control
  • Another clinical sample from the same source experimental
  • DS6A mycobacteriophages can be used to confer specificity for M. tuberculosis onto L5 mycobacteriophages.
  • Figure 31 represents an outline of a method which can be used to diagnose tuberculosis and determine drug susceptibility using reporter mycobacteriophage DS6A.
  • mycobacteriophages are different, except that one of the mycobacteriophages from France had a considerable similarity to the L5 mycobacteriophage, which was originally isolated in Japan.
  • the host range of the mycobacteriophages varies greatly, some being able to infect only M. smegmatis and others being able to infect M. smegmatis, BCG and M. tuberculosis, but not M. avium.
  • These mycobacteriophages may be developed into reporter mycobacteriophages and cosmid cloning systems, and may provide a source of useful transcriptional translation initiating sequences, transcriptional terminators, or host-range specificity genes.
  • reporter gene In addition, the choice of reporter gene and its method of expression are critical. It is necessary to choose a reporter gene whose product would not normally be found in clinical samples, but whose product is also easily detectable.
  • Luciferase reporter genes have been used in many diversified biological systems, including E. coli, cyanobacteria, phytopathogenic bacteria and Bacillus.
  • the presence of luciferase reporter genes can be detected by the emission of photons in the presence of a substrate, such as luciferin or decanal.
  • Luciferin and decanal can permeate mycobacteria, and thereby allow for the detection of gene products, such as photons. Since one molecule of the luciferase gene product can yield 0.85 photons of light, it is the most sensitive biological reporter molecule known.
  • the preferred reporter genes of this invention are luciferase reporter genes, such as the Firefly lux gene (FFlux), the Vibrio fischeri lux genes and the Xenorhabdus luminescens lux genes, as well as the E. coli ⁇ -galactosidase (lacZ) genes.
  • Luciferase genes especially the Firefly lux gene, generate a high amount of luminescence activity. They generate photons. the detection of which is simple and sensitive, using commercially available luminometers that can detect 100-1000 molecules of luciferase with a linear relationship to enzyme concentration. In addition, it is unlikely that clinical samples will contain significant levels of endogenous luciferase activity.
  • promoter candidates currently available are the BCG hsp60 promoter and the L5 gene 71 promoter, which are of comparable strength.
  • the hsp ⁇ o promoter gives good levels of luciferase expression from plasmid recombinants, but lower levels of luciferase expression where the mycobacteriophage is TM4. It is possible that the reason for this is that the hsp60 promoter is shut off by the TM4 enzymes following infection, thus producing only a modest level of luciferase.
  • the gene 71 promoter may behave in a similar manner with the TM4 phage since the gene 71 product is a good candidate for the L5 repressor and is expressed at high levels in the absence of other mycobacteriophage functions. Knowing the sequence of the mycobacteriophage used will help in identifying, characterizing and cloning the appropriate promoter to be used in the reporter mycobacteriophages of this invention. There are several methods which can be utilized to introduce the reporter genes and transcriptional promoters into mycobacterial species-specific mycobacteriophages. One method is the utilization of shuttle phasmids.
  • the shuttle phasmids which consist of the E. coli cosmid, the reporter genes and mycobacteriophage promoters, may then be characterized.
  • Shuttle phasmids can be propagated in E. coli as plasmids, and propagated in mycobacteria as mycobacteriophages.
  • a second method of introducing the reporter genes and transcriptional promoters into mycobacteriophages is by homologous recombination.
  • non-essential regions of a mycobacteriophage must be determined. Again, in order to do this, it is necessary to know the sequence of the mycobacteriophage. Consequently, L5 is an ideal phage to use with this method as its genome has already been sequenced and characterized by the inventors.
  • plasmids are constructed wherein reporter genes hooked to transcriptional promoters are flanked by mycobacteriophage non-essential region sequences in mycobacterial plasmids.
  • homologous recombination systems may be utilized in M. smegmatis or E. coli to perform gene replacement whereby the plasmid constructs containing the reporter genes are put into mycobacteriophages.
  • a third method of introducing reporter genes and transcriptional promoters into mycobacteriophages is by use of transposons.
  • transposon IS1096 may be utilized.
  • reporter genes and transcriptional promoters are put into transposons, and the transposons containing the reporter genes and transcriptional promoters are delivered on plasmids in mycobacteria.
  • the transposons will hop into non-essential regions of the mycobacteriophages, thereby introducing themselves therein.
  • the mycobacteriophages are still viable, and contain the reporter genes and transcriptional promoters.
  • a fourth method of introducing reporter genes and transcriptional promoters into mycobacteriophages is by debilitated phages packaged into phage heads and tails (phage particles).
  • helper phage systems which allow for pieces of DNA containing pac sites to be packaged.
  • helper phages allow for the synthesis of head and tail genes at will in mycobacteria, prevent themselves from being packaged into phage heads and tails, and facilitate packaging of pacmids into phage heads and tails.
  • Helper phage systems may be generated from the L5 mycobacteriophage. The genome of the helper phage is put into the mycobacterial chromosome, at which time the mycobacteria are grown up.
  • pacmids which comprise phages which have pac sites, reporter genes, transcriptional promoters and mycobacterial replicons are transformed onto the mycobacterial strain.
  • the production of head and tail proteins may be induced, for example, through an increase in temperature, and the pacmids are then packaged into phage heads and tails.
  • the L5 genome has cohesive (cos) termini. This suggests the possibility of constructing L5 cosmid vectors, which could be packaged through the cos sites into L5 particles either in vivo or in vitro. Then, a large number of genes could be easily and efficiently delivered to mycobacteria.
  • Packaging into phage heads and tails may also be utilized in a fifth methodology wherein the pacmid is a plasmid.
  • the methodology is similar to the methodology wherein a debilitated phage is used, however, instead of using phage pacmids, the pacmids comprise plasmids which have pac sites, reporter genes, transcriptional promoters, and plasmid replicons.
  • direct cloning using recombinant DNA techniques in vitro may be used to introduce reporter genes and transcriptional promoters into mycobacteriophages.
  • This methodology consists of ligating a mycobacteriophage, identifying or introducing unique restriction enzyme sites in non-essential regions of the mycobacteriophage, cleaving the mycobacteriophage with the restriction enzyme sites, and cleaving DNA which encodes the promoter and the reporter gene so that it has the unique sites flanking it on either side.
  • ligation is set up in vitro between the cleaved mycobacteriophage with the unique restriction enzyme sites and the reporter gene cassette. The result is a circular DNA molecule which consists of the mycobacteriophage, the reporter genes and the transcriptional promoters.
  • the circular DNA may then be electroporated directly into mycobacteria.
  • a promoter probe vector was constructed which incorporated a truncated E. coli ⁇ -galactosidase (lacZ) gene as a reporter probe into a shuttle plasmid vector that replicated in either mycobacteria or E. coli. Random DNA fragments from the three mycobacteriophages Ll, TM4 and Bxbl were cloned into a unique BamHI site immediately upstream of the lacZ gene and screened for their ability to produce ⁇ -galactosidase. This established that lacZ could be used as a reporter gene in the mycobacteria, and identified the DNA sequences which could effectively express foreign genes in both M. smegmatis and M. tuberculosis.
  • lacZ E. coli ⁇ -galactosidase
  • ⁇ -galactosidase activity could be detected from lysed cells using OMPG, or from unlysed cells using either X-gal or a fluorescent methylumbelliferyl ⁇ -galactosidase derivative.
  • the promoter hsp60 gene highly expressed the lacZ gene in both M. smegmatis and BCG.
  • the FFlux gene was cloned into pMV261 downstream from the hsp60 promoter in plasmid pYUB180 (see Figure 2), which plasmid was shown to express the FFlux gene in M. smegmatis, BCG and M. tuberculosis H37Ra.
