Simultaneous identification of CSN3 and LGB genotypes in cattle by high-resolution melting curve analysis

Kyselová, J; Rychtářová, Jana; Sztankóová, Z.; Czerneková, V.

doi:10.1016/j.livsci.2011.12.018

Cited by 5 publications

(3 citation statements)

References 22 publications

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“…Along with the widely used method for assessing the allelic polymorphism of the CSN3 gene in cattle based on PCR-RFLP analysis [6][7][8], SSCP-analysis [9][10][11], ARMS-PCR [12,13], allele-specific PCR (AS-PCR) [14,15], HRM analysis [16][17][18], and TaqMan real-time PCR [19,20].…”

Section: Thematic Scope: Publication Of Priority Research In the Fielmentioning

confidence: 99%

Real-Time PCR Technology for Cattle Genotyping by a and B Kappa-Casein Gene Alleles

Vafin

Gilmanov

2021

Series Chemistry and Technology

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The main goal of the study was to develop and test an effective technology for cattle genotyping by the CSN3 gene based on real-time PCR with hybridization-fluorescence detection. There was developed a method for real-time PCR for cattle genotyping by A and B alleles of the CSN3 gene in the format of hybridization-fluorescence detection, involving the use of two 5/-fluorescence-labeled forward allele-specific primers, one reverse common primer, and one anti-primer labeled with a fluorescence quencher at the 3/-end of the oligonucleotide. As a result of practical studies aimed at testing the developed method, we obtained the technical result provided by the proposed technology, expressed in the effective identification of the desired genotypes due to correct interpretation of these curves of increasing fluorescence intensity, the results reliability of which was also confirmed by the well-known PCR-RFLP analysis technique for Bos taurus genotyping for similar allelic variants of the kappa-casein gene.

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Section: Thematic Scope: Publication Of Priority Research In the Fielmentioning

confidence: 99%

Real-Time PCR Technology for Cattle Genotyping by a and B Kappa-Casein Gene Alleles

Vafin

Gilmanov

2021

Series Chemistry and Technology

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“…A wide range of techniques have been previously used for cattle genotyping, such as Restriction Fragment Length Polymorphism-PCR (RFLP-PCR) 19 , Single Stranded Conformation Polymorphism-PCR (SSCP-PCR) 24 , Amplification-Refractory Mutation System-PCR (ARMS-PCR) 11 , 25 , Allele Specific-PCR (AS-PCR) 26 or High Resolution Melting Analysis -qPCR (HRMA-qPCR) 27 – 29 . Moreover, qPCR assays are highly specific and sensitive DNA-based methods with special interest for genotyping cattle breeds.…”

Section: Introductionmentioning

confidence: 99%

Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection

Jiménez-Montenegro

Mendizábal

Alfonso

et al. 2022

Sci Rep

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Milk proteins determine important milk technological characteristics. Among caseins, Ƙ-casein has been correlated with fat and protein content and cheese yield. Fourteen Ƙ-caseins variants have been described but the alleles A, B and E are the most important ones due to their frequency and/or influence on the technological aptitudes of milk. Therefore, in the present study two different duplex qPCR assays with locked nucleic acid probes (for positions 13104 and 13124 of the Ƙ-casein gene) were developed for the detection of A, B and E variants. Firstly, DNA isolation method from milk somatic cells and hair was optimised. The developed 13124-qPCR assay showed an increased sensitivity reaching up to 6.7 copies DNA copies/reaction at a 95% confidence level with A, B and E alleles reference samples. The 13104-qPCR assay reached up to 6.7 DNA copies/reaction for A allele reference sample and 67 DNA copies/reaction for B and E samples. Intra-assay variation results were below 6%. Applicability was determined using DNA samples from animals with known genotype for Ƙ-casein (AA, AB, BB, BE, AE, EE) and both assays were able to discriminate among the six genotypes with 100% accuracy. Thus, this qPCR method represents a sensitive and rapid option for the detection of Ƙ-casein alleles in both hair and milk samples.

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“…), animal breeding (Kyselová et al . ), microbiology (Ruskova & Raclavsky ) and genotyping of wild populations (Henri & Mouton ).…”

Section: Introductionmentioning

confidence: 99%

High‐resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples

Morinha

Travassos

Seixas

et al. 2013

Molecular Ecology Resources

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High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex-specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.

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Simultaneous identification of CSN3 and LGB genotypes in cattle by high-resolution melting curve analysis

Cited by 5 publications

References 22 publications

Real-Time PCR Technology for Cattle Genotyping by a and B Kappa-Casein Gene Alleles

Real-Time PCR Technology for Cattle Genotyping by a and B Kappa-Casein Gene Alleles

Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection

High‐resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples

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