Amplification-Free Detection of MiRNA Signatures in Milk: A Noninvasive Sensing Approach for Early Diagnosis of Bovine Mastitis

Abstract

Bovine mastitis is an infectious disease with a significant impact on the dairy industry. It causes reduced milk yield and quality, and occasional mortality. Early and accurate diagnosis is challenging, being especially critical for subclinical mastitis lacking visible symptoms. MicroRNAs (miRNAs) are emerging as promising biomarkers for mastitis. Herein, using next-generation sequencing, a distinct miRNA signature (miRNA-146b, miRNA-221, miRNA-223) is identified, which is differentially expressed in healthy and naturally infected subclinical cows. Next, electrochemical DNA sensors are developed by self-assembling methylene blue-tagged oligonucleotides, complementary to each miRNA, on gold electrodes. MiRNA detection is achieved by monitoring hybridization-induced conformational changes using square-wave voltammetry. The sensors exhibit a sigmoidal response (dynamic range 0.1–100 nM), with a limit of detection of 0.1 nM and excellent selectivity. Importantly, they demonstrate strong diagnostic performance in milk-derived RNA extracts, with receiver operating characteristic–area under the curve values of 0.88, 0.80, and 0.70 for miRNA-221, miRNA-146b, and miRNA-223, respectively. Moreover, the responses are biologically meaningful, as correlation analysis highlights distinct associations of miRNA-146b and miRNA-221 with inflammation, and of miRNA-221 and miRNA-223 with microbial burden. The amplification-free detection of clinically relevant miRNAs in milk extracts represents a significant advancement toward noninvasive testing for the early diagnosis of mastitis. The development of simple, sensitive, and specific sensors targeting unique signatures of host immune-derived biomarkers promises a new framework to improve disease management and antimicrobial stewardship.