Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen's kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.
- MeSH
- bakteriální geny genetika MeSH
- DNA gyráza genetika MeSH
- DNA primery genetika MeSH
- endodeoxyribonukleasy genetika MeSH
- mastitida skotu diagnóza mikrobiologie MeSH
- mléko mikrobiologie MeSH
- Mycoplasma bovis genetika MeSH
- RNA ribozomální 16S genetika MeSH
- skot MeSH
- techniky amplifikace nukleových kyselin normy MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338 Da originated from the proteolysis of aS1-casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the determination of a biomarker for cow mastitis in milk by MALDI-TOF mass spectrometry.
- MeSH
- biologické markery analýza MeSH
- mastitida skotu diagnóza MeSH
- mléčné bílkoviny analýza MeSH
- mléko chemie MeSH
- molekulární modely MeSH
- nanočástice chemie MeSH
- peptidy analýza MeSH
- proteinová korona analýza MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- syrovátka chemie MeSH
- železité sloučeniny chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Detection of mastitis-associated bacteria can be accomplished by culturing or by molecular techniques. On the other hand, rapid and inexpensive methods to enumerate bacterial load without culturing can be better achieved by molecular methods. Staphylococcus aureus and Escherichia coli are the predominant bacterial pathogens associated with bovine mastitis. Here, we describe the application of conventional PCR for the limit of detection (LOD) of genomic DNA of S. aureus and E. coli based on single-copy genes. The selected genes were thermonuclease (nuc), aureolysin (aur), and staphopain A (scpA) for S. aureus and β-D-glucuronidase A (uidA), cytochrome d oxidase (cyd), and rodA (a gene affecting cell shape and methicillin sensitivity) for E. coli. The LOD was 5.3, 15.9, and 143 pg for aur, nuc, and scpA genes, corresponding to S. aureus genomic copies of 1.75 × 10(3), 5.16 × 10(3), and 4.71 × 10(4), respectively. The LOD was 0.45, 12.3 and 109 pg for uidA, rodA and cyd genes, corresponding to E. coli genome copies of 8.91 × 10(1), 2.43 × 10(3), and 2.16 × 10(4), respectively. Application of uidA and aur PCRs to field strains revealed that as low as approximately 100 genome copies of E. coli and 1000-10,000 copies of S. aureus could be detected. This study is the first to report LOD of genomic DNA using conventional PCR for aur and scpA genes of S. aureus, and rodA and cyd genes of E. coli. The results should be useful for developing assays to assess bacterial load in milk and to determine the load that contributes to subclinical or clinical mastitis.
- MeSH
- bakteriální proteiny genetika MeSH
- DNA bakterií genetika MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- infekce vyvolané Escherichia coli diagnóza mikrobiologie veterinární MeSH
- limita detekce MeSH
- mastitida skotu diagnóza mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- skot MeSH
- stafylokokové infekce diagnóza mikrobiologie veterinární MeSH
- Staphylococcus aureus genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- MeSH
- Bacteria MeSH
- bakteriální léková rezistence imunologie účinky léků MeSH
- bakteriologie trendy MeSH
- enterotoxiny imunologie izolace a purifikace MeSH
- infekce spojené se zdravotní péčí * diagnóza epidemiologie komplikace MeSH
- kontaminace potravin * analýza prevence a kontrola MeSH
- lidé MeSH
- mastitida skotu * diagnóza epidemiologie etiologie MeSH
- stafylokokové infekce diagnóza epidemiologie prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
