Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1993-05-27
1996-02-13
Jones, W. Gary
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 243, 536 2433, 435 6, 435 911, 435 912, 435810, 935 8, 935 22, C07H 2104, C12Q 168, C12P 1934, C12N 1500
Patent
active
054912253
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention.
This invention provides for superior nucleic acid primers for amplification of select target regions of the genome of the genus Legionella. The invention facilitates detection of pathogenic and nonpathogenic forms of this genus. The invention further provides for processes for using the primers in template dependent nucleic acid polymerase extension reactions to amplify select target regions. Kits for the use of these primers are also provided.
This invention further provides for methods of controlling the intensity of visual signal for detection of duplex formation in nucleic acid hybridization assays under high stringent conditions. This method involves the blending of different capture probes onto a solid support.
2. Information Disclosure.
Legionella species are known as both pathogenic and nonpathogenic microorganisms. In the pneumonic form, they are intracellular pathogens of lung macrophage cells. Legionellaceae, Chpt. 9260 J. in Standard Methods of the Examination of Water and Wastewater, Eds. Clesceri, Greenberg and Trussell, 17th Ed. 1989 pages 9-149 to 9-153 and Muraca, P. W. et al., 1988, Environmental Aspects of Legionnaires' Disease, J. Amer. Water Works Assoc. 80:78-86.
A surface antigen of Legionella has been implicated as a requirement for intracellular pathogenicity and is called a macrophage infectivity potentiator or mip. Cianciotto, N. P. et al., 1989, A Legionella pneumophila Gene Encoding a Species-specific Surface Protein Potentiates Initiation of Intracellular Infection, Infection and Immunity, 57:1255-1262.
The nucleotide sequence of the 5S rRNA has been reported by MacDonnell, M. T. and R. R. Colwell, 1987, The Nucleotide Sequence of the 5S rRNA From Legionella pneumophila, Nucleic Acid Research, 15:1335; and, by Chumakov, K. M et al., 1986, Use of 5S Ribosomal RNA Nucleotide Sequence Analysis for the Study of Phylogeny of the Genus Legionella, Mol. Genet., Mikrobiol Virusol., 8:38-40.
The nucleotide and amino acid sequence of the macrophage infectivity protein is known and reported by Engleberg, N. C. et al., 1989, DNA Sequence of mip, a Legionella pneumophila Gene Associated With Macrophage Infectivity, Infection and Immunity, 57:1263-1270.
Standard culture techniques for Legionella are not adequate to properly assess risks from this deadly pathogen. Hussong, D. et al, 1987, Viable Legionella pneumophila Not Detectable by Culture on Agar Media, Bio/Technology 5:947-950.
Nucleic acid probes for detection of Legionella pneumophila have been reported. Grimont, P. A. D. et al., 1985, DNA Probe Specific for Legionella pneumophila, J. Clin. Micro., 21:431-437; and Engleberg, N. C. et al., 1986, A Legionella-Specific DNA Probe Detects Organisms in Lung Tissue Homogenates from Intranasally Inoculated Mice, Israel J. of Med. Sciences, 22:703-705.
The use of polymerase chain reaction amplification methods to detect Legionella species has been disclosed by Starnbach, M. N. et al., 1989, Species-Specific Detection of Legionella pneumophila in Water by DNA Amplification and Hybridization, J. of Clin. Microbiol., 27:1257-1261; in U.S. Ser. No. 07/467,813 filed on Jan. 1, 1990, U.S. Pat. No. 5,286,934, issued Oct. 26, 1993; and, also in Detection of Legionella in Environmental Samples Using Polymerase Chain Reaction (PCR), Biotechnology Bulletin, May 1990 pages 11-12.
SUMMARY OF THE INVENTION
This invention provides for superior amplification primers for use in assays for detecting pathogenic Legionella species. Described primers are able to amplify select regions of the 5 S RNA gene that are uniquely common to most species of Legionella. Other primers are able to discriminate between Legionella having the mip gene and those without the mip gene. The primers are further advantageous in that they have similar thermal melting points and can be used in combination to efficiently and equally amplify more than one region of the Legionella genome in a single amplification reaction mixture.
In particular, this invention provides for a compos
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Johnson et al., "Bergy's Manual of Systematic Bacteriology" vol. 1 published 1984 by Williams and Wilkins (Baltimore) pp. 88-11.
Grimont et al., 1985, "DNA Probe Specific for Legionella pneumophila" J. Microbiol. 21(3):431-437.
Chumakov et al., 1986, "Use of 5S Ribosomal RNA Nucleotide Sequence Analysis for the Study of Phylogeny of the Genus Legionella" Mol. Genet. Mikrobiol. Birusol. 8:38-40.
Engleberg et al., 1986, "A Legionella-Specific DNA Probe Detects Organisms in Lung Tissue Homogenates From Intranasally Inoculated Mice" Isreal J. of Med. Sciences 22:703-705.
Hussong et al., 1987, "Viable Legionella Pneumophila Not Detectable by Culture on Agar Media" Bio/Technology 5:947-950.
MacDonell et al., 1987, "The Nucleotide Sequence of the 5S rRNA From Legionella pneumophila" Nucleic Acids Research 15(3):1335.
Muraca et al., 1988, "Environmental Aspects of Legionnaires' Disease" J. Amer. Water Works Assoc. 80:78-86.
Steffan et al. 1988, "DNA Amplification to Enhance Detection of Genetically Engineered Bacteria in Environmental Samples" Appl. Environ. Microbiol. 54(9):2185-2191.
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Cianciotto et al., 1989, "A Legionella pneumophila Gene Encoding a Species-Specific Surface Protein Potentiates Initiation of Intracellular Infection" INfection and Immunity 57:1255-1262.
Clesceri et al., "Legionellaceae" Standard Methods for the Examination of Water and Wastewater 17th Ed. Chapter 9260 J. pp. 9-149 through 9-153 (1989).
Engleberg et al., 1989, "DNA Sequence of mip, a Legionella pneumophila Gene Associated With Macrophage Infectivity" Infection and Immunity 57(4):1263-1270.
Starnbach et al., 1989, "Species-Specific Detection of Legionella pneumophila in Water by DNA Amplification and Hybridization" J. Clin. Microbiol. 27(6):1257-1261.
"Detection of Legionella in Environmental Samples Using Polymerase Chain Reaction (PCR)" Biotechnology Bulletin pp. 11-12 (May, 1990).
Cianciotta et al., 1990, "A Mutation in the mip Gene Results in an Attenuation of Legionella pneumophila Virulence" J. Infect. Diseases 161:121-126.
Cianciotto et al., 1990, "Identification of mip-Like Genes in the Genus Legionella" Infection and Immunity 58(9):2912-2918.
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McCallum Theresa M.
Picone Teresa K. H.
Zoccoli Michael A.
Gould George M.
Hoffmann-La Roche Inc.
Jones W. Gary
Petry Douglas A.
Sias Stacey R.
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