PCR-RFLP molecular confirmation of color dilution alopecia in dogs in Brazil

2021 ◽  
pp. 104063872110223
Author(s):  
Silvana M. Caramalac ◽  
Simone M. Caramalac ◽  
Veronica J. Babo-Terra ◽  
Carlos A. N. Ramos ◽  
Mariana I. P. Palumbo
Keyword(s):  
Statistical Difference ◽  
Fragment Length Polymorphism ◽  
Coat Color ◽  
Molecular Technique ◽  
Nucleotide Variation ◽  
Single Nucleotide ◽  
Pcr Rflp ◽  
Clinical Characteristic ◽  
Yorkshire Terrier ◽  
Restriction Fragment Length

Color dilution alopecia (CDA) is a dermatopathy observed exclusively in animals having a diluted coat color. In dogs, color dilution occurs as a result of a single-nucleotide variation (SNV) c.-22G  >  A in the melanophilin gene. We standardized a PCR–restriction-fragment length polymorphism (PCR-RFLP) technique to identify this mutation and determine its frequency in dogs in Brazil. The standardized PCR-RFLP technique could efficiently identify the SNV c.-22G  >  A in the melanophilin gene, with mutated allele frequencies of 0.1, 0.1, and 0.0875 in Dachshund, Miniature Pinscher, and Yorkshire Terrier breeds, respectively, with no statistical difference among the breeds ( p = 0.252). The mutation was identified in 2 homozygous Dachshund dogs with alopecia, confirming the clinical characteristic of CDA. The standardization of a simpler and more accessible molecular technique for recognition of the SNV c.-22G  >  A in the melanophilin gene allows identification of heterozygous (phenotypically normal) dogs that can be excluded from reproduction, to avoid the birth of dogs with diluted coat color and consequently CDA.

scholarly journals Identifikasi Keragaman Gen DGAT1 serta Asosiasinya terhadap Karakteristik Karkas dan Sifat Perlemakan Domba

2019 ◽  
Vol 6 (2) ◽  
pp. 259
Author(s):  
Asep Gunawan ◽  
Ratna Sholatia Harahap ◽  
Kasita Listyarini ◽  
Cece Sumantri
Keyword(s):  
Polymerase Chain Reaction ◽  
Single Nucleotide Polymorphism ◽  
Fragment Length Polymorphism ◽  
Nucleotide Polymorphism ◽  
Chain Reaction ◽  
Single Nucleotide ◽  
Dgat1 Gene ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Restriction Fragment Length

ABSTRAK Karakteristik karkas dan sifat perlemakan pada daging domba dikontrol oleh banyak gen salah satunya gen DGAT1 (Diacylglycerol Acyltransferasel 1). Penelitian ini bertujuan mengidentifikasi SNP (Single Nucleotide Polymorphism) gen DGAT1 pada titik mutasi g.8539 C>T dan asosiasinya terhadap karakteristik karkas dan sifat perlemakan pada domba Indonesia. Total sampel domba yang digunakan sebanyak 150 buah terdiri dari 35 sampel domba compass agrinak (DCA), 36 sampel domba barbados cross (DBC), 41 sampel domba komposit garut (DKG), 20 sampel domba ekor gemuk (DEG), dan 18 sampel domba ekor tipis (DET). Karakteristik karkas dan sifat perlemakan diukur dari domba jantan berumur 10-12 bulan. Identifikasi keragaman DGAT1|ALuI dianalisis dengan metode PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Hasil keragaman gen DGAT1 bersifat polimorfik dalam DET dan DEG, sedangkan DCA, DBC, dan DKG bersifat monomorfik. Dua genotipe disebut CC dan  CT ditemukan dalam DET dan DEG. Titik mutasi gen DGAT1 berasosiasi (P<0.05) dengan karakteristik karkas, yaitu bobot dan panjang karkas. Selain itu, keragaman gen DGAT1 juga berasosiasi signifikan (P<0.05) dengan asam lemak jenuh, yaitu asam stearat (C18:0) dan asam arakidat (C20:0) dan asam lemak tak jenuh tunggal, yaitu asam oleat (C18:1n9c). Gen DGAT1 memiliki kontribusi dalam karakteristik karkas dan komposisi asam lemak pada domba.Kata Kunci: domba, gen DGAT1, karakteristik karkas, PCR-RFLP, sifat perlemakan                                                              ABSTRACT            Characteristic of carcass and fatness traits of sheep is regulated by many genes such as DGAT1 (Diacylglycerol Acyltransferasel 1) gene. The research was aimed to investigate SNP (Single Nucleotide Polymorphism) of DGAT1 and its association with characteristic of carcass and fatness traits in Indonesian sheep. A total sample of sheeps used 150 rams of 10–12 months consisted 35 samples of compas agrinak sheep (CAS), 36 of barbados cross (BCS), 41 of garut composite (GCS), 20  of javanese fat tailed (JFT), and 18 of javanese thin tailed (JTT). Identification variant of DGAT1|ALuI were performed by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). The results of polymorphism of DGAT1 were found in JTT and JFT. However, SNP of DGAT1 in CAS, BCS and GCS were monomorfic. Two genotype namely CC and CT were found in JTT and JFT populations. A SNP of the DGAT1 was associated (P<0.05) with characteristic of carcass, including weight and length of carcass. The variant of DGAT1 was associated too with saturated fatty acids (SFA) including stearic acid (C18:0) and arachidic acid (C20:0), and mono unsaturated fatty acid (MUFA) including oleic acid (C18:1n9c). The DGAT1 gene was contribute to characteristic carcass and fatty acid composition in sheep.Keywords: DGAT1 gene, characteristic carcass, fatness traits, PCR-RFLP, sheep

