Patent ID: 6867021

Claim:
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 Cryptosporidium parvum oocysts; (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 Cryptosporidium parvum from the calf fecal sample to provide an isolate; (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 Cryptosporidium parvum , a second oligonucleotide primer pair consisting of an upstream primer of SEQ ID NO: 3 and a downstream primer of SEQ ID NO: 4, which anneals to a second target nucleic acid sequence of bovine coronavirus, and third oligonucleotide primer pair consisting of an upstream primer of SEQ ID NO: 5 and a downstream primer of SEQ ID NO: 6, which anneals to a third target nucleic acid sequence of bovine rotavirus, wherein each primer pair flanks its respective target nucleic acid sequence for PCR amplification of the target nucleic acid, and four deoxynucleotide triphosphates selected from the group consisting of adenosine deoxynucleotide triphosphate, guanosine deoxynucleotide triphosphate, thymidine deoxynucleotide triphosphate, cytosine deoxynucleotide triphosphate, and nucleotide analogs thereof; (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 Cryptosporidium parvum , the second target nucleic acid from the bovine coronavirus cDNA, and the third target nucleic acid from the bovine rotavirus cDNA in the reaction mixture under suitable PCR reaction mixture temperature conditions by a repetitive series of PCR thermal cycling steps comprising: (1) denaturing the DNA and cDNA into denatured strands; (2) annealing the oligonucleotide primers provided in step (d) to the denatured strands of the DNA and cDNA, and (3) extending the hybridized primers with the four deoxynucleotide triphosphates and the nucleic acid polymerase to provide amplified PCR products; and (i) following amplification, screening for the first, second, and third target nucleic acids in the amplified PCR products so as to simultaneously detect the Cryptosporidium parvum , bovine coronavirus and bovine rotavirus, respectively, in the calf fecal sample.