The present disclosure is in the technical field of DNA based pathogen analysis. More particularly, the present disclosure is in the technical field of pathogen analysis on plant, agriculture, food and water material using DNA based microarray technology.
Prevailing techniques used to identify microbial pathogens rely upon established clinical microbiology monitoring. Pathogen identification is conducted using standard culture and susceptibility tests. These tests require a substantial investment of time, effort, cost as well as labile products. Further, such techniques are not ideal for testing large numbers of samples. Culture-based testing is fraught with inaccuracies which include both false positives and false negatives, as well as unreliable quantification of colony forming units (CFUs). There are issues with the presence of viable but non-culturable microorganisms which do not show up using conventional culture methods. Certain culture tests are very non-specific in terms of detecting both harmful and harmless species which diminishes the utility of the test to determine if there is a threat present in the sample being tested.
In response to challenges including false positives and culturing of microorganisms, DNA-based diagnostic methods such as polymerase chain reaction (PCR) amplification techniques were developed. For use of PCR, the pathogen DNA to be analyzed is extracted from the material prior to analysis, this is a time-consuming and costly step in the process. In an attempt to eliminate the preanalysis extraction step of PCR, Colony PCR was developed. Using cells directly from colonies from plates or liquid cultures, Colony PCR allows PCR!of bacterial cells without sample preparation. This technique was a partial success, as it was not as sensitive as culture which indicated a possible issue with interference of the PCR by constituents in the specimens. The possible interference issue was deemed not significant enough to invalidate the utility of the testing performed. However, such interference can be significant for highly sensitive detection of pathogens for certain types of tests. Consequently, Colony PCR did not eliminate the pre-analysis extraction step for use of PCR, especially for highly sensitive detection of pathogens.
It is known from the literature that 16S DNA in bacteria and the ITS2 DNA in yeast or mold can be PCR amplified, and once amplified can be analyzed to provide information about the specific bacteria or specific fungal contamination in or on plant material. Further, for certain samples such as blood, fecal matter and others, PCR may be performed on the DNA in such samples absent any extraction of the DNA. However, for blood it is known that the result of such direct PCR is prone to substantial sample to sample variation due to inhibition by blood analytes. Additionally, attempts to perform direct PCR analysis on plant matter have generally been unsuccessful, due to heavy inhibition of PCR by plant constituents.
Over time, additional methods and techniques were developed to improve on the challenges of timely and specific detection and identification of pathogens. Immunoassay techniques provide specific analysis, however, the technique is costly in the use of chemical consumables and has a long response time. Optical sensor technologies produce fast real-time detection, however, such sensors lack identification specificity, as they offer a generic detection capability as the pathogen is usually optically similar to its benign background. Quantitative Polymerase Chain Reaction (qPCR) technique is capable of amplification and detection of a DNA sample in less than an hour. However, qPCR is largely limited to the analysis of a single pathogen. Consequently, if many pathogens are to be analyzed concurrently, as is the case with plant, agriculture, food and water material, a relatively large number of individual tests are performed in parallel.
Biological microarrays have become a key mechanism in a wide range of tools used to detect and analyze DNA. Microarray-based detection combines DNA amplification with the broad screening capability of microarray technology. This results in a specific detection and improved rate of process. DNA microarrays can be fabricated with the capacity to interrogate, by hybridization, certain segments of the DNA in bacteria and eukaryotic cells such as yeast and mold. However, processing a large number of PCR reactions for downstream microarray applications is costly and requires highly skilled individuals with complex organizational support. Because of these challenges, microarray techniques have not led to the development of downstream applications.
We have found, that there is a need for a method of DNA based pathogen analysis that interrogates a large number of samples, uses fewer chemical and labile products, and provides faster results while maintaining accuracy, specificity and reliability.
Embodiments of the present disclosure for a microarray based multiplex pathogen analysis method include two steps. One step is DNA amplification of the pathogen DNA of interest. For example, PCR amplification of the sample is conducted prior to biochemical or physical extraction of the pathogen DNA. In this step, the DNA amplification reaction itself provides enrichment of the pathogen DNA(s) of interest. By bypassing the DNA extraction and purification steps, the test procedure is made markedly faster. Further, the test procedure improves sensitivity as the circumvention of DNA extraction procedures mitigates the DNA loss and DNA dilution that accompany DNA extraction. In short, the embodiments do not require pre-analysis DNA extraction nor purification because a microbial pellet obtained from the material is subjected to DNA amplification without purification, the resulting PCR-amplified material is then suitable for analysis.
A second step is DNA microarray analysis of highly repetitive DNA segments in the plant borne pathogens; DNA segments in bacteria or DNA segments in eukaryotic pathogens (yeast and mold), or amplification of specific sequences from unique single copy gene. The repetitive DNA segments are used primarily for highly sensitive detection of specific organisms or for unique biomarker genes or genomic sequences with significant value towards identifying pathogens or even genetic variations within the genome of the host organism (i.e., a plant). In bacteria, the highly repetitive DNA segments are the 16S rDNA gene.
In eukaryotes, the highly repetitive DNA segments are the Internal Transcribed Spacer—2 region, (ITS2). These two types of highly repetitive DNA are known to harbor DNA sequence changes that can be used to distinguish bacteria from each other (16S) and yeast or mold from each other (ITS2). In the present disclosure, a panel of nucleic acid probes is assembled that are capable of recognizing (by DNA hybridization) those DNA sequence changes in bacteria (within 16S), and also within the genes which encode specific pathogen genes and the corresponding sequence changes in yeast or mold (within ITS2). Therefore, subsequent to DNA amplification of non-extracted samples, the amplified 16S DNA can be interrogated on a single microarray, as a single hybridization test, thereby resolving a panel of bacteria which may be present in the plant, agriculture, food, or water specimens. Similarly, subsequent to DNA amplification of non-extracted samples, the amplified ITS2 DNA can be interrogated on a single microarray, as a single hybridization test, thereby resolving a panel of yeast and mold which may be present in the plant, agriculture, food, or water material. The embodiments of the present disclosure with a microarray component of the present disclosure allow a number of individual tests to be performed as a single multiplex test.