scholarly journals Variability of ACOX1 Gene Polymorphisms across Different Horse Breeds with Regard to Selection Pressure

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2225
Author(s):  
Grzegorz Myćka ◽  
Adrianna D. Musiał ◽  
Monika Stefaniuk-Szmukier ◽  
Katarzyna Piórkowska ◽  
Katarzyna Ropka-Molik
Keyword(s):  
Gene Polymorphisms ◽  
Selection Pressure ◽  
Fragment Length Polymorphism ◽  
Living Conditions ◽  
Rna Seq ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Pcr Rflp ◽  
Acyl Coenzyme A ◽  
Restriction Fragment Length

The ACOX1 gene encodes peroxisomal acyl-coenzyme A oxidase 1, the first enzyme in the fatty acid β-oxidation pathway, which could be significant for organisms exposed to long periods of starvation and harsh living conditions. We hypothesized that variations within ACOX1, revealed by RNA Sequencing (RNA-Seq), might be based on adaptation to living conditions and had resulted from selection pressure. There were five different horse breeds used in this study, representing various utility types: Arabian, Thoroughbred, Polish Konik, draft horses, and Hucul. The single-nucleotide polymorphism (SNP) located in the ACOX1 (rs782885985) was used as a marker and was identified using the PCR restriction fragment length polymorphism method (PCR-RFLP). Results indicated extremely different genotype and allele distributions of the ACOX1 gene across breeds. A predominance of the G allele was exhibited in horses that had adapted to difficult environmental conditions, namely, Polish Konik and Huculs, which are considered to be primitive breeds. The prevalence of the T allele in Thoroughbreds indicated that ACOX1 is significant in energy metabolism during flat racing.

scholarly journals Effect of porcine IL-6 polymorphism on litter size traits in commercial pig breeds

2020 ◽  
Vol 19 (2) ◽  
pp. 237-246
Keyword(s):  
Polymerase Chain Reaction ◽  
Litter Size ◽  
Fragment Length Polymorphism ◽  
Nucleotide Polymorphisms ◽  
Chain Reaction ◽  
Large White ◽  
Single Nucleotide ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Restriction Fragment Length

This study aimed to verify the polymorphisms in the porcine IL-6 gene and to elucidate its effects on litter size traits in Large White and Landrace sows. Four single nucleotide polymorphisms (SNPs) of the porcine IL-6 gene (g.91506415A>G, g.91507983A>G, g.91508173C>T, and g.91508716C>T) were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. There was no polymorphism observed on the three SNPs (g.91506415A>G, g.91507983A>G, and g.91508716C>T) of the porcine IL-6 gene. The porcine IL-6 g.91508173C>T polymorphism was found to be segregating in Large White and Landrace sows. The porcine IL-6 g.91508173C>T polymorphism was significantly associated with the total number born (TNB) and the number of piglets weaned alive (NWA) traits in Large White sows (P<0.05). Moreover, the porcine IL-6 g.91508173C>T polymorphism was significantly associated with the TNB, number born alive (NBA), and NWA traits in Landrace sows (P<0.05). These results indicated that the porcine IL-6 g.91508173C>T polymorphism was associated with litter size traits. These findings confirmed the importance of the IL-6 gene as a candidate gene for litter size traits in pigs.

