Comparative Analysis of Rapid and Less Invasive Methods for A2A2 Dairy Cattle Genotyping and A2 Milk Purity Detection

The lateral flow immunoassay (LFIA) is one of the most successful sensing platforms for real-world point-of-care (POC) testing. However, achieving PCR-level sensitivity without compromising the inherent advantages of LFIA, such as rapid and robust operation, affordability, and naked-eye detection, has remained a primary challenge. In this study, a plasmonic scattering-utilising LFIA was proposed, created by transparentising a nitrocellulose membrane and placing a light-absorbing backing card under the membrane. This LFIA minimised the background signal from its matrix, leading to substantially enhanced sensitivity and enabling naked-eye detection of the plasmonic scattering signal from gold nanoparticles without optics. Our plasmonic scattering-utilising LFIA showed an approximately 2600–4400 times higher detection limit compared with that of commercial LFIAs in influenza A assays. In addition, it exhibited 90% sensitivity in clinical validation, approaching PCR-level sensitivity, while commercial LFIAs showed 23–30% sensitivity. The plasmonic scattering-utilising LFIA plays a ground-breaking role in POC diagnostics and significantly boosts follow-up research.

The global pandemic of COVID-19 continues to be an important threat, especially with the fast transmission rate observed after the discovery of novel mutations. In this perspective, prompt diagnosis requires massive economical and human resources to mitigate the disease. The current study proposes a rational design of a colorimetric lateral flow immunoassay (LFA) based on the repurposing of human samples to produce COVID-19-specific antigens and antibodies in combination with a novel dye-loaded polymersome for naked-eye detection. A group of 121 human samples (61 serums and 60 nasal swabs) were obtained and analyzed by RT-PCR and ELISA. Pooled samples were used to purify antibodies using affinity chromatography, while antigens were purified via magnetic nanoparticles-based affinity. The purified proteins were confirmed for their specificity to COVID-19 via commercial LFA, ELISA, and electrochemical tests in addition to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polymersomes were prepared using methoxy polyethylene glycol-b-polycaprolactone (mPEG-b-PCL) diblock copolymers and loaded with a Coomassie Blue dye. The polymersomes were then functionalized with the purified antibodies and applied for the preparation of two types of LFA (antigen test and antibody test). Overall, the proposed diagnostic tests demonstrated 93 and 92.2% sensitivity for antigen and antibody tests, respectively. The repeatability (92–94%) and reproducibility (96–98%) of the tests highlight the potential of the proposed LFA. The LFA test was also analyzed for stability, and after 4 weeks, 91–97% correct diagnosis was observed. The current LFA platform is a valuable assay that has great economical and analytical potential for widespread applications.

Approximately 30% of milk protein is β-casein. We aimed to determine whether lactose maldigesters who chronically consumed two cups of A1/A2 milk (containing 75% A1 β-casein and 25% A2 β-casein) would adapt to have fewer intolerance symptoms, lower serum inflammatory markers, and/or altered glutathione levels similar to those consuming A2 milk (containing 100% A2 β-casein). A double-blinded, randomized, crossover trial was conducted. Sixteen confirmed lactose maldigesters consumed 250 mL of A1/A2 milk and A2 milk twice daily with meals for two weeks. At the end of the adaptation period on day 15, lactose maldigestion was measured after a challenge with the same milk used for adaptation (0.5 g of lactose per kg of body weight) with a hydrogen breath test. Fecal urgency was higher during the two-week consumption of A1/A2 milk compared to A2 milk (p = 0.04, n = 16). Bloating (p = 0.03, n = 16) and flatulence (p = 0.02, n = 16) were also higher on the 15th day with A1/A2 milk compared to A2 milk challenge. However, day-to-day symptoms, hydrogen, serum inflammatory markers, and antioxidant concentrations were not different after A1/A2 and A2 milk consumption adaptation periods. Adaptation over two weeks did not improve lactose digestion or tolerance of A1/A2 milk to match that of A2 milk.

Rapid identification of A1 and A2 milk based on the combination of mid-infrared spectroscopy and chemometrics

Milk contributes a vital role in nutritional security in most of the countries of the world. Recently a debate is going on worldwide that, A1 milk is harmful and A2 milk is beneficial. Though there was no supporting clinical evidence to claim these, milk is selling in the name of A2 milk for high cost without proper identification in the field level. To address this issue to both consumer and producers, scientists are in a position to rule out the A1 and A2 milk. In this study TANUVAS A1A2 detect kit was used in the A1 or A2 type milk with dried blood or milk spot. 31 samples were collected from farms and individual farmers from different places of Tiruchirappalli district. All the samples were analysed with the ready to use primer based PCRÂ test TANUVAS A1A2 detect kit. This study revealed that 20% of the crossbred cows, 78.95% of the indigenous breed cows, 66.67 % of non descriptive cows and 100 percent of the buffaloes had A2A2 genotype. The results of this study is giving the fact that, milk needs to be checked out with proper test like TANUVAS A1A2 detect before claiming for A2 milk.

This study explores the adoption of laser-induced breakdown spectroscopy (LIBS) for the analysis of lateral-flow immunoassays (LFIAs). Gold (Au) nanoparticles are standard biomolecular labels among LFIAs, typically detected via colorimetric means. A wide diversity of lanthanide-complexed polymers (LCPs) are also used as immunoassay labels but are inapt for LFIAs due to lab-bound detection instrumentation. This is the first study to show the capability of LIBS to transition LCPs into the realm of LFIAs, and one of the few to apply LIBS to biomolecular label detection in complete immunoassays. Initially, an in-house LIBS system was optimized to detect an Au standard through a process of line selection across acquisition delay times, followed by determining limit of detection (LOD). The optimized LIBS system was applied to Au-labeled Escherichia coli detection on a commercial LFIA; comparison with colorimetric detection yielded similar LODs (1.03E4 and 8.890E3 CFU/mL respectively). Optimization was repeated with lanthanide standards to determine if they were viable alternatives to Au labels. It was found that europium (Eu) and ytterbium (Yb) may be more favorable biomolecular labels than Au. To test whether Eu-complexed polymers conjugated to antibodies could be used as labels in LFIAs, the conjugates were successfully applied to E. coli detection in a modified commercial LFIA. The results suggest interesting opportunities for creating highly multiplexed LFIAs. Multiplexed, sensitive, portable, and rapid LIBS detection of biomolecules concentrated and labeled on LFIAs is highly relevant for applications like food safety, where in-field food contaminant detection is critical. Graphical abstract.

Simple SummaryEnhancing the concentration of individual fatty acids (FA) in milk has been, for a long time, a major aim for researchers because certain FAs are linked with several health benefits in humans as well as improving the processing quality of milk products. It is well documented that diet, management regime, and extent of biohydrogenation in the rumen are critical in determining the composition of FA in the milk of dairy cows. This study investigated the effects of including chicory into the traditional feeding regime of ryegrass/white clover, and time of its allocation on milk production, rumen fermentation, and FA composition of milk and rumen digesta of dairy cows. Our findings show that allocation of mature chicory herbage to dairy cows at 50% of their ration modified rumen fermentation and improved both milk yield and the FA profile of the milk. Allocating chicory herbage during the afternoon is a useful strategy that can translate to improved milk production and quality. These findings reflect not just the feasibility of including chicory as part of a feeding regime, but also the role of chicory in rumen fermentation and biohydrogenation.The goals of the current study were to investigate the effects of including chicory (Cichorium intybus L.) into the traditional feeding regime of ryegrass/white clover (Lolium perenne L./Trifolium repens L.), and time of its allocation on milk production, rumen fermentation, and FA composition of milk and rumen digesta of dairy cows. Nine groups of four cows were allocated one of three replicated feeding regimes: (1) ryegrass/white clover only (RGWC), (2) ryegrass/white clover + morning allocation of chicory (CHAM), and (3) ryegrass/white clover + afternoon allocation of chicory (CHPM). One cow per group had a rumen cannulae fitted. Treatment did not affect total grazing time or estimated dry matter intake, but cows ruminated more when fed RGWC than chicory. Allocating chicory in the afternoon elevated milk production compared with RGWC and CHAM. Milk from cows grazing chicory contained greater concentrations of polyunsaturated FA (PUFA) such as C18:3 c9, 12, 15 and C18:2 c9, 12 than those on RGWC. As with milk, rumen digesta concentration of PUFA increased when cows grazed on chicory rather than RGWC, which corresponded with lower concentrations of intermediate vaccenic and biohydrogenation end-product stearic acid for cows grazing on chicory. Mean ruminal pH was lower for cows offered chicory than those on RGWC, reflecting greater rumen concentrations of volatile fatty acids (VFA) for cows fed chicory. Allocating chicory during the afternoon is a useful strategy that can translate to improved milk production. The lower rumen pH, lower concentration of vaccenic and stearic acids, and elevated concentration of PUFA in the rumen of cows fed chicory suggest reduced biohydrogenation and may explain the elevated concentration of PUFA in the milk of cows fed chicory compared with those fed RGWC.

Lactose maldigesters report an increase in abdominal pain due to the consumption of milk containing a mixture of A1 and A2 β-casein as compared to milk containing only A2 β-casein. Gastric transit affects gastrointestinal symptoms and rapid transit has been associated with an increase in abdominal pain. We conducted a double-blinded, randomized, crossover trial in 10 lactose maldigesters. Subjects consumed each of the two types of milk: conventional milk containing 75% A1 β-casein and 25% A2 β-casein and A2 milk containing 100% A2 β-casein. Magnetic resonance images were acquired, and abdominal pain was rated and recorded at 0, 10, 30, 60 and 120 min after milk consumption. The volume of milk in the stomach was calculated using FSL software. The volume of milk in the stomach after consuming milk with 75% A1 β-casein and 25% A2 β-casein was significantly lower at 30 (p = 0.01), 60 (p = 0.002) and 120 (p < 0.001) minutes as compared to milk with 100% A2 β-casein in the 10 lactose maldigesters. The transit of New-World milk containing A1 and A2 β-casein was more rapid as compared to Old-World milk containing only A2 β-casein. This difference in transit may mediate symptoms of lactose intolerance.

Background: The most frequent infectious complication in transfusion therapy in developed countries is related to the bacterial contamination of platelet concentrates (PCs). Rapid and cultural screening methods for bacterial detection in platelets are available, but external performance evaluation, especially of rapid methods, has been difficult to realize so far. Here we summarize the results of three individual collaborative trials using an external quality assessment program (EQAP) for the application of current rapid and cultural screening methods. Methods: Three different modules were available for the detection of bacterial contamination: module 1: rapid methods, module 2: culture methods, module 3: bacterial identification methods. The sample set-up included up to six different bacterial strains, 1-2 negative samples and 4-6 positive samples with stabilized bacterial cell counts (approximately 10³/10⁴/10⁵ CFU/ml). Time schedule for testing was limited (module 1: 6 h, module 2 and 3: 7 days). Results: Samples of module 1 were analyzed with two different rapid methods (BactiFlow, NAT). The results of the three individual collaborative trials showed that all participants detected the negative samples with both assays correctly. Samples spiked with 10⁴ to 10⁵ CFU/ml of bacteria obtained positive results with both rapid screening methods, whereas samples spiked with only 10³ CFU/ml disclosed a lower number of correctly identified positive results by NAT (86.6-93.8% sensitivity) compared to BactiFlow (100% sensitivity). The results for modules 2 and 3 revealed a 100% diagnostic sensitivity and specificity in all three collaborative trials. Conclusion: This proficiency panel facilitates the verification of the analytical sensitivity of rapid and cultural bacterial detection systems under controlled routine conditions. The concept of samples provided in this EQAP has three main advantages: i) samples can be examined by both rapid and culture methods, ii) the provided material is matrix-equivalent, and iii) the sample material is ready-to-use.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy

Mid-infrared spectroscopy can be applied to authenticate A2 milk.

The perceived health benefits of β-casein (β-CN) A2 milk for humans have driven increased selection for homozygous β-CN A2 dairy cattle. However, little is known about the effects of feeding milk with different β-CN variants to calves, or about the influence of β-CN genotypes on calf health and performance. We aimed to evaluated the performance, daily fecal score, and body composition of calves that carried β-CN A1A1, A1A2, or A2A2 genotypes and different κ-CN genotypes, fed homozygous β-CN A1 or A2 milk. Every day, during the first 14 days of life, calves were offered 7.5L/day of either homozygous β-CN A1 or A2 milk (27 females and 25 males per group), and total energy-corrected milk intake was recorded for each calf. On Day 15, the body composition of all 104 calves was assessed using magnetic resonance imaging and dual-energy X-ray absorptiometry. No significant differences were observed in performance, daily fecal scores, or body composition between calves fed β-CN A1 milk and those fed β-CN A2 milk. Compared with β-CN A2A2 calves, calves with the β-CN A1A1 and A1A2 genotypes presented greater final body weights, average daily gains, and feed efficiencies and fewer days with diarrhea. These findings suggest that the exclusive selection of β-CN A2A2 animals should be reconsidered because of the potential impact of this genotype on calf health and growth performance.

β-Casein, a major protein in cow's milk, is divided into the A1 and A2 type variants. Digestion of A1 β-casein yields the peptide β-casomorphin-7 which could cause gastrointestinal (GI) discomfort but A2 milk containing only A2 β-casein might be more beneficial than A1/A2 (regular) milk. The aim of this study was to evaluate the differences in GI discomfort after ingestion of A2 milk and A1/A2 milk. A randomized, double-blind, cross-over human trial was performed with 40 subjects who experienced GI discomfort following milk consumption. For each intervention period, either A2 milk first (A2→A1/A2) or A1/A2 milk was first consumed for 2 weeks (A1/A2→A2) following a 2-week washout period. GI symptom rating scale (GSRS) scores, questionnaire for digestive symptoms, and laboratory tests including fecal calprotectin were evaluated. For symptom analysis, generalized estimating equations gamma model was used. A2 milk increased bloating (P = 0.041) and loose stools (P = 0.026) compared to A1/A2 milk in GSRS. However, A2 milk caused less abdominal pain (P = 0.050), fecal urgency (P < 0.001) and borborygmus (P = 0.007) compared to A1/A2 milk in questionnaire for digestive symptoms. In addition, fecal calprotectin also decreased or less increased after consumption of A2 milk compared to A1/A2 milk (P = 0.030), and this change was more pronounced in males (P = 0.005) than in females. There were no significant adverse reactions during the trial. A2 milk alleviated digestive discomfort in Koreans following A2 milk consumption (ClinicalTrials.gov NCT06252636 and CRIS KCT0009301).

Chromaflo buys Tint-Ayd colorants business from Elementis

Worldwide research on the health effects of bovine milk containing A1 and A2 β-casein: Unraveling the current scenario and future trends through bibliometrics and text mining