  • the expression of the FFlux gene was detected by observing luminescence of mycobacterial clones containing the cloned gene in the dark room, and verified use in photographic film. This demonstrated that the luciferase was expressed in the mycobacteria, and that luciferin, the substrate used, was able to penetrate mycobacterial cell walls and yield photons expressed by the mycobacteria.
  • M. smegmatis provided a model with which to determine a minimal number of individual cells detectable with the luciferase assay.
  • M. smegmatis containing pYUB180 were grown in the presence of kanamycin to ensure that every cell contained the plasmid.
  • the cells were diluted 10-fold serially and the amount of luciferase activity was determined using a luminometer.
  • Figure 3 shows that the amount of luciferase activity from 5 X 10 7 cells approached 108 luciferase units, though at this level of activity the luminometer was unable to yield an accurate measurement.
  • the activity decreased in a linear manner down to 1200 units for 500 cells.
  • 5000 cells expressing the FFlux gene can be clearly discerned above the background measurement, which approaches the number of cells that one would expect to observe in clinical samples.
  • FIG. 12 shows a schematic diagram of the extrachromosomal plasmid pYUB180 and the integration plasmid pGS16. Both of the luciferase constructs were electroporated into the M. smegmatis strain mc 2 155.
  • Kan r transformants were grown to a density of approximately 5 ⁇ 10 8 cells/ml and 10-fold serial dilutions were prepared. 100 ⁇ l samples were mixed with 250 ⁇ l of 0.1 M Na citrate, pH5 in a 13 ⁇ 75 mM polystyrene tube. This mixture was placed in the monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) and 100 ⁇ l of 1 mM luciferin (Sigma, St. Louis, MO) was injected into the tube and the luciferase activity was measured as relative light units.
  • monolight 2010 luminometer Analytical Luminescence Laboratory, San Diego, CA
  • 1 mM luciferin Sigma, St. Louis, MO
  • luciferase activity was readily measured from intact mycobacterial cells infected with both the extrachromosomal and the integrating vectors. Serial dilutions indicated that it was possible to detect as few as 500 to 5,000 M. smegmatis cells expressing firefly luciferase, thereby establishing that the luciferase-luciferin system could be developed as a sensitive reporter system for ATP in mycobacteria. Distinguishing Drug-Resistant Mycobacteria From
  • luciferase is a powerful indicator of the metabolic abilities of a bacterial cell. Since anti-tuberculosis drugs are likely to significantly decrease the metabolic activity of a cell, the measurement of luciferase activity should provide a sensitive means of distinguishing drug-resistant mycobacteria from drug-sensitive mycobacteria.
  • the levels of luciferase production were 100 to 1000 times less at eight hours after the addition of the drugs compared to the untreated control.
  • the first vectors developed to introduce recombinant DNA into mycobacteria were shuttle phasmid phage vectors.
  • Shuttle phasmids have the ability to replicate in E. coli as cosmids and then replicate in mycobacteria as phages.
  • Shuttle phasmids of TM4 which contained the FFlux and lacZ genes transcribed from hsp60 and L1 promoters, respectively, were constructed (see Figure 5).
  • FIG. 7 represents a flow chart for cloning different promoters into the TM4::lux shuttle phasmid phAE39.
  • E. coli cosmid pYUB216 was inserted into a non-essential region of the mycobacteriophage TM4.
  • the pYUB216 cosmid contained FFlux in a transcriptional fusion with the hsp60 promoter of BCG, a ColEl origin and an ampicillin-resistant gene (AP) for replication and selection in E. coli, and a bacteriophage lambda cos sequence as well as a unique Bc/l site.
  • the phAE39 shuttle phasmid was constructed with Bc/l-digested pYUB216 being ligated to Sau3A-partially digested TM4 DNA. As shown in Figure 8, the shuttle phasmid phAE39 readily forms plaques of M. tuberculosis, but does not efficiently plaque on BCG. A spontaneous host range mutant of phAE39 was isolated at a frequency of 10 to 10 , and designated phAE40. Mutant shuttle phasmid phAE40 was found to be capable of infecting BCG vaccine strains, in addition to being capable of infecting M. smegmatis and M. tuberculosis strains.
