for detecting Mycobacterium paratuberculosis in a sample using nucleic acid molecules, polypeptides, and antibodies; for immunization and prevention of mycobacterial animal infections
Inventors
Kapur, Vivek • Bannantine, John P. • Li, Ling-Ling • Zhang, Qing • Amonsin, Alongkorn
Assignees
US Department of Agriculture USDA • University of Minnesota System
Publication Number
US-7867704-B2
Publication Date
2011-01-11
Expiration Date
2023-03-06
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Abstract
The present invention provides nucleic acid molecules unique to M. paratuberculosis. The invention also provides the polypeptides encoded by the M. paratuberculosis-specific nucleic acid molecules of the invention, and antibodies having specific binding affinity for the polypeptides encoded by the M. paratuberculosis-specific nucleic acid molecules. The invention further provides for methods of detecting M. paratuberculosis in a sample using nucleic acid molecules, polypeptides, and antibodies of the invention. The invention additionally provides methods of preventing a M. paratuberculosis infection in an animal.
Core Innovation
The invention provides nucleic acid molecules unique to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), alongside the polypeptides encoded by these nucleic acid molecules and antibodies with specific binding affinity for such polypeptides. It includes methods for detecting M. paratuberculosis in samples using these nucleic acids, polypeptides, or antibodies, as well as methods for preventing M. paratuberculosis infection in animals by administering these molecules or their derivatives.
The problem addressed is the difficulty in identifying nucleic acids and polypeptides specific to M. paratuberculosis due to its close genetic relationship to M. avium. Current detection and prevention methods face sensitivity and specificity challenges, especially to distinguish M. paratuberculosis from genetically similar organisms. The invention offers isolated nucleic acid molecules from M. paratuberculosis genomes that generate amplification products under standard conditions and do not generate amplification products from a wide range of other organisms' nucleic acids, including closely related mycobacteria.
Further, the invention details isolated polypeptides encoded by these unique nucleic acids and antibodies against these polypeptides. The nucleic acids range from at least 10 nucleotides and show a minimum of 75% sequence identity to sequences unique to M. paratuberculosis. These molecules are used to develop specific diagnostic assays, including PCR and hybridization-based methods, methods employing polypeptides and antibodies, as well as immunization strategies to prevent M. paratuberculosis infection in various animal species.
Claims Coverage
The patent contains two main independent claims directed to methods for detecting M. paratuberculosis presence or absence in biological samples by nucleic acid-based methods: one employing amplification techniques and the other hybridization techniques.
Use of isolated nucleic acid molecules for detecting M. paratuberculosis by amplification
A method comprising contacting a biological sample with an isolated nucleic acid molecule at least 10 nucleotides in length with at least 75% sequence identity to an aligned portion of SEQ ID NO:1355 or its complement under standard amplification conditions. An amplification product is generated if M. paratuberculosis nucleic acid is present, enabling detection. The method includes detecting the amplification product, where its presence or absence indicates the presence or absence of M. paratuberculosis, respectively. The nucleic acid molecule can be selected from sequences including SEQ ID NOs: 46-101, 1343-1354. Typical samples include fecal, blood, or milk, from various animal species.
Use of isolated nucleic acid molecules for detecting M. paratuberculosis by hybridization
A method comprising contacting a biological sample with an isolated nucleic acid molecule at least 10 nucleotides in length with at least 75% sequence identity to an aligned portion of SEQ ID NO:1355 or its complement under hybridization conditions to produce a hybridization complex if M. paratuberculosis nucleic acid is present. The method includes detecting the presence or absence of the hybridization complex, correlating with M. paratuberculosis presence or absence. The nucleic acid molecule can be selected from sequences including SEQ ID NOs: 46-101, 1343-1342. The method encompasses electrophoretic separation and use of solid supports like nylon or nitrocellulose membranes. Suitable biological samples include fecal, milk, or blood from various animals.
The claims focus on nucleic acid-based methods for detecting M. paratuberculosis utilizing isolated nucleic acids unique to M. paratuberculosis with high specificity and sensitivity. The inventive features emphasize the sequence identity requirements, sample types, nucleic acid lengths, and conditions under which amplification or hybridization results are diagnostic for M. paratuberculosis infection.
Stated Advantages
The disclosed nucleic acid molecules, polypeptides, and antibodies enable high sensitivity and specificity for detecting M. paratuberculosis, distinguishing it from genetically related organisms such as M. avium.
The invention provides improved diagnostic methods that allow early and accurate detection of M. paratuberculosis in biological samples, including fecal, milk, or blood from various animals.
Methods and compositions disclosed allow for immunization and prevention of M. paratuberculosis infection in animals, potentially reducing disease incidence and spread.
The use of multiple unique nucleic acid sequences and polypeptides reduces false positives and enhances robustness of diagnostic and preventive approaches.
Documented Applications
Detection of M. paratuberculosis in biological samples from animals such as cattle, sheep, goats, rabbits, deer, antelopes, and bison by nucleic acid amplification methods (e.g., PCR) using unique nucleic acid molecules.
Detection of M. paratuberculosis in biological samples using nucleic acid hybridization methods with labeled probes.
Detection of M. paratuberculosis-specific polypeptides and antibodies in biological samples using immunoassays, including ELISA and immunoblotting.
Use of purified M. paratuberculosis-specific polypeptides to generate antibodies for diagnostic or research purposes.
Immunization of animals against M. paratuberculosis infection by administering compounds comprising M. paratuberculosis-specific nucleic acid molecules or polypeptides.
Developing kits and articles of manufacture comprising nucleic acids, polypeptides, or antibodies for the diagnosis or prevention of M. paratuberculosis infection.
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