We used polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis to screen the most frequent variants (A, B, C, and E) found in the bovine kappa-casein gene. The PCR products (453 bp) were heat-denatured, loaded onto nondenaturing polyacrylamide gels, and silver-stained. Each variant yielded patterns clearly distinguishable from the others. Optimal conditions for the simultaneous detection of the four variants were 12% polyacrylamide gels (100:1 acrylamide:bis-acrylamide ratio) with 5% glycerol and a constant running temperature of 10 degrees C. Eight reference samples initially used for this purpose and 40 anonymous samples of different cattle breeds diagnosed by PCR-RFLP and PCR-SSCP showed no discrepancies between the two methods and confirmed previous results. Because it is cost-effective, sensitive, and fast, PCR-SSCP is strongly recommended to routinely screen kappa-casein variants for industrial purposes or in cattle selection schemes.

We have optimized the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique to screen the most frequent variants (A1, A2, A3, and B) of the bovine beta-casein gene. Five partly overlapping PCR products (233, 234, 265, 466, and 498 bp) of Exon VII of the beta-casein gene that encompass the target point mutations were heat-denatured, separated on nondenaturing polyacrylamide gels, and silver-stained. Simultaneous detection of all variants in reference samples of known genotypes (A1A2, A2A2, A1A3, A1B, and A2B) was best achieved on 17% polyacrylamide (100:1 acrylamide:bis-acrylamide ratio) gels with the PCR product of 234 bp. These results were confirmed by sequencing the allele-specific SSCP bands directly excised from polyacrylamide gels. A population of 65 anonymous samples belonging to various breeds was then analyzed twice, without discrepancies in a blind trial. Routine beta-casein genotyping using PCR-SSCP is proposed as a cost-effective, fast, and sensitive technique.

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Background There are limited data about the role of endoscopic ultrasound-guided tissue acquisition (EUS-TA), by fine needle aspiration (EUS-FNA) or biopsy (EUS-FNB), in the evaluation of the adrenal glands (AG). The primary aim was to assess the diagnostic yield and safety. The secondary aims were the malignancy predictors, and to create a predictive model of malignancy. Methods This was a retrospective nationwide study involving all Spanish hospitals experienced in EUS-TA of AGs. Inclusion period was from April-2003 to April-2016. Inclusion criteria: all consecutive cases that underwent EUS-TA of AGs. EUS and cytopathology findings were evaluated. Statistical analyses: diagnostic accuracy of echoendoscopist’s suspicion using cytology by EUS-TA, as gold standard; multivariate logistic regression model to predict tumor malignancy. Results A total of 204 EUS-TA of AGs were evaluated. Primary tumor locations were lung70%, others19%, and unknown11%. AG samples were adequate for cytological diagnosis in 91%, and confirmed malignancy in 60%. Diagnostic accuracy of the endosonographer's suspicion was 68%. The most common technique was: a 22-G (65%) and cytological needle (75%) with suction-syringe (66%). No serious adverse events were described. The variables most associated with malignancy were size>30mm (OR2.27; 95%CI, 1.16–4.05), heterogeneous echo-pattern (OR2.11; 95%CI, 1.1–3.9), variegated AG shape (OR2.46; 95%CI, 1–6.24), and endosonographer suspicion (OR17.46; 95%CI, 6.2–58.5). The best variables for a predictive multivariate logistic model of malignancy were age, sex, echo-pattern, and AG-shape. Conclusions EUS-TA of the AGs is a safe, minimally invasive procedure, allowing an excellent diagnostic yield. These results suggest the possibility of developing a pre-EUS procedure predictive malignancy model.

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