Embodiments of the present disclosure are described herein by reference to a microarray based multiplex pathogen analysis. The disclosure is not, however, limited by the #! advantages of the aforementioned embodiment. The present method may also be applied to many types of material capable of generating DNA based pathogen analysis. Further, it should be understood that the disclosed embodiments may be combined with one another. In addition, features of particular embodiments may be exchanged with features of other embodiments.
These and other features, aspects, and advantages of the embodiments of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawing, wherein:
The present disclosure provides an improved method for DNA based pathogen analysis. The embodiments of the present disclosure contemplate the use of DNA amplification methodologies, including but not limited to loop-mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR) tests that can selectively amplify the DNA complement of that plant material using unpurified plant and pathogen material. The embodiments are also based on the use of aforementioned PCR-amplified DNA as the substrate for microarray-based hybridization analysis, wherein the hybridization is made simple because the DNA probes used to interrogate the DNA of such pathogens is optimized to function at room temperature. This enables the use of the above mentioned microarray test at ambient temperature, thus bypassing the previously established requirement that testing be supported by an exogenous temperature-regulating device.
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Representative Microarray Hybridization Data Obtained from Purified Bacterial DNA Standards
Aeromonas
Bacillus
Campylobactor
hydrophila
subtilus
E. coli
E. Coli specific gene
E. Coli Stx1
E. Coli Stx2
Enterobacteriacea
Salmonella/
Enterobacter
Salmonella specific
Aeromonas
Pseudomonas
Pseudomonas
aeriginosa
Xanthomonas
Listeria
Campylobacter
Bacillus Group 2
E. coli
E. coli
Listeria ssp.
E. coli
E. Coli specific gene
E. Coli Stx1
E. Coli Stx2
Enterobacteriacea
Salmonella/
Enterobacter
Salmonella specific
Aeromonas
Pseudomonas
Pseudomonas
aeriginosa
Xanthomonas
Listeria
Campylobacter
Bacillus Group 2
Pseudomonas
Salmonella
Xanthomonas
aeruginosa
enterica
E. coli
E. Coli specific gene
E. Coli Stx1
E. Coli Stx2
Enterobacteriacea
Salmonella/
Enterobacter
Salmonella specific
Aeromonas
Pseudomonas
Pseudomonas
aeriginosa
Xanthomonas
Listeria
Campylobacter
Bacillus Group 2
Representative Microarray Hybridization Data Obtained from Purified Bacterial DNA Standards
A.
A.
Fusarium
Penicillium
fumigatus
flavus
A. niger
Mucor
A.
fumigatus
A. flavus
A. niger
Botrytis
Penicillium
F. solani
Mucor
The data of
At least two methods of sample collection are possible. A simple rinsing of the fruit, exactly as described for cannabis, above. Another method of sample collection is a “tape pull”, wherein a piece of standard forensic tape is applied to the surface of the fruit, then pulled off. Upon pulling, the tape is then soaked in the standard wash buffer described above, to suspend the microbes attached to the tape. Subsequent to the tape-wash step, all other aspects of the processing and analysis (i.e. raw sample genotyping, PCR, then microarray analysis) are exactly as described above.
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Embodiments for analysis of industrial washes derived from food processing. The above teaching shows by example that unprocessed leaf and bud samples in cannabis and hops may be washed in an aqueous water solution, to yield a water-wash containing microbial pathogens which can then be analyzed via the present combination of RSG and microarrays. The above data also show that environmentally-derived well water samples may be analyzed by an embodiment. Further, if a water sample containing microbes were obtained from industrial processing sources (such as the water effluent from the processing of fruit, vegetables, grain, meat) and then analyzed directly, or after ordinary water filtration to concentrate the microbial complement onto the surface of the filter, that the present combination of RSG and microarray analysis would be capable of recovering and analyzing the DNA complement of those microbes.
Embodiments for analysis of air filtrates. The above teaching shows by example that unprocessed leaf and bud samples in cannabis and hops may be washed in an aqueous water solution, to yield a water wash containing microbial pathogens which can then be analyzed via the present combination of RSG and microarrays. Further, if an air sample containing microbes as an aerosol or adsorbed to airborne dust were obtained by air filtration onto an ordinary air-filter (such as used in the filtration of air in an agricultural or food processing plant, or on factory floor, or in a public building or a private home) that such air-filters could then be washed with a water solution, as has been demonstrated for plant matter, to yield a microbe-containing filter eluate, such that the present combination of RSG and microarray analysis would be capable of recovering and analyzing the DNA complement of those microbes.
While the foregoing written description of an embodiments enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The present disclosure should therefore not be limited by the above described embodiments, methods, and examples, but by all embodiments and methods within the scope and spirit of the present disclosure.
This is a continuation under 35 U.S.C. § 120 of pending non-provisional application U.S. Ser. No. 15/388,561, filed Dec. 22, 2016, which claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Ser. No. 62/271,371, filed Dec. 28, 2015, now abandoned, the entirety of both of which are hereby incorporated by reference.
Number | Date | Country | |
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62271371 | Dec 2015 | US |
Number | Date | Country | |
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Parent | 15388561 | Dec 2016 | US |
Child | 16819564 | US |