Methods and compositions to evaluate and determine inactivation of hazardous biological material
Inventors
PEREZ DIAZ, Ilenys M. • CALDWELL, Jane M.
Assignees
North Carolina State University • US Department of Agriculture USDA
Publication Number
US-10718032-B2
Publication Date
2020-07-21
Expiration Date
2034-09-09
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Abstract
Novel time and temperature integrator (TTI) assays, kits containing the components of the assays, and the novel components for those assays are provided herein. These novel TTI assays evaluate and/or determine the inactivation of biological material in/on a sample by quantifying the degradation of DNA using qPCR. The sample can be a food product (e.g., fruits, vegetables, meat from animals, or eggs) while the item can be any object (e.g., medical equipment, especially reusable medical equipment) for which one needs to determine that the amount of inactivation of specific hazardous biological material on the object or in a sample is at or below a pre-determined amount.
Core Innovation
This invention provides novel time and temperature integrator assays, including kits and components, that evaluate and determine the inactivation of hazardous biological material in or on a sample by quantifying DNA degradation using quantitative PCR (qPCR). The samples can be food products such as fruits, vegetables, meat, or eggs, or items like reusable medical equipment where confirmation of biological material inactivation is necessary. The assays measure degradation of intrinsic mitochondrial DNA (mtDNA) or total DNA fragmentation using qPCR, or use extrinsic mtDNA as an indicator added to the process.
The invention addresses the problem of the inadequacy of traditional methods such as culturing bacteria or surrogate spores to timely and accurately assess inactivation of biological hazards. Culturing suffers from long incubation times and issues with recovery and tracking, while existing molecular methods cannot reliably distinguish between live and dead microorganisms as DNA can remain detectable after inactivation. The invention offers a more rapid, simple, and accurate means to measure the effect of inactivation processes by correlating DNA degradation, as measured by qPCR or DNA integrity assays, with the inactivation of hazardous biological material.
Claims Coverage
The claims include three independent methods covering determination of inactivation of hazardous biological material in food matrices or on items using nucleic acid degradation assessment, the efficacy determination of inactivation protocols via nucleic acid fragmentation, and quality control of hazardous material inactivation using nucleic acid analysis.
Method for determining inactivation of hazardous biological material in food matrix using intrinsic nucleic acid qPCR
A method comprising exposing intrinsic nucleic acid from a food matrix to first and second polynucleotides and an optional label, amplifying to produce an amplicon of approximately 80 to 250 base pairs which has sequences at the 5' and 3' ends at least 95% identical to the polynucleotides. The food matrix has undergone prior inactivation processing. The threshold cycle (Ct) value of amplified nucleic acid is determined and compared to known Ct values equivalent to desired inactivation levels. The polynucleotide sequences are within approximately 15 to 45 bases of SEQ ID NO: 9 or its reverse complement.
Method for determining efficacy of inactivation protocol via intrinsic nucleic acid fragmentation
A method to assess the efficacy of a protocol to inactivate hazardous biological material by determining the amount of intrinsic nucleic acid fragmentation in the food matrix post-treatment. This involves exposing intrinsic nucleic acid to first and second polynucleotides and optionally a label, amplifying, determining Ct values to identify nucleic acid fragmentation, and comparing this to known fragmentation levels indicative of inactivation. The amplicon length is between about 80 and 250 base pairs and primers bind to different strands of mtDNA within approximately 15 to 45 contiguous bases.
Method for assessing efficacy of inactivation protocol using extrinsic nucleic acid fragmentation
A method to assess inactivation efficacy by subjecting extrinsic nucleic acid to the inactivation protocol, exposing it to first and second polynucleotides and optionally a label, amplifying to produce an amplicon of between about 80 and 250 base pairs, determining the threshold cycle value of amplified extrinsic nucleic acid, and comparing to known fragmentation levels equivalent to inactivation of hazardous biological material. Polynucleotides bind to different strands of the extrinsic DNA.
Quality control method for hazardous biological material inactivation using nucleic acid analysis
A quality control method comprises processing a sample containing intrinsic or extrinsic nucleic acid according to a predetermined inactivation protocol, optionally isolating the degraded nucleic acid, exposing to first and second polynucleotides and optionally a label, amplifying to produce an amplicon between approximately 80 and 250 base pairs, determining Ct value, and comparing to known Ct values indicating desired nucleic acid degradation that correlates with hazardous biological material inactivation.
The independent claims focus on methods for using qPCR amplification of intrinsic or extrinsic nucleic acids and comparing threshold cycle values to known standards to determine the amount of inactivation of hazardous biological material in food matrices or on items, as well as for quality control of inactivation processes. They specify polynucleotide primer lengths, amplicon size ranges, and optional labeling agents, establishing assays that quantify DNA degradation as a surrogate for biological material inactivation.
Stated Advantages
Provides a rapid and simple method to determine inactivation of hazardous biological material, significantly faster than culturing methods.
Offers more accurate determination of viable biological material reduction compared to prior PCR detection methods that cannot differentiate live from dead organisms.
Can be implemented using commercially available reagents and apparatus, making the assay inexpensive and accessible.
Enables evaluation of processing efficacy and deviations, as well as quality control for inactivation processes on foods and medical or other items.
Documented Applications
Evaluating and determining the inactivation of hazardous biological material such as pathogens or spores in food matrices including fruits, vegetables, meat, eggs, nuts, and composite food products.
Assessing decontamination and sterilization efficacy on reusable medical and dental devices including endoscopes, catheters, sponges, scalpels, drills, and suction tubes.
Testing the efficacy of inactivation or sterilization protocols on packaging containers for food products, such as glass jars undergoing pasteurization, ultraviolet light, high pressure, or radiation treatments.
Quality control and validation of sterilization processes for biological material inactivation in foods, food containers, medical equipment, and other items requiring monitoring for hazardous biological material reduction.
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