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  Nucleotide sequences specific to brucella and methods for the detection of brucellaUSPTO Application #: 20060019255Title: Nucleotide sequences specific to brucella and methods for the detection of brucella Abstract: Described herein are the identification of nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed. (end of abstract) Agent: John H. Lee Assistant Laboratory Counsel - Livermore, CA, US Inventors: Paula M. McCready, Lyndsay Radnedge, Gary L. Andersen, Linda L. Ott, Thomas R. Slezak, Thomas A. Kuczmarski USPTO Applicaton #: 20060019255 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060019255. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No. 60/400,891, filed Aug. 1, 2002, and entitled, "DNA Diagnostics Brucella Species," which is incorporated herein by this reference. GENE SEQUENCE LISTING IN COMPUTER READABLE FORM [0003] The sequence listing information recorded in computer readable form is identical to the written on paper sequence listing. BACKGROUND [0004] Brucella is the species of bacteria known to cause what is commonly known as Brucellosis, a serious and sometimes fatal disease. Since the attack on the World Trade Center in New York of Sep. 11, 2001, there has been a growing concern that terrorists or rogue governments will use the Brucella bacterium as a weapon of mass destruction and instrument of terror. Since the events of Sep. 11, 2001, the United States Government has been developing reliable methods and systems to detect the Brucella bacterium so that immediate and effective counter measures can be undertaken. The existing methods for detecting the Brucella bacterium are considered inadequate because of the higher than acceptable rate of false positive and false negative results. False positive results lead to confusion regarding whether the Brucella bacterium is actually present and whether protective measures should immediately be implemented. Conversely, false negative results would allow the Brucella bacterium to remain undetected with consequent adverse impacts. A more reliable method of detecting the Brucella bacterium would reduce the occurrence of false positive and false negative results and provide decision makers with greater confidence in implementing appropriate counter measures. SUMMARY OF THE INVENTION [0005] This invention includes the nucleotide sequences that are identified in SEQ ID NOs:4, 8, 12, 16, 20 and 24 that are specific to Brucella. [0006] Another aspect of the invention includes a Forward Primer, the nucleotide sequences that are identified in SEQ ID NOs:1, 5, 9, 13, 17 and 21, and any primers that are derived from these nucleotide sequences. [0007] A further aspect of the invention is a Reverse Primer, the nucleotide sequences that are identified in SEQ ID NOs:2, 6, 10, 14, 18 and 22, and any primers that are derived from these nucleotide sequences. [0008] A further aspect of the invention includes a Hybridization Probe, the nucleotide sequences that are identified in SEQ ID NOs:3, 7, 11, 15, 19 and 23, and any probes that are derived from these nucleotide sequences. [0009] This invention also includes a method for the detection of Brucella using the bacterium specific nucleotide sequence comprising: providing a sample in an environment that is suitable for isolating genomic DNA for amplification using PCR and under conditions suitable for hybridization with a least one group of nucleotides consisting of a forward primer, a reverse primer and a hybridization probe and detecting the existence of Brucella specific nucleotide sequences by a nucleotide detection method, such as PCR and flurogenic 5' nuclease PCR assay, wherein the existence of the nucleotide sequence indicates the presence of Brucella in the sample. BRIEF DESCRIPTION OF THE SEQUENCES [0010] SEQ ID NO:1 Primer [0011] SEQ ID NO:2 Primer [0012] SEQ ID NO:3 Probe [0013] SEQ ID NO:4 Amplicon [0014] SEQ ID NO:5 Primer [0015] SEQ ID NO:6 Primer [0016] SEQ ID NO:7 Probe [0017] SEQ ID NO:8 Amplicon [0018] SEQ ID NO:9 Primer [0019] SEQ ID NO:10 Primer [0020] SEQ ID NO:11 Probe [0021] SEQ ID NO:12 Amplicon [0022] SEQ ID NO:13 Primer [0023] SEQ ID NO:14 Primer [0024] SEQ ID NO:15 Probe [0025] SEQ ID NO:16 Amplicon [0026] SEQ ID NO:17 Primer [0027] SEQ ID NO:18 Primer [0028] SEQ ID NO:19 Probe [0029] SEQ ID NO:20 Amplicon [0030] SEQ ID NO:21 Primer [0031] SEQ ID NO:22 Primer [0032] SEQ ID NO:23 Probe [0033] SEQ ID NO:24 Amplicon DETAILED DESCRIPTION [0034] Disclosed herein are six nucleotide sequences located on different loci of the Brucella bacterium genome. Also disclosed, are primers and the hybridization probes used in detecting the specific nucleotide sequences as well as a method for identifying Brucella by analyzing samples taken from monitoring devices, such as air monitors, for the nucleotide sequences that are specific to Brucella. By using the primers and hybridization probes developed from the nucleotide