US 12,270,076 B2
Method for identifying mastitis-causing microbes
Michael E. Hogan, Stony Brook, NY (US); Frederick H. Eggers, Sahuarita, AZ (US); and Melissa R. May, Tucson, AZ (US)
Filed by Michael E. Hogan, Stony Brook, NY (US); Frederick H. Eggers, Sahuarita, AZ (US); and Melissa R. May, Tucson, AZ (US)
Filed on Nov. 2, 2020, as Appl. No. 17/087,099.
Claims priority of provisional application 62/928,899, filed on Oct. 31, 2019.
Prior Publication US 2021/0130898 A1, May 6, 2021
Int. Cl. C12Q 1/68 (2018.01); C12N 1/14 (2006.01); C12N 1/20 (2006.01); C12Q 1/6832 (2018.01); C12Q 1/6834 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6888 (2018.01); C12N 1/12 (2006.01)
CPC C12Q 1/6874 (2013.01) [C12N 1/14 (2013.01); C12N 1/20 (2013.01); C12Q 1/6832 (2013.01); C12Q 1/6834 (2013.01); C12Q 1/6888 (2013.01); C12N 1/12 (2013.01); C12Q 2600/124 (2013.01)] 8 Claims
 
1. A method for diagnosing a bovine mastitis infection in a dairy cow, comprising the steps of:
obtaining a raw milk sample from the dairy cow;
emulsifying a fat complement of the raw milk sample;
centrifuging the milk sample twice to separate a microbial pellet from the raw milk sample;
isolating total nucleic acids from the pellet comprising DNA and non-DNA nucleic acids;
amplifying in a first PCR reaction unpurified microbial DNA in the total nucleic acids with at least one primer pair selective for at least one bovine mastitis-causing microbe-specific DNA to yield at least one bovine mastitis-causing microbe-specific amplicons; said bovine mastitis-causing microbe-specific DNA comprises bovine mastitis-causing bacterial DNA, bovine mastitis-causing fungal DNA or a combination thereof;
amplifying in a second PCR reaction the bovine mastitis-causing microbe-specific amplicons as templates with at least one fluorescent labeled primer pair to yield fluorescent-labeled amplicons;
hybridizing the fluorescent-labeled amplicons with mastitis-causing microbe species-specific gene probes selected from the group consisting of mastitis-causing bacterial oligonucleotide probes selected from the group consisting of SEQ ID NOS: 37-53, mastitis-causing fungal oligonucleotide probes selected from the group consisting of SEQ ID NOS: 54-61 and a combination thereof, said mastitis-causing microbe species-specific gene probes immobilized at specific known positions on the microarray;
imaging the microarray to detect fluorescent signals from the hybridized fluorescent-labeled amplicons at the specific known positions; and
correlating the position of the fluorescent signals on the microarray to a presence of the specific bovine mastitis-causing microbe in the raw milk, thereby diagnosing the bovine mastitis infection in the dairy cow.