  • the shuttle phasmid phAE40 was deposited with the American Type Culture Collection on April 29, 1993 and catalogued as ATCC No. 75457.
  • the reporter mycobacteriophages were mixed with M. smegmatis cells and then exposed at different times to luciferin.
  • high titers of phAE40 were prepared as described above for TM4 phages. Both M. smegmatis, mc 2 155 cells and
  • BCG-Pasteur cells were grown in roller bottles to approximately 5 ⁇ 10 7 cells per ml in M-ADC-TW broth at 37oC. Either the M. smegmatis or the BCG cells were harvested by centrifugation and washed two times in M-ADC broth, containing no tween. The resulting pellet was resuspended in the original volume of M-ADC broth. The cells were then diluted into fresh M-ADC broth and allowed to incubate overnight standing at 37oC. Tween-80 appeared to remove the receptors, and it was determined that the optimal activities were achieved if the cells were given a chance to grow in the absence of tween.
  • washed cells approximately 5 ⁇ 10 7 cells
  • 0.1 ml phAE40 particles 5 ⁇ 10 8 pfu/ml
  • the cells phage mixture was incubated at 37oC.
  • 0.1 ml samples were removed. Luciferase activity was measured as described in Figure 13. Light signals were detected within minutes following infection using a luminometer and increased 1,000 fold within 2 hours.
  • mutants of BCG were selected that were resistant to streptomycin, isoniazid and rifampicin.
  • Spontaneous mutants of BCG-Pasteur were isolated on Middlebrook 7H10 agar containing either 50 ⁇ g/ml rifampicin, 250 ⁇ g/ml streptomycin or 50 ⁇ g/ml isoniazid.
  • the rifampicin-, streptomycin-, or isoniazid-resistant mutants were purified and designated mc 2 768, mc 2 767 and mc 2 765, respectively. All three mutants and the BCG parent were grown to midlog phase, harvested and washed. As shown in Figure 10C, the mc 2 768 cells and the BCG cells were incubated standing at
  • M. tuberculosis strains were grown in a biological safety level 3 containment facility: (i) the virulent drug-sensitive M. tuberculosis Erdman strain; (ii) strain 92-2025, a singly isoniazid-resistant strain; and (iii) an MDR strain of tuberculosis that has been shown to be resistant to rifampicin, streptomycin, isoniazid, ethambutol and ethionamide and the cause of several nosocomial outbreaks in New York City.
  • the Erdman strain was subcultured from the starter culture by inoculation of 0.4 ml into 20 ml of Middlebrook 7H9 broth containing OADC enrichment (Difco Laboratories, Detroit, MI) plus 0.5 Tween-80 (M-OADC-TW broth).
  • OADC enrichment Difco Laboratories, Detroit, MI
  • M-OADC-TW broth 0.5 Tween-80
  • L5::FFlux phages a plasmid (pGS12) was constructed in which a DNA segment of the L5 genome was inserted into the E. coli-mycobacterial shuttle plasmid pMD31.
  • pMD31 is described by Donnelly-Wu et al. in "Superinfection Immunity of Mycobacteriophage L5: Applications for Genetic Transformation of Mycobacteria", Molecular Microbiology, Vol. 7, No. 3, pages 407-417 (1993).
  • This DNA segment contained the tRNA gene cluster from L5 as described by Hatfull et al. in "DNA Sequence, Structure and Gene Expression of Mycobacteriophage L5: A Phage System for Mycobacterial Genetics", Molecular Microbiology, Vol.
  • this plasmid was further manipulated by insertion of a segment of DNA containing the FFlux gene between the second and third tRNA, to produce pGS24.
  • the resulting plasmid DNA was introduced into M. smegmatis by electroporation, and an L5 lysate was prepared by growth of L5 phage on this plasmid-containing strain.