scholarly journals MOLECULAR TECHNIQUE FOR DERMATOPHYTE IDENTIFICATION ISOLATED FROM PETS IN JAKARTA AND BOGOR

2021 ◽  
Vol 22 (1) ◽  
pp. 56-67
Author(s):  
Dwi Endrawati ◽  
Eko Sugeng Pribadi ◽  
Agustin Indrawati ◽  
Eni Kusumaningtyas
Keyword(s):  
Fragment Length Polymorphism ◽  
Molecular Techniques ◽  
Molecular Technique ◽  
Microsporum Canis ◽  
Chain Reaction ◽  
Molecular Arrangement ◽  
Pcr Rflp ◽  
Examination Techniques ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Dermatophytosis is one of the superficial mycoses which causes skin health problems in pet animals. This study conducted molecular characterization using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on specimens obtained from patients suspected of dermatophytosis in several clinics in DKI Jakarta Province and Bogor City. Fifty samples of skin scrapings from patients suspected of clinically dermatophytosis were collected and analyzed by conventional and molecular techniques. The Research aimed to identify dermatophyte that were isolated from pet animals using PCR-RFLP technique. The primers of ITS 1, ITS 4, Chytin Synthase, and cutting enzymes of Dde1 were used in this Research. Four specimens off 50 spesimens were tested positive using direct and culture PCR examination techniques. Based on the sequencing results, Microsporum canis was identified in four spesimens. Specimens that were positive for dermatophytes followed by RFLP using the Dde 1 enzyme. The results of the study showed that molecular techniques were a reliable way to determine the high-precision dermatophytes in diagnosing dermatophytosis. The Results also showed that molecular arrangement of B1 isolate was different from three other isolates.

scholarly journals Το κληρονομικό αγγειοοίδημα στην Ελλάδα και ο ρόλος των γενετικών βλαβών γονιδίων που κωδικοποιούν ένζυμα της οδού καλλικρεϊνης -βραδυκινίνης στην κλινική έκφρασή του

2016 ◽  
Author(s):  
Νικόλαος Κουτσοστάθης
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Pcr Rflp ◽  
Restriction Fragment Length

ΣΚΟΠΟΣ Η παρουσίαση των πρώτων αποτελεσμάτων της πανελλήνιας καταγραφής ασθενών με κληρονομικό αγγειοοίδημα (ΚΑΟ) κατά την τελευταία τετραετία (Ιούλιος 2010 – Ιούνιος 2014) και η αναζήτηση της συσχέτισης των πολυμορφισμών γονιδίων, τα οποία παράγουν πρωτεΐνες της οδού του συστήματος κινινών- καλλικρεΐνης, με φαινοτυπικά χαρακτηριστικά του ΚΑΟ.ΥΛΙΚΟ-ΜΕΘΟΔΟΙ Έγινε πανελλήνια συστηματική καταγραφή των περιπτώσεων ΚΑΟ μέσω ευρείας συνεργασίας με ιατρούς και νοσοκομεία σε όλη τη χώρα. Σε όσους ασθενείς συμμετείχαν στην μελέτη συμπληρώθηκε: α) τυποποιημένο ερωτηματολόγιο για την καταγραφή δημογραφικών - κλινικών και θεραπευτικών χαρακτηριστικών της νόσου και β) γενεαλογικό δέντρο σε κάθε οικογένεια. Σε κάθε συμμετέχοντα ασθενή έγινε λήψη περιφερικού αίματος για τον προσδιορισμό των πολυμορφισμών F12-5046T>C (rs1801020), BDKRB1-(-699G>C) (rs4905475), SERPING1-21963G>A (p.V480M, rs4926) και ACE I/D (rs1799752). Η ανίχνευση των πολυμορφισμών έγινε με την τεχνική PCR-RFLP (restriction fragment length polymorphism), δηλ. μέσω ενίσχυσης τμημάτων DNA με PCR και εν συνεχεία πέψης με ένζυμα περιορισμού για τους τρεις πρώτους και με απλή PCR για τον τελευταίο. Σε ορισμένα δείγματα χρησιμοποιήθηκε ανάλυση αλληλουχίας βάσεων για την επιβεβαίωση των αποτελεσμάτων. Ως μάρτυρες χρησιμοποιήθηκαν δείγματα περιφερικού αίματος από υγιείς δότες. ΑΠΟΤΕΛΕΣΜΑΤΑ Καταγράφηκαν 128 ασθενείς με ΚΑΟ τύπου Ι και ΙΙ που ανήκουν σε 42 μη συγγενείς οικογένειες (επιπολασμός της νόσου στην Ελλάδα 1:84.000). Επιπρόσθετα καταγράφηκε και μια οικογένεια με 3 μέλη με ΚΑΟ με φυσιολογικό C1-INH. Σε 85 από τους ως άνω ασθενείς, από 34 οικογένειες, έγινε γενετική ανάλυση. Η μέση καθυστέρηση της διάγνωσης της νόσου ήταν τα 17.5 έτη (εύρος 0-58 έτη) , αλλά η διάγνωση ασθενών με ΚΑΟ έχει σαφώς αυξηθεί την τελευταία δεκαετία. 16.7% των ασθενών είχαν υποβληθεί σε διασωλήνωση της τραχείας ή τραχειοτομή λόγω οιδήματος λάρυγγα και 23.5% είχαν υποβληθεί σε άσκοπες χειρουργικές επεμβάσεις λόγω κρίσεων αγγειοοιδήματος στο πεπτικό. Η συχνότητα των θανάτων λόγω ΚΑΟ ήταν περίπου 1 ανά 3 οικογένειες και αφορούσε στην πλειοψηφία τους μη διαγνωσμένους ασθενείς. Το 10.4% των ασθενών δήλωσαν ότι δεν είχε οποιοδήποτε φάρμακο για την αντιμετώπιση των επεισοδίων ΑΟ και 84.7% ανέφεραν ότι η ζωή τους ήταν επηρεασμένη από μέτρια ως σοβαρά λόγω της νόσου. Η παρουσία του πολυμορφισμού F12-5046T>C και του BDKRB1-(-699G>C) καθυστερούσε την έναρξη του ΚΑΟ κατά 4.6 έτη (p=0.03) και κατά 9.4 έτη (p=0.02) αντίστοιχα. Στους ομόζυγους ή ετερόζυγους με πολυμορφισμό V480M στο SERPING1 κυριαρχούσαν οι κρίσεις από το δέρμα. ΣΥΜΠΕΡΑΣΜΑΤΑ Η παρούσα διατριβή, η οποία αποτέλεσε την πρώτη προσπάθεια πανελλαδικής καταγραφής ασθενών με ΚΑΟ, κατάφερε να καλύψει το μεγαλύτερο τμήμα της χώρας. Στην Ελλάδα υπήρχε σημαντική καθυστέρηση της διάγνωσης του ΚΑΟ, από τις μεγαλύτερες παγκοσμίως, η οποία την τελευταία δεκαετία φαίνεται να αναστρέφεται θεαματικά. Το γεγονός αυτό σε συνδυασμό με την θεραπευτική αντιμετώπιση, η οποία σαφώς υπολείπεται της διεθνούς πραγματικότητας, έχουν δυσμενείς συνέπειες για τους ασθενείς. Έτσι αναδεικνύεται η ανάγκη για την προώθηση πρακτικών αντιμετώπισης του προβλήματος. Τα υπόλοιπα επιδημιολογικά και κλινικά χαρακτηριστικά του ΚΑΟ στη χώρα μας δε διαφέρουν από τα αναφερόμενα διεθνώς. Οι πολυμορφισμοί F12-5046T>C και του BDKRB1-(-699G>C) σχετίζονται σημαντικά με καθυστερημένη έναρξη της νόσου και άρα ηπιότερη νόσηση από ΚΑΟ, ενώ ο SERPING1-21963G>A με εντόπιση κρίσεων αγγειοοιδήματος στο δέρμα. Σε πολύ πρόσφατη πολυκεντρική Ευρωπαϊκή μελέτη, προέκταση της παρούσας μελέτης, επιβεβαιώθηκε ότι ο πολυμορφισμός F12-5046T>C αποτελεί τον μοναδικό, μέχρι στιγμής, ανεξάρτητο γενετικό παράγοντα με τόσο ισχυρή επίδραση στον φαινότυπο και την πρόγνωση της νόσου.

scholarly journals Characterisation of avian Pasteurella multocida strains with PCR-RFLP analysis of the ompH gene

2013 ◽  
Vol 61 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Boglárka Sellyei ◽  
Éva Ivanics ◽  
Tibor Magyar
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Pasteurella Multocida ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
The Other ◽  
Pcr Rflp ◽  
H Gene ◽  
Geographic Regions ◽  
Type Strains ◽  
Restriction Fragment Length

The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

scholarly journals Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 196 ◽  
Author(s):  
García-Suárez ◽  
González-Rodríguez ◽  
Cima-Cabal ◽  
Yuste ◽  
Vazquez ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Dna Sequences ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

scholarly journals Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae

2014 ◽  
Vol 63 (5) ◽  
pp. 667-673 ◽  
Author(s):  
Sharda Prasad Awasthi ◽  
Masahiro Asakura ◽  
Sucharit Basu Neogi ◽  
Atsushi Hinenoya ◽  
T. Ramamurthy ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Vibrio Cholerae ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Pcr Rflp ◽  
Cholix Toxin ◽  
Rflp Assay ◽  
Restriction Fragment Length

Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

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