sequences identified as unique to the Brucella bacterium to detect the presence of the Brucella bacterium, far more reliable results are obtained than by using existing methods. False positive and false negative results are greatly reduced. [0035] Brucella is the bacterium that causes what is commonly known as Brucellosis, a disease that can cause can cause significant adverse health effects if not detected early and treated with appropriate antibiotics. Brucellosis is characterized by "a range of symptoms that are similar to the flu and may include fever, sweats, headaches, back pains, and physical weakness. Sever infections of the central nervous systems or lining of the heart may occur. Brucellosis cab [sic] also causes long-lasting or chronic symptoms that include recurrent fevers, joint pain, and fatigue." This information can be found at the Internet address www.cdc.gov/ncidod/dbmd/diseaseinfo/brucellosis_g.htm#whatis. It is on the Center for Disease Control and Prevention (CDC) list of possible bacteria that has potential as a biological warfare weapon. The CDC has developed a list of possible pathogens that may be used as bioterrorism weapons. Brucella has been listed in Category B of possible diseases and agents. Those diseases and agents in Category B are considered a high risk to national security because they "are moderately easy to disseminate; result in moderate morbidity rates and low mortality rates; and require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance." This information can be found at the internet address www.bt.cdc.gov/agent/agentlist-category.asp. [0036] A key element in developing defenses against the use of Brucella is the ability to quickly and accurately detect the presence of the bacterium. Early detection will allow for the implementation of effective counter measures. Additionally, early detection will allow for the identification and treatment of those that may have been exposed to the bacterium. Early detection and treatment is essential for the treatment of Brucellosis because significant adverse health effects, including death, may occur if it is not detected early and treated with antibiotics. [0037] Existing detection methods have resulted in a higher than acceptable rate of false positive and false negative results. Such results are inadequate and can create confusion regarding the appropriate countermeasures, if any, that should be undertaken because it is unclear whether the bacterium is present or not. If the bacterium is not present, undertaking counter measures may cause undue expense and create unwarranted concern among those that may incorrectly believe they have been exposed. [0038] Although the genome for Brucella has already been mapped, this alone was not sufficient to develop a reliable and accurate detection mechanism because the current methods use nucleotide sequences that may be common to many different bacteria. Thus, existing detection methods could not distinguish between various bacteria, which resulted in higher than acceptable false positive detection rates. Similarly, some existing detection methods resulted in false negative results because they were not sensitive enough to detect the bacterium. [0039] Using a nucleotide sequence that is specific to the Brucella bacterium results in a more reliable detection method. [0040] Six nucleotide sequences contained in SEQ ID NOs:4, 8, 12, 16, 20 and 24 are specific to Brucella. These sequences are known as "amplicons." The existence of these nucleotide sequences in a sample is conclusive proof that the bacterium Brucella is present. In order to detect any of the six amplicons specific to Brucella, a series of forward and reverse primers and hybridization probes were developed for each of the six amplicons. [0041] The typical assay determines the presence of SEQ ID NOs:4 and 8 using the sequence specific primers and the hybridization probes. If there is a positive result for the presence of Brucella then an assay is run to determine the presence of additional amplicon sequences, as a means to double check for the presence of Brucella. [0042] Once the Brucella specific nucleotide sequences were identified, the presence of the bacteria could be detected from environmental samples using PCR assay analysis and detection. PCR is a technique utilized to amplify genomic DNA. Typical PCR reactions include appropriate PCR buffers, nuclease polymerase and one or more oligonucleotide primers and hybridization probes. Various modifications of PCR techniques are possible as detailed in Current Protocols in Molecular Biology ed. F. M. Ausubel, R. Brent, D. D. Moore, K. Struhle, Massachusetts General Hospital and Harvard Medical School (1987), which is hereby incorporated by reference. The following US patents describe PCR and are incorporated herein by reference: U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159. Continue reading... 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