Development of a Novel Biochip for Rapid Multiplex Detection of Seven Mastitis-Causing Pathogens in Bovine Milk Samples

Abstract

Abstract. To efficiently prevent and treat bovine mastitis and minimize its effect on the dairy industry, a sensitive, rapid, and specific test is required for identifying the mastitis-causing pathogens. In this study, a biochip capable of detecting 7 common species of mastitis-causing pathogens, including Corynebacterium bovis, Mycoplasma bovis, Staphylococcus aureus, and the Streptococcus spp. S. agalactiae, S. bovis, S. dysgalactiae, and S. uberis, within 6 hr was developed. The technique is based on DNA amplification of genes specific to the target pathogens and consists of 4 basic steps: DNA extraction of bacteria, polymerase chain reaction, DNA hybridization, and colorimetric reaction. To examine the accuracy and specificity of this biochip, a preliminary test with 82 random quarter milk samples were analyzed and compared with results from conventional microbiological methods conducted simultaneously. Results from all but 1 sample analyzed by the biochip were in agreement with those analyzed by bacteriology. The biochip could be a feasible tool for rapidly diagnosing mastitis-causing pathogens in milk and providing information for a more effective treatment to cure mastitis.

Development of a microarray for simultaneous detection and differentiation of different tospoviruses that are serologically related to Tomato spotted wilt virus

“…It also provides an advantage of customized probe spotting. The reliability of the PVC biochip has been proven in several applications; e.g., the simultaneous detection and differentiation of seven mastitis-causing pathogens in bovine milk samples [37], Newcastle disease and avian influenza in the poultry industry [42], and Cymbidium mosaic virus , Odontoglossum ringspot virus and CaCV for orchid inspection (Dr. Orchid-3™ Kit, Dr. Chip), and the identification of insect species [43] and the sex of owls [44]. …”

“…Twenty micromolar of each probe were spotted on the surface of PVC microchips with an automatic spotting machine (DR. Chip Biotechnology Inc., Miaoli, Taiwan) and immobilized by an ultraviolet crosslinker (UV Light Enterprise Co., Taichung, Taiwan) with 1.2 J for 5 min following the previous report [37]. In addition to the spots for specific tospoviruses, 100 μM of the commercial probe Anti-VP1 (Dr. Chip) was spotted for positive control of hybridization.…”

Gene Technology: Applications and Techniques

“…Song et al (2005) reported a portable biochip system based on complementary metal oxide semiconductor (CMOS) technology that has great potential as a device for single bacteria detection. Lee et al (2008) reported a biochip, which could be a feasible tool for rapidly diagnosing mastitis causing pathogens in milk and providing information for a more effective treatment to cure mastitis. Bacillus globigii spores, which are a surrogate species for B. anthracis spores, were used as the test sample.…”

Identification of pathogens in mastitis milk samples with fluorescent in situ hybridization

“…17 Various culture-independent methods have been published for the rapid detection of mastitis pathogens in milk samples, as well as various polymerase chain reaction (PCR) approaches including real-time PCR methods. 6,15,16 A culture-independent molecular method allowing the direct detection of pathogens in clinical samples is fluorescent in situ hybridization (FISH). 24,25,29 Fluorescence-labeled oligonucleotide probes bind specific regions of the target RNA/DNA.…”