Method for assessing juice/cider quality and/or safety
Inventors
Zhao, Wei • Baldwin, Elizabeth A • Bai, Jinhe • Plotto, Anne • Irey, Michael S
Assignees
SOUTHERN GARDEN CITRUS • SOUTHERN GARDENS CITRUS • US Department of Agriculture USDA
Publication Number
US-10047389-B2
Publication Date
2018-08-14
Expiration Date
2034-09-29
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Abstract
A novel method for isolating DNA from juices and ciders is described. This method is low cost and yield large quantities of highly purified DNA even though one uses a small quantity of juice or cider. A method for determining if a juice or cider is safe to consume and/or the quality of the juice or cider are also described. For these methods, one can perform qPCR on the DNA which can be obtained using the disclosed method or any other prior art method, and comparing the amount of DNA from microorganisms is present in the juice and/or cider to determine the safety and/or quality of the juice and/or cider. These methods work even if the liquid was pasteurized.
Core Innovation
This invention relates to a novel method for isolating nucleic acids from juices and ciders, which can include other liquids such as wine or fermented beverages. The method involves separating solid components present in a sample of the liquid from liquid soluble components, lysing the cells present in the solid components to release nucleic acids, proteins, polysaccharides, lipids, and non-polar material, and then separating proteins, lipids, and non-polar material from nucleic acids and polysaccharides. It further involves separating polysaccharides from nucleic acids by mixing an aqueous solution of cetyl trimethyl ammonium bromide (CTAB) and salt such that nucleic acids precipitate and polysaccharides remain dissolved. This method yields large quantities of highly purified DNA from small quantities of juice or cider, overcoming prior difficulties in achieving high yield and purity due to interfering substances like pectin and sugars.
The invention also includes a method for determining the quality and/or safety of a juice or cider by quantifying the amount of nucleic acids belonging to microorganisms present in the liquid. Quantitative polymerase chain reaction (qPCR) is used to amplify and quantify microorganism DNA, obtaining a cycle threshold value (Ct) that indicates the amount of microorganism DNA present. By comparing the obtained Ct values to known values, it is possible to determine if the juice or cider is safe for consumption or if its quality—reflected by flavor, taste, smell, color, mouth feel, and aftertaste—has been adversely impacted by microorganisms. This determination works both for pasteurized and unpasteurized liquids.
The problem addressed is that isolation of sufficient quantities of highly pure DNA from juices and ciders, especially those rich in polysaccharides like pectin, has been difficult. Existing DNA isolation methods from plant tissues or juices were either too complex, yielded low DNA quantities, or did not effectively separate DNA from interfering compounds. Since prior methods failed to provide a cost-effective, reliable method for use with small sample volumes, there was a need for a simple method to isolate nucleic acids free of contamination and suitable for downstream qPCR-based analysis to assess juice quality and safety.
Claims Coverage
The patent includes two independent claims covering methods for detecting poor taste quality in pectin-containing juice or cider samples caused by Candidatus Liberibacter asiaticus (CLas) and for detecting CLas DNA in pectin-containing juice via DNA isolation and qPCR amplification.
Method for detecting poor taste quality caused by CLas DNA in juices or ciders
A method that includes separating solid from liquid components of a pectin-containing juice or cider, lysing cells from solids, generating and separating aqueous, organic, and interface phases to isolate nucleic acids in the aqueous phase, mixing CTAB and salt solution to precipitate nucleic acids while keeping polysaccharides dissolved, separating nucleic acids from polysaccharides, amplifying CLas DNA using primers and DNA polymerase, and quantifying the amplified DNA to detect when greater amounts of CLas DNA correlate to poor taste quality.
Use of specific CLas primers and Ct value thresholds to indicate poor taste quality
CLas primers consisting essentially of SEQ ID NO: 3 and 4 with Ct value approximately 35 or less indicating poor taste quality and greater CLas presence. Alternatively, CLas primers comprising SEQ ID NO: 1 and 2 with Ct value approximately 30 or less indicating poor taste quality due to CLas presence.
Salt concentration range in CTAB aqueous solution for nucleic acid precipitation
The aqueous solution of CTAB and salt used in the method has a salt concentration between approximately 10 mM and 400 mM to enable nucleic acids to precipitate while polysaccharides remain in solution.
Method steps for washing nucleic acids to remove CTAB
A step of washing the nucleic acids in ethanol to remove remaining CTAB, followed by separating the washed nucleic acids from ethanol.
Method for detecting CLas DNA in pectin-containing juices by applying specific buffers and organic separation
A method involving centrifugation to separate solid components, exposing solids to solutions with Tris-base, EDTA, salt, PVP40, β-mercaptoethanol, NaOH, and a surfactant to lyse cells, optional heating, chloroform addition to generate phases separating lipids, proteins, and nucleic acids/polysaccharides, mixing CTAB to precipitate nucleic acids, subsequent nucleic acid isolation and amplification with CLas primers.
The independent claims cover methods integrating a novel nucleic acid isolation process—employing CTAB and controlled salt concentration—to purify DNA from pectin-containing juices and ciders, combined with qPCR amplification using specific CLas primers and specified Ct value thresholds, enabling detection of CLas amounts correlated with poor taste quality or juice safety.
Stated Advantages
The method is low cost, yielding large quantities of highly purified DNA from small volumes of juice or cider.
It provides a simple and reliable assay to detect CLas and other microorganisms in juices and ciders.
The method works effectively even with pasteurized juices and ciders.
It allows quantitative assessment of juice/cider quality and safety based on microorganism DNA levels.
The method improves DNA purity by efficiently separating DNA from interfering polysaccharides like pectin.
Documented Applications
Isolating nucleic acids from juices and ciders, including pectin-containing liquids like orange juice.
Determining the quality of juice or cider, including flavor, taste, smell, color, mouth feel, aftertaste, and safety, by quantifying microorganism DNA.
Detecting infection of citrus trees by Candidatus Liberibacter species associated with Huanglongbing disease via DNA quantification.
Detecting and quantifying other microorganisms in juices and ciders such as lactic acid bacteria, acetic acid bacteria, Alicyclobacillus spp., Zygosaccharomyces spp., Rhodotorula ruba, Escherichia coli, Listeria spp., Shigella spp., Salmonella spp., and Klebsiella spp.
Rapid quality control of commercial juice brands by measuring Ct values of microorganisms' DNA presence.
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