  • Plasmids pGS11, pGS12 and pGS22 were constructed as described below and then used to construct plasmid pGS24.
  • L5 DNA was cleaved with Xba I and Bcl I and the 3,993bp fragment was purified. This DNA fragment represents the L5 segment defined by the coordinates 3,150-7,143.
  • Figure 14 is a segment of L5 DNA used for FFlux insertion which shows the left arm of the L5 genome with genes 1-33 indicated. The segment of L5 taken to make FFlux inserts is between the Xba I and Bcl I sites indicated. The Nhe I site that defines the position of insertion of FFlux is shown.
  • FIG. 15 is a map of plasmid pGS11 which contains the Xba I - Bcl I segment of L5 inserted into pMV2611acZ.
  • the Bcl I end was inserted into the Bam HI site of the vector and both the Bcl I and Bam HI sites were destroyed.
  • the Hind III and Xba I sites that were used to construct pGS12 are indicated.
  • Plasmid pGS1l DNA was cleaved with Xba I and Hind III and the 4,013bp fragment was purified and inserted into plasmid pMD31 (Donnelly-Wu et al., 1993) cleaved with Xba I and Hind III.
  • This plasmid was named pGS12.
  • Figure 16 is a map of plasmid pGS12 showing the location of the Xba I and Hind III sites used to insert the Xba I - Hind III piece from pGS11 into pMD31. The unique Nhe I site used for the insertion of FFlux is also shown.
  • Plasmid pGS12 contains a unique Nhe I restriction site which corresponds to the Nhe I site at position 4,441 in the L5 genome which is located between the tRNA-trp and tRNA-gln genes (genes 8 and 9).
  • Plasmid pYUB216 was cut with Hind III, the sticky ends converted to blunt ends by Klenow enzyme and dNTP's and the DNA religated. The resulting plasmid was named pGS22.
  • Figure 17 is a map of plasmid pGS22 which shows the two Nhe I sites that flank the FFlux gene. This procedure was followed to generate an additional Nhe I site upstream of the FFlux gene in pYUB216.
  • pGS12 was digested with Nhe I.
  • pGS22 was also digested with Nhe I which produces a fragment of approximately 2.4kb.
  • the DNA's were mixed, ligated and a recombinant recovered in which the Nhe I fragment derived from pGS22 was inserted into the Nhe I site of pGS12.
  • This plasmid was named pGS24.
  • Figure 18 is a map of plasmid pGS24 which contains the Nhe I FFlux DNA fragment inserted into the unique Nhe I site of pGS12. The two Nhe I sites are indicated.
  • the orientation of the inserted DNA was determined by restriction enzyme digestion and found to be in the appropriate orientation for FFlux to be expressed from the same DNA strand as the L5 tRNA's.
  • pGS24 is thus a E. coli-mycobacterial shuttle plasmid that contains the FFlux gene flanked upstream by approximately 1,291bp of L5 DNA and downstream by approximately 2,702b
  • Figure 19 shows the strategy for recombination between pGS24 and L5. Specifically, Figures 19A and 19B show the left arm of L5 and the position of genes 1-33. Figure 19A shows the segment of L5 DNA present in pGS24 and the location of FFlux inserted between the tRNA-trp and tRNA-gln genes.
  • Plasmid pGS24 DNA was introduced into M. smegmatis mc 2 -155 by electroporation, and transformants recovered by selection with kanamycin.
  • a lysate of phage L5 was prepared by infection of approximately 0.5 ml late-log phage M. smegmatis cells containing plasmid pGS24 with approximately 10 L5ts11 particles and incubation on solid media at 37°C. [L5ts11 is a poorly characterized temperature-sensitive mutant of L5].
  • the phages were harvested and shown to have a titer of approximately 10 plaque forming particles/ml (pfu/ml).
  • Phage DNA's were prepared from high titer stocks of phGS1 and phGS5 using standard methods.
