AUTHOR=Shrinet Garima , Chhabra Rajesh , Sharma Archana , Batra Kanisht , Talukdar Saurabh Jyoti , Maan Sushila TITLE=High throughput Luminex beads based multiplex assay for identification of six major bacterial pathogens of mastitis in dairy animals JOURNAL=Frontiers in Cellular and Infection Microbiology VOLUME=13 YEAR=2023 URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1125562 DOI=10.3389/fcimb.2023.1125562 ISSN=2235-2988 ABSTRACT=Introduction

Bovine mastitis is caused by over 150 different microorganisms. Specific identification and quantification of multiple bacteria in a single milk sample becomes essential for rapid intervention.

Methods

In the present study a Luminex beads based multiplex assay emphasizing on the precise identification of six major bacterial pathogens of mastitis was developed. Assay was developed in two triplex sets, triplex 1 comprised of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis while triplex 2 consisted of Staphylococcus aureus, E. coli and Klebsiella pneumoniae.

Results

The analytical sensitivity was 10 6 copies per reaction mixture for all the six bacteria. A 100% analytical specificity was observed for simultaneous detection of these bacteria. Clinical milk samples from 100 bovine quarters were tested for validation.

Discussion

The analytical sensitivity was similar to the findings reported earlier in real time PCR multiplex assay targeting the DNA of the 11 most common bacterial species or groups in mastitis. The analytical specificity of the optimized assay was 100% similar to reported earlier for simultaneous detection of Mycoplasma spp. and for seven entric viruses of humans.The developed assay indicates a concept proof of a rapid, cost effective high throughput diagnostic tool for identification of major bacteria causing mastitis.