Multiplex RT-PCR/PCR for simultaneous detection of bovine coronavirus, bovine rotavirus, Cryptosporidium parvum, and Escherichia coli

Abstract

The present invention provides a multiplex RT-PCR/PCR method, which enables in a single assay the simultaneous detection of any combination of bovine rotavirus, bovine coronavirus, Cryptosporidium parvum , and optionally, Escherichia coli strains producing K99 pili or heat-stable enterotoxin STa.

1 - 11 . (canceled)  
     
     
         12 . A kit for simultaneous screening of a fecal sample for at least two pathogens selected from the group consisting of bovine coronavirus, bovine rotavirus, and  Cryptosporidium parvum  comprising in three or more containers: 
 (a) a first oligonucleotide primer pair which hybridizes to opposite strands of a target nucleic acid sequence of  Cryptosporidium parvum;      (b) a second oligonucleotide primer pair which hybridizes to opposite strands of a target nucleic acid sequence of bovine coronavirus; and    (c) a third oligonucleotide primer pair which hybridizes to opposite strands of a target nucleic acid sequence of bovine rotavirus, wherein each primer pair flanks its target nucleic acid sequence for PCR amplification of the target nucleic acid sequence.    
     
     
         13 . The kit of  claim 12  wherein the first primer pair includes an upstream primer with the nucleic acid sequence in SEQ ID NO:1 and a downstream primer with the nucleic acid sequence in SEQ ID NO:2, the second primer pair includes an upstream primer with the nucleic acid sequence in SEQ ID NO:3 and a downstream primer with the sequence in SEQ ID NO:4, and the third primer pair includes an upstream primer with the nucleic acid sequence in SEQ ID NO:5 and a downstream primer with the nucleic acid sequence in SEQ ID NO:6.  
     
     
         14 . The kit of  claim 12  which further comprises in one or more containers at least one of an optimized buffer for the reaction, a control nucleic acid comprising the target nucleic acid sequence, a mixture of deoxynucleotide triphosphates comprising adenosine deoxynucleotide triphosphate, guanosine deoxynucleotide triphosphate, thymidine deoxynucleotide triphosphate, cytosine deoxynucleotide triphosphate, and nucleotide analogs thereof, a thermostable DNA polymerase, and a reverse transcriptase.  
     
     
         15 . The kit of  claim 12  wherein the kit further includes at least one additional oligonucleotide primer pair, which hybridizes to opposite strands of a target nucleic acid sequence of  Escherichia coli , selected from the group of primer pairs consisting of an upstream primer that includes the nucleic acid sequence in SEQ ID NO:7 and a downstream primer that includes the nucleic acid sequence in SEQ ID NO:8 for detecting a target sequence encoding K99 pili, and an upstream primer that includes the nucleic acid sequence in SEQ ID NO:9 and a downstream primer that includes the nucleic acid sequence in SEQ ID NO:10 for detecting a target encoding heat-stable enterotoxin STa.  
     
     
         16 . The kit of  claim 12  or  15  which further includes a container comprising immunomagnetic beads which enable immunomagnetic separation of bovine coronavirus, bovine rotavirus,  Cryptosporidium parvum , and  Escherichia coli  from the fecal sample.  
     
     
         17 . The kit of  claim 12  that further includes one or more probes wherein each probe is complementary to a sequence within the target sequence of one of the pathogens.  
     
     
         18 . The kit of  claim 17  wherein the probes are labeled at its 5′ end with an energy transfer donor fluorophore and labeled at its 3′ end with an energy transfer acceptor fluorophore.  
     
     
         19 . The kit of  claim 18  wherein the fluorophore is selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′, 5′-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL), tetrachloro-6-carboxy-fluorescein (TET), VIC, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).  
     
     
         20 . The method of  claim 17  wherein the probes are blocked against chain extension at its 3′ end.  
     
     
         21 . A primer pair comprising an upstream primer having the nucleic acid sequence in SEQ ID NO: 3 and a downstream primer having the nucleic acid sequence in SEQ ID NO:4.  
     
     
         22 . A primer pair comprising an upstream primer having the nucleic acid sequence in SEQ ID NO: 3 and a downstream primer having the nucleic acid sequence in SEQ ID NO:4.