  • phGS1 and phGS5 DNA's were digested with several different restriction enzymes (including Bam HI, Nhe I, Bst E II, Asp718, Cla I, Bgl II) and the patterns observed compared with those obtained from wild-type L5, using agarose gel electrophoresis.
  • restriction enzymes including Bam HI, Nhe I, Bst E II, Asp718, Cla I, Bgl II
  • Several differences were observed between phGS1 and phGS5 as compared to L5 DNA. Some of these changes were consistent with a double crossover recombination event inserting FFlux onto the L5 genome as anticipated. Other differences were consistent with deletion of some of the L5 DNA close to the right end of the genome.
  • Figures 21A-1 and 21A-2 maps show the expected restriction products from FFlux insertion - Bam HI.
  • the location of the L5 probe (from pGS12) used for hybridization is shown (labeled 'probe'). This probe is expected to hybridize to two comigrating Bam HI fragments (3.010bp and 3,104bp) in wild-type L5 DNA (shown as 'labeled BAM HI fragments' in the top part of the figure).
  • Figure 21A-2 shows the anticipated structure of the FFlux insertion and the expected fragments resulting from digestion with either Bam HI or BAM HI + EcoRI that hybridize with the probe.
  • Figures 21B-1 and 21B-2 maps show the expected restriction products from FFlux insertion - Asp718/Cla I.
  • the pGS12 probe is anticipated to hybridize to 2,690bp, 1,148bp and 8,078bp fragments resulting from Asp718 digestion of L5 and 2,690bp, 2,981bp and 8,078bp from the FFlux recombinants.
  • This probe is also expected to hybridize to 645bp, 1,148bp and 8,078bp L5 fragments from Asp718 + Cla I digestion and 645bp, 1,078bp and 8,078bp fragments from this digestion of the FFlux recombinants.

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Abstract

L'invention se rapporte à des mycobactériophages rapporteurs spécifiques d'espèces mycobactériennes (mycobactériophages rapporteurs), à des procédés de production et d'utilisation desdits mycobactériophages rapporteurs pour établir un diagnostic rapide des infections mycobactériennes et pour évaluer les sensibilités médicamenteuses de souches mycobactériennes contenues dans des échantillons cliniques. L'invention se rapporte en particulier à la production et à l'utilisation de mycobactériophages contenant des gènes rapporteurs de luciférase pour diagnostiquer la tuberculose. Ces mycobactériophages rapporteurs comprennent des mycobactériophages spécifiques d'espèces mycobactériennes contenant des gènes rapporteurs et des promoteurs transcriptionnels. Lorsque les mycobactériophages rapporteurs sont incubés avec des échantillons cliniques qui peuvent contenir les mycobactéries à étudier, le produit génique des gènes rapporteurs sera exprimé si l'échantillon contient lesdites mycobactéries, ce qui permet de diagnostiquer l'infection mycobactérienne.
PCT/US1994/004788 1993-04-29 1994-04-29 Mycobacteriophages rapporteurs specifiques d'especes mycobacteriennes WO1994025572A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
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US5922282A (en) * 1995-06-07 1999-07-13 Ledley; Robert S. Super fast tuberculosis diagnosis and sensitivity testing method
WO2006075996A2 (fr) * 2004-04-07 2006-07-20 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Systeme d'encapsidation de phage de plasmide rapporteur utile pour detecter les bacteries
EP2142667A1 (fr) * 2007-04-05 2010-01-13 Sequella, Inc. Procédés perfectionnés et compositions pour déterminer l'état pathogène des agents infectieux
US8501400B2 (en) 2007-04-05 2013-08-06 Sequella, Inc. Methods and compositions for determining the pathogenic status of infectious agents
US9133497B2 (en) 2013-03-13 2015-09-15 GeneWeave Biosciences, Inc. Systems and methods for detection of cells using engineered transduction particles
US9388453B2 (en) 2013-03-13 2016-07-12 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US9540675B2 (en) 2013-10-29 2017-01-10 GeneWeave Biosciences, Inc. Reagent cartridge and methods for detection of cells
US10351893B2 (en) 2015-10-05 2019-07-16 GeneWeave Biosciences, Inc. Reagent cartridge for detection of cells
WO2021220302A1 (fr) * 2020-04-29 2021-11-04 Indian Council Of Medical Research Procédé pour déterminer la sensibilité du bacille de koch à un médicament sur un disque en papier
CN114164183A (zh) * 2021-12-17 2022-03-11 中国科学院大学 一株南非诺卡氏菌噬菌体p69及其应用

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US4861709A (en) * 1985-05-31 1989-08-29 Technicon Research A.G. Detection and/or identification of microorganisms in a test sample using bioluminescence or other exogenous genetically-introduced marker

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5922282A (en) * 1995-06-07 1999-07-13 Ledley; Robert S. Super fast tuberculosis diagnosis and sensitivity testing method
WO2006075996A2 (fr) * 2004-04-07 2006-07-20 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Systeme d'encapsidation de phage de plasmide rapporteur utile pour detecter les bacteries
WO2006075996A3 (fr) * 2004-04-07 2006-10-26 Us Gov Health & Human Serv Systeme d'encapsidation de phage de plasmide rapporteur utile pour detecter les bacteries
EP2142667A1 (fr) * 2007-04-05 2010-01-13 Sequella, Inc. Procédés perfectionnés et compositions pour déterminer l'état pathogène des agents infectieux
JP2010531636A (ja) * 2007-04-05 2010-09-30 セケラ インコーポレイテッド 感染性病原体の病原性状態を決定するための改良された方法および組成物
EP2142667A4 (fr) * 2007-04-05 2011-08-10 Sequella Inc Procédés perfectionnés et compositions pour déterminer l'état pathogène des agents infectieux
US8501400B2 (en) 2007-04-05 2013-08-06 Sequella, Inc. Methods and compositions for determining the pathogenic status of infectious agents
AU2008236615B2 (en) * 2007-04-05 2013-10-03 Sequella, Inc. Improved methods and compositions for determining the pathogenic status of infectious agents
US9481903B2 (en) 2013-03-13 2016-11-01 Roche Molecular Systems, Inc. Systems and methods for detection of cells using engineered transduction particles
US10240212B2 (en) 2013-03-13 2019-03-26 GeneWeave Biosciences, Inc. Systems and methods for detection of cells using engineered transduction particles
US9133497B2 (en) 2013-03-13 2015-09-15 GeneWeave Biosciences, Inc. Systems and methods for detection of cells using engineered transduction particles
EP3699592A1 (fr) * 2013-03-13 2020-08-26 Geneweave Biosciences Inc. Particules de transduction non réplicative et systèmes de rapporteur basé sur des particules de transduction
US9546391B2 (en) 2013-03-13 2017-01-17 GeneWeave Biosciences, Inc. Systems and methods for detection of cells using engineered transduction particles
US9752200B2 (en) 2013-03-13 2017-09-05 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US9771622B2 (en) 2013-03-13 2017-09-26 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US9388453B2 (en) 2013-03-13 2016-07-12 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US10227662B2 (en) 2013-03-13 2019-03-12 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US10227663B2 (en) 2013-03-13 2019-03-12 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US10125386B2 (en) 2013-10-29 2018-11-13 GeneWeave Biosciences, Inc. Reagent cartridge and methods for detection of cells
US9540675B2 (en) 2013-10-29 2017-01-10 GeneWeave Biosciences, Inc. Reagent cartridge and methods for detection of cells
US10351893B2 (en) 2015-10-05 2019-07-16 GeneWeave Biosciences, Inc. Reagent cartridge for detection of cells
WO2021220302A1 (fr) * 2020-04-29 2021-11-04 Indian Council Of Medical Research Procédé pour déterminer la sensibilité du bacille de koch à un médicament sur un disque en papier
CN114164183A (zh) * 2021-12-17 2022-03-11 中国科学院大学 一株南非诺卡氏菌噬菌体p69及其应用

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