Patent ID: 6867021
Filing Date: 2005-03-15
Classification: C12Q

Abstract:
1. A method of screening to simultaneously detect Cryptosporidium parvum , bovine coronavirus and bovine rotavirus in a calf fecal sample, the method comprising: (a) providing a calf fecal sample; (b) performing a freeze-thaw procedure for the disruption of (c) performing a procedure for isolating nucleic acids consisting of single-stranded RNA of the bovine coronavirus, double-stranded RNA of the bovine rotavirus and DNA of the (d) providing in an RT-PCR/PCR reaction mixture the isolate, a first oligonucleotide primer pair consisting of an upstream primer of SEQ ID NO: 1 and a downstream primer of SEQ ID NO: 2, which anneals to a first target nucleic acid sequence of (e) denaturing the double-stranded RNA of the bovine rotavirus to provide denatured double-stranded RNA; (f) providing a thermostable DNA polymerase, and a reverse transcriptase; (g) synthesizing bovine coronavirus cDNA from the single-stranded RNA of the bovine coronavirus, and bovine rotavirus cDNA from the denatured double-stranded RNA of the bovine rotavirus under suitable reverse transcription reaction conditions with the deoxynucleotide triphosphates and reverse transcriptase; (h) amplifying by a PCR reaction the first target nucleic acid from the DNA of the (i) following amplification, screening for the first, second, and third target nucleic acids in the amplified PCR products so as to simultaneously detect the