Quantitative Real-Time Assay for Noroviruses and Enteroviruses with Built in Quality Control Standard

Abstract

A method is provided for reverse transcription-polymerase chain reaction (RT-PCR) comprising a) amplifying a reverse transcribed cDNA in a mixture comprising Norovirus Genogroup I and Norovirus Genogroup II primers and probes, wherein said Norovirus primers and probes distinguish between Genogroup I and Genogroup II viruses; b) quantifying virus; and c) normalizing data based on a universal internal RNA control. Optionally, the method may also include primers and probes for Enteroviruses. A reaction mixture comprising Norovirus Genogroup I and Norovirus Genogroup II primers and probes, wherein said Norovirus primers and probes distinguish between Genogroup I and Genogroup II viruses and universal internal RNA control primers and probes are also included.

1 . A method of reverse transcription-polymerase chain reaction (RT-PCR) comprising:
 a) amplifying a reverse transcribed cDNA in a mixture comprising
 i) an oligonucleotide probe and primers specific for Norovirus Genogroup I; and 
 ii) an oligonucleotide probe and primers specific for Norovirus Genogroup II; 
   b) quantifying Norovirus Genogroup I and Norovirus Genogroup II virus; and   c) normalizing data to amplification of a universal internal RNA control in the same reaction mixture.   
     
     
         2 . The method of RT-PCR of  claim 1 , wherein the primers specific for Norovirus Genogroup I comprise SEQ ID NO: 1 and SEQ ID NO:2. 
     
     
         3 . The method of RT-PCR of  claim 1 , wherein the primers specific for Norovirus Genogroup II comprise SEQ ID NO: 5 and SEQ ID NO:6. 
     
     
         4 . The method of RT-PCR of  claim 1 , wherein the oligonucleotide probe specific for Norovirus Genogroup I comprise SEQ ID NO: 3 and SEQ ID NO:4. 
     
     
         5 . The method of RT-PCR of  claim 1 , wherein the oligonucleotide probe specific for Norovirus Genogroup II comprises SEQ ID NO:7. 
     
     
         6 . The method of RT-PCR of  claim 1 , wherein the universal internal RNA control primers comprise SEQ ID NO:11 and SEQ ID NO:12. 
     
     
         7 . The method of RT-PCR of  claim 1 , wherein the universal internal RNA control oligonucleotide probe comprises SEQ ID NO: 13. 
     
     
         8 . The method of RT-PCR of  claim 1 , wherein the RT-PCR reaction mixture further comprises an oligonucleotide probe and primers specific for a RNA virus. 
     
     
         9 . The method of RT-PCR of  claim 8 , wherein the RNA virus comprises rotavirus or a positive strand RNA virus. 
     
     
         10 . (canceled) 
     
     
         11 . The method of RT-PCR of  claim 9 , wherein the positive strand RNA virus comprises Hepatitis A virus or an Enterovirus group virus. 
     
     
         12 . The method of RT-PCR of  claim 11 , wherein the Enterovirus group virus comprises poliovirus, coxsackievirus, echovirus, or enterovirus. 
     
     
         13 . The method of RT-PCR of  claim 12 , wherein the coxsackievirus comprises coxsackievirus Group A or coxsackievirus Group B. 
     
     
         14 . The method of RT-PCR of  claim 1 , wherein said cDNA comprises reverse transcribed from RNA in a biological sample. 
     
     
         15 . The method of RT-PCR of  claim 14 , wherein said biological sample comprises blood, urine, or a stool sample. 
     
     
         16 . The method of RT-PCR of  claim 1 , wherein said cDNA comprises reverse transcribed from RNA in a food sample. 
     
     
         17 . The method of RT-PCR of  claim 16 , wherein said food sample comprises shellfish. 
     
     
         18 . The method of RT-PCR of  claim 1 , wherein said cDNA comprises reverse transcribed from RNA in a water sample. 
     
     
         19 . The method of RT-PCR of  claim 18 , wherein said water sample comprises wastewater, ocean water, lake water, river water, groundwater, or recreational water. 
     
     
         20 . A reverse transcription polymerase chain reaction (RT-PCR) mixture comprising:
 a) an oligonucleotide probe and primers specific for Norovirus Genogroup I;   b) an oligonucleotide probe and primers specific for Norovirus Genogroup II;   c) a universal internal nucleic acid molecule, wherein said molecule contains at least one forward primer annealing site, at least one reverse primer annealing site, and at least one amplifiable region;   d) a universal internal control oligonucleotide probe; and   e) a universal internal control primer.   
     
     
         21 - 33 . (canceled) 
     
     
         34 . A RT-PCR kit comprising:
 a) an oligonucleotide probe and primers specific for Norovirus Genogroup I;   b) an oligonucleotide probe and primers specific for Norovirus Genogroup II;   c) a universal internal nucleic acid molecule, wherein said molecule contains at least one forward primer annealing site, at least one reverse primer annealing site, and at least on amplifiable region;   d) a universal internal RNA control probe; and   e) a universal internal RNA control primer in a package.   
     
     
         35 . (canceled)