Method for assessing juice/cider quality and/or safety
Inventors
Zhao, Wei • Baldwin, Elizabeth A • Bai, Jinhe • Plotto, Anne
Assignees
US Department of Agriculture USDA
Publication Number
US-11046994-B2
Publication Date
2021-06-29
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 provides a novel method for isolating DNA from juices and ciders that is low cost and yields large quantities of highly purified DNA even from small quantities of liquid. The invention also describes methods for determining if a juice or cider is safe to consume and/or the quality of the juice or cider by performing quantitative PCR (qPCR) on the isolated DNA and comparing the amount of DNA from microorganisms present in the sample. These methods are effective even if the liquid has been pasteurized.
The problem addressed is the difficulty of isolating high quality and quantity of DNA from fruit juices and ciders, especially those containing high levels of pectin and other interfering compounds, which inhibit DNA liberation and PCR amplification. Standard methods either yield low amounts of DNA or suffer from contamination from polysaccharides and secondary metabolites that inhibit downstream molecular assays, making it challenging to accurately assess microbial contamination or quality attributes associated with microorganism presence in juices and ciders.
Claims Coverage
The patent contains several independent claims covering methods of isolating nucleic acids from pectin-containing juices or ciders, and methods for determining safety and quality of such liquids using nucleic acid quantification.
Method for isolating nucleic acids from pectin containing juice or cider
This method comprises: (a) separating solid components from liquid components of the juice or cider; (b) lysing cells in solid components to release nucleic acids and other cell materials; (c) creating an organic phase, interface, and aqueous phase where lipids, proteins, nucleic acids and polysaccharides distribute accordingly; (d) separating the aqueous phase; (e) mixing aqueous CTAB and salt solution with nucleic acids and polysaccharides so that nucleic acids precipitate and polysaccharides remain dissolved; and (f) separating nucleic acids from dissolved polysaccharides.
Salt concentration range for nucleic acid isolation
The concentration of salt in the aqueous solution containing CTAB and nucleic acids is between approximately 10 mM and approximately 400 mM.
Enhanced nucleic acid isolation method using specific buffers and steps
A method involving: (a) separating solid components from liquid of juice or cider; (b) exposing solids to a solution containing Tris-base, EDTA, salt, PVP40, β-mercaptoethanol, NaOH, and a surfactant to dissolve solids and lyse cells; (c) optional heating to lyse cells; (d) adding chloroform to generate separate phases; (e) separating aqueous phase; (f) adding CTAB to cause nucleic acid precipitation; (g) optional heating; (h) separating nucleic acids; (i) optionally washing nucleic acids in ethanol; (j) optionally separating washed nucleic acids from ethanol.
Method for assessing juice or cider safety by nucleic acid quantification
Isolating nucleic acids from pectin containing juice or cider using the nucleic acid isolation method; quantifying microbial nucleic acids; and comparing the quantity to known values indicating maximum safe microbial load for consumption.
Quantifying microbial nucleic acids via nucleic acid amplification and Ct determination
Exposing isolated nucleic acids to specific primers complementary to microorganism DNA, amplifying to generate amplicons, and determining Ct value to quantify microbial nucleic acids.
Use of fluorescent and other labels in quantification
The labeling for quantification includes intercalating dyes, fluorescent compositions, spectroscopic, photochemical, biochemical, immunochemical, or chemical labels.
Microorganism species for safety and quality assessment
The microorganisms targeted include lactic acid bacteria, acetic acid bacteria, Alicyclobacillus spp., Zygosaccharomyces spp., Rhodotorula ruba, Escherichia coli, Listeria spp., Shigella spp., Salmonella spp., Klebsiella spp., Saccharomyces spp., and Aspergillus spp.
The claims disclose novel methods for isolating nucleic acids from pectin-containing juices or ciders using specific buffer compositions and CTAB precipitation to separate nucleic acids from polysaccharides, as well as methods for determining the safety and quality of these liquids by quantifying microbial nucleic acids through PCR and qPCR with specific primers and labels.
Stated Advantages
The method yields large quantities of highly purified DNA from small quantities of juice or cider at low cost.
The DNA isolation method efficiently separates nucleic acids from polysaccharides such as pectin, resulting in DNA free from contamination that inhibits downstream assays.
The method works with both pasteurized and unpasteurized juices and ciders.
The quantitative PCR methods allow objective, fast, and quantitative assessment of juice or cider quality and safety, replacing subjective and labor-intensive sensory panels.
Documented Applications
Isolating DNA from juices and ciders obtained from various fruits and vegetables including citrus fruits, apple, grape, pear, peach, plum, pomegranate, celery, cucumber, onion, carrot, lettuce, spinach, beets, watercress, rhubarb, pumpkin, and tomato.
Assessing flavor quality of juice or cider by quantifying microbial nucleic acids and correlating Ct values from qPCR to flavor descriptors such as off-flavors stemming from microbial contamination or infection such as Candidatus Liberibacter spp.
Determining safety of juice or cider for consumption by quantifying nucleic acids of harmful microorganisms including Escherichia coli, Listeria spp., Shigella spp., Salmonella spp., Klebsiella spp., Candidatus Liberibacter asiaticus, africanus, americanus.
Quantifying and detecting presence of specific microorganisms like Alicyclobacillus spp., Zygosaccharomyces spp., Rhodotorula ruba, Saccharomyces spp., Aspergillus spp., and E. coli in juices or ciders using qPCR with microorganism-specific primers.
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