DNA Isolation from Recalcitrant Materials Such as Tree Roots, Bark, and Forest Soil for the Detection of Fungal Pathogens by Polymerase Chain Reaction

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

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A novel buffer system, AnyDirect, can improve polymerase chain reaction from whole blood without DNA isolation

Clinica Chimica Acta ◽

10.1016/j.cca.2007.01.019 ◽

2007 ◽

Vol 380 (1-2) ◽

pp. 112-117 ◽

Cited By ~ 25

Author(s):

Young Geun Yang ◽

Jong Yeol Kim ◽

Young-Han Song ◽

Doo-Sik Kim

Keyword(s):

Polymerase Chain Reaction ◽

Whole Blood ◽

Dna Isolation ◽

Buffer System ◽

Chain Reaction ◽

Polymerase Chain

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New Method of DNA Isolation from Two Food Additives Suitable for Authentication in Polymerase Chain Reaction Assays

Journal of Agricultural and Food Chemistry ◽

10.1021/jf0482919 ◽

2005 ◽

Vol 53 (9) ◽

pp. 3345-3347 ◽

Cited By ~ 7

Author(s):

Mercedes Urdiaín ◽

Antonio Doménech-Sánchez ◽

Sebastián Albertí ◽

V. Javier Benedí ◽

Josep A. Rosselló

Keyword(s):

Polymerase Chain Reaction ◽

Dna Isolation ◽

Food Additives ◽

New Method ◽

Chain Reaction ◽

Polymerase Chain

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Survival of Îº-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

FEMS Microbiology Ecology ◽

10.1111/j.1574-6941.1995.tb00270.x ◽

1995 ◽

Vol 16 (1) ◽

pp. 71-82 ◽

Cited By ~ 3

Author(s):

Kamtin Leung ◽

Michael B. Cassidy ◽

Steve B. Holmes ◽

Hung Lee ◽

Jack T. Trevors

Keyword(s):

Polymerase Chain Reaction ◽

Pseudomonas Aeruginosa ◽

Forest Soil ◽

Chain Reaction ◽

Polymerase Chain

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Polymerase Chain Reaction Detection and Phylogenetic Characterization of an Agent Associated with Yellow Vine Disease of Cucurbits

Phytopathology ◽

10.1094/phyto.1998.88.5.428 ◽

1998 ◽

Vol 88 (5) ◽

pp. 428-436 ◽

Cited By ~ 12

Author(s):

Francisco J. Avila ◽

Benny D. Bruton ◽

Jacqueline Fletcher ◽

J. L. Sherwood ◽

Sam D. Pair ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Nucleotide Sequence ◽

16S Rdna ◽

Fungal Pathogens ◽

Pcr Amplification ◽

Diagnostic Techniques ◽

Chain Reaction ◽

Phylogenetic Characterization ◽

South Central ◽

Polymerase Chain

Diagnosis of yellow vine disease (YVD) in cucurbits, an important disease in the south-central United States, relies on external symptom appearance, phloem discoloration, and the presence of bacterium-like organisms (BLOs) in phloem. Polymerase chain reaction (PCR) amplification of BLO nucleotide sequences was explored as a means to improve diagnostic techniques. PCR, using a primer pair based on sequences of the citrus-greening BLO, amplified a 0.15-kilobase (kb) fragment from the DNA of symptomatic plants, but not from that of asymptomatic plants. Its nucleotide sequence suggested that the DNA amplified was of pro-karyotic origin. A primer pair, designed to amplify nonspecific prokaryotic 16S rDNA, amplified a 1.5-kb DNA fragment in both the symptomatic and asymptomatic plants. The 1.5-kb fragment from the asymptomatic plants corresponded to chloroplast 16S rDNA, and the band from the symptomatic plants was composed of 16S rDNAs from both chloroplasts and a prokaryote. The nucleotide sequence of the prokaryotic DNA was determined and used to design three primers (YV1, YV2, and YV3). Fragments of 0.64 and 1.43 kb were amplified with primers YV1-YV2 and primers YV1-YV3, respectively, from symptomatic plants. Neither primer set yielded fragments from asymptomatic plants, unrelated bacteria, or selected soilborne fungal pathogens of cucurbits. Phylogenetic analysis indicated that the prokaryote is a gamma-3 proteobacterium. The consistent association of the 0.64- and 1.43-kb fragments with symptomatic plants suggests that the gamma-3 proteobacterium may be the causal agent of YVD of cantaloupe, squash, and watermelon.

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