Background Mastitis is an inflammation of the mammary glands caused by a microbial infection. The common bacteria causing this infection in dairy farms are Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. The aptamer is a new biosensor platform for detecting pathogens; however, its use for simultaneous detection of S. aureus, S. agalactiae, and E. coli bacteria has not been reported. This study’s objective is to isolate and characterize polyclonal DNA aptamer with broad reactivity to the mastitis bacteria S. aureus, S. agalactiae, and E. coli using a sequential toggle cell-SELEX. Methods and results The DNA aptamer pool from SELEX 15 was inserted into the pGEM-T easy plasmid. Furthermore, the transformant clones were selected by PCR colony, plasmid isolation, and sequencing. Six DNA aptamers, consisting of S15K3, S15K4, S15K6, S15K13, S15K15, and S15K20 with a constant region and the right size of 81 bp were derived from the sequencing analysis. The secondary structure of the DNA was predicted using Mfold software. The DNA was analyzed with binding characteristics, including binding capacity and affinity (Kd), using qPCR. The results indicated aptamer S15K15 has the highest binding ability into S. agalactiae, while S15K13 performed binding capacity most to E. coli EPEC 4, and S15K3 has the highest capacity of binding to S. aureus BPA-12. Conclusion Aptamer S15K3 has the best binding characteristics on all three bacterial targets.

Mastitis is a complex disease in cattle that it involves interactions between management practices and infectious agents. The common microorganisms causing mastitis are bacteria, besides this disease can be caused by mycoplasma, algae, and yeast. Pathogen microorganisms in milk can be obtained from cattle, human hands, equipment and the environment. This study aims to analyze the metagenomic of pathogen mastitis in cow’s milk from Cicurug, Sukabumi, West Java. ZymoBIOMICSTM DNA Miniprep Kit was used for genome isolation to metagenomic analysis. The 16S rRNA PCR amplification was used for analysis the results of miniprep. Metagenomic analysis from subclinical mastitis milk showed that bacteria in cow’s milk were the genera of Corynebacterium_1 (20.53 %), Corynebacterium (11.67%), Solibacillus (8.78%), Romboutsia (5.45%), Micrococcus (4.18%), Acinetobacter (3.64%), Aerosphaera (1.94%), Ignavigranum (1.90%), Lysinibacillus (1.49%), and Staphylococcus (1.38%).

There is a protease produced by bateria that has characteristics similar to rennin from a calf.  Rennin has the ability to clot casein in milk. Rennin-like protease (RLP) is produced by bacteria extracellularly. Lactic Acid Bacteria (LAB) have the potential to be developed for RLP production because they are safe and non-pathogenic bacteria. Rennin is needed in the process of milk coagulation to subsequently obtain a curd in the process of making cheese. In this study, the LAB isolated from Ettawa goat milk (isolate 2.12) which produced RLP was 99% identical to Lactobacillus paracasei based on 16S rRNA gene sequence analysis. The purification of the RLP L. paracasei 2.12 with 60% ammonium sulfate deposition, dialysis, and filtration gel chromatography Sephadex G-50 showed a single 38 kDa protein band with SMCA/SPA was 4.48 higher than that of the calf rennet with a ratio value of 1, therefore in this study, RLP L. paracasei 2.12  was developed as an alternative to renin in cheese making.