Novel LNA probe-based assay for the A1 and A2 identification of β-casein gene in milk samples

Giglioti, Rodrigo; Okino, Cíntia Hiromi; Azevedo, Bianca Tainá; Gutmanis, Gunta; Katiki, Luciana Morita; Oliveira, M. C. de S.; Filho, Aníbal Eugênio Vercesi

doi:10.1016/j.fochms.2021.100055

Cited by 7 publications

(7 citation statements)

References 35 publications

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“…At present, there are many milk protein allergen detection methods, which can be divided into two categories in terms of detection objects. One category chooses DNA as the object, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) [ 4 , 5 , 6 , 7 ]. However, since these methods detect the non-allergenic DNA but not the allergen protein itself, it may be independent of the amount of protein in an unknown substrate.…”

Section: Introductionmentioning

confidence: 99%

Identification of Characteristic Peptides of Casein in Cow Milk Based on MALDI-TOF MS for Direct Adulteration Detection of Goat Milk

Dai

Zhang

et al. 2023

Foods

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It is widely acknowledged that casein is an important allergenic protein in milk which may cause danger to customers. The identification and confirmation of caseins through mass spectrometry requires the selection of suitable characteristic peptides. In this study, by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the three most representative specific peptides of caseins in cow milk were screened out with mass-to-charge ratios (m/z) of 830, 1195, and 1759, respectively. By comparing 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) MALDI matrices, it was found that DHB was more suitable for peptide detection with the limits of detection (LODs) of 0.1 mg/L for α, β-casein. Furthermore, on the basis of verifying the characteristic peptides of casein from cow milk, this protocol was applied to goat milk authentication. Cow milk addition in goat milk was investigated by using the screened specific peptides. The results showed that the adulteration could be identified when the proportion of cow milk was 1% or more. When applied to inspect adulteration in five brands of commercial goat milk, specific peptides of bovine casein were detected in four of them. The method has the advantages of strong reliability, high throughput, simple preprocessing, and fast speed, which can provide powerful help for prewarning dairy allergen.

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Section: Introductionmentioning

confidence: 99%

Identification of Characteristic Peptides of Casein in Cow Milk Based on MALDI-TOF MS for Direct Adulteration Detection of Goat Milk

Dai

Zhang

et al. 2023

Foods

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“…The alternative elaborated variant was found in the methods of allele-specific PCR (AS-PCR), real-time probes for PCR, etc. (Manga et al, 2010;Rahimi et al, 2015;Giglioti et al, 2021;Ristanic et al, 2022). Sequencing is conducted in some cases, but regardless of its efficiency, at this stage, it is not always reasonable for routine large-scale population studies (Dai et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Comparative analysis of A1 and A2 allele detection efficiency for bovine CSN2 gene by AS-PCR methods

2023

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This study was aimed at conducting a comparative analysis of the efficiency in genotyping cattle by beta-casein locus using the allele-specific PCR methods. The results of the study have demonstrated the necessity to optimize the protocol for the use of AS-PCR to detect alleles A1 and A2. It was found that the use of non-optimized PCR protocols led to the genotyping errors, manifested regarding beta-casein locus (CSN2). The impossibility of using the touchdown PCR as an optimization instrument for AS-PCR was proven. The elaborated typing protocols were used to study the genetic structure of the cattle populations of different breeds, reared in Ukraine – Ukrainian Red-and-White dairy, Ukrainian Black-and-White dairy (two populations), and Charolais. It was found that locus CSN2 was polymorphic in all the cattle populations. The frequencies of allele A2 varied within 0.34–0.91 depending on the population of the animals, which may be conditioned by the specificities in the selection work. No deviation from the Hardy-Weinberg equilibrium was found in any investigated population of cattle.

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“…A2 milk authentication using molecular methods such as chromatographic (Oglobline et al, 2022) and isoelectric focusing techniques have been recently developed (Mayer et al, 2021). Alternatively, DNA-based methods for β-casein A1 and A2 allele detection can be employed based on the analysis of DNA of milk somatic cells in cows (Giglioti et al, 2020(Giglioti et al, , 2021Mayer et al, 2021). Moreover, to increase the specificity of the qPCR assays, locked nucleic acid (LNA) probes can be used (Giglioti et al, 2021(Giglioti et al, , 2022Puente-Lelievre & Eischeid, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…Alternatively, DNA-based methods for β-casein A1 and A2 allele detection can be employed based on the analysis of DNA of milk somatic cells in cows (Giglioti et al, 2020(Giglioti et al, , 2021Mayer et al, 2021). Moreover, to increase the specificity of the qPCR assays, locked nucleic acid (LNA) probes can be used (Giglioti et al, 2021(Giglioti et al, , 2022Puente-Lelievre & Eischeid, 2021). LNA probes improve stability and mismatch discrimination thankful for the formation of a methylene bridge between the 2 ′ oxygen and the 4' carbon of the pentose ring that results in higher affinity for the complementary DNA region (You et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

et al. 2022

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Novel LNA probe-based assay for the A1 and A2 identification of β-casein gene in milk samples

Cited by 7 publications

References 35 publications

Identification of Characteristic Peptides of Casein in Cow Milk Based on MALDI-TOF MS for Direct Adulteration Detection of Goat Milk

Identification of Characteristic Peptides of Casein in Cow Milk Based on MALDI-TOF MS for Direct Adulteration Detection of Goat Milk

Comparative analysis of A1 and A2 allele detection efficiency for bovine CSN2 gene by AS-PCR methods

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

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