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;  
 ii) an oligonucleotide probe and primers specific for Norovirus Genogroup I;  
   b) quantifying 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.  
     
     
         10 . The method of RT-PCR of  claim 8 , wherein the RNA virus comprises a positive strand RNA virus.  
     
     
         11 . The method of RT-PCR of  claim 10 , