Rapid mastitis detection assay on porous nitrocellulose membrane slides

Abstract

We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase at their surface. The antibodies were inkjet printed onto three different nitrocellulose membrane slides, Unisart (Sartorius), FAST (GE Whatman), and Oncyte-Avid (Grace-Biolabs), and the final assay signals on these slides were compared. The blackness of the spots was determined by flatbed scanning and assessment of the pixel gray volume using TotalLab image analysis software. The black spots could be easily read by the naked eye. We successfully demonstrated the detection of specific amplicons from mastitis-causing pathogens in less than 3 h. Using a similar protocol, we also showed that it was possible to detect specific amplicons from four different mastitis-causing pathogens (six strains) on the same pad. The influence of two different printing buffers, phosphate-buffered saline (pH 7.4) and carbonate buffer (pH 9.6), on the functionality of the primary antibodies was also compared.

Detection of Single-Nucleotide Polymorphisms in Plasmodium falciparum by PCR Primer Extension and Lateral Flow Immunoassay

“…Even if it is possible, it would result in a complicated readout. An alternative to performing all visualization reactions of the PEXT products in one assay is to develop a nucleic acid microarray immunoassay (NAMIA), as was done for the detection of genes encoding virulence factors of Escherichia coli O157 and mastitis pathogens (27,28). In the microarray assay, the number of analyzed PEXT products can be increased due to the higher level of complexity, e.g., a microarray of 100 spots on a 6-by 6-mm nitrocellulose pad.…”

“…Another possibility to reduce the total assay time is to apply a PCR machine that makes use of thin-walled PCR tubes, like the PIKO thermal cycler (Thermo Fisher Scientific, Vantaa, Finland) (17,27,28). In particular, performing the PCR step in the PIKO, using an accompanying fast and robust DNA polymerase, would save a lot of time.…”

Amplified Fluorescence by ZnO Nanoparticles vs. Quantum Dots for Bovine Mastitis Acute Phase Response Evaluation in Milk

“…Bovine mastitis (BM) is a prominent inflammatory disease affecting the dairy industry worldwide, originated by pathogenic agent invasion onto the mammary gland [1][2][3]. Decreased milk production yield, unsatisfying product quality and increased treatment costs are all proportionally correlated to the severity of the occurring BM [4,5]. The disease is classified as chronic, clinical and subclinical based on the induced pathogenesis [2,6,7].…”

“…[1,2]. Conventional techniques measure somatic cell counts (SCC), identify causative pathogenic bacteria by selective cell culture diagnosis and assess enzymatic activity [3,5,8,9]. Lactate dehydrogenase and N-acetyl-β-d-glucosaminidase (NAGase) are predominant indicators of BM, which correlate with the SCC levels found in milk [3].…”

Hexamethyldisilazane Modified Paper as an Ultra-sensitive Platform for Visual Detection of Hg2+, Co2+, Zn2+ and the Application to Semi-quantitative Determination of Hg2+ in Wastewater

“…On the other hand, the application of HMDS modified filter paper has been limited for phase separator applications. Hence, the present article is focused on: A) the application of HMDS modified paper as a platform for rapid detection of Hg 2+ using 6-hydroxy-3-(2-oxoindolin-3-ylideneamino)-2-thioxo-2H-1,3-thiazin-4(3H)-one (HOTT), B) digitalizing the color intensity of the assay spots by flatbed scanning and scoring the pixel gray volumes 21,22 (PGV) using image analysis software, and C) demonstrating the application of the developed method for detection of the tested metal ions in water samples.…”