title: Comparison of six commercial DNA extraction kits for detection of Brucella neotomae in Mexican and Central American-style cheese and other milk products.
abstract: Raw or inadequately pasteurized milk from infected animals and cheese made with such milk are a frequent vehicle for human brucellosis infection. Also, biological terrorism is a concern with certain Brucella spp. Due to matrix-associated real-time polymerase chain reaction (qPCR) inhibitors, robust sample preparations are crucial. We compared six commercial nucleic acid extraction kits using nine Mexican and Central American-style soft cheeses or creams and three liquid milk products inoculated with Brucella neotomae, a surrogate for pathogenic Brucella spp. Kits were evaluated by purity and quantity of DNA as determined by qPCR Ct values, reproducibility across cheese and milk types, and cost. At 10(7) CFU/g in four different cheeses, Qiagen statistically outperformed all other kits. When two cheese styles were inoculated at dual levels, Qiagen and High Pure kit extracted samples at 1.5 × 10(5) CFU/g produced average Ct values of 34-39, while PrepSEQ and MagMAX kit extracted samples exhibited higher or no Ct values. High Pure and Qiagen kits excelled also with liquid milk products. Considering matrices, inoculation levels, and kits evaluated, High Pure and Qiagen products produced Brucella DNA of high quality and quantity indicated by the lowest Ct values and were the least expensive.
authors.affiliation: Oak Ridge Institute for Science and Education, Oak Ridge, TN 38730, USA. tina.lusk@fda.hhs.gov
authors.forename: Tina S
authors.initials: TS
authors.lastname: Lusk
authors.forename: Errol
authors.initials: E
authors.lastname: Strain
authors.forename: Julie A
authors.initials: JA
authors.lastname: Kase
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000295:instrumentation / Q000379:methods*; D000818:Animals; D002002:Brucella / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D002417:Cattle; D002611:Cheese / Q000382:microbiology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis; D008892:Milk / Q000382:microbiology*; D011933:Reagent Kits, Diagnostic
chemical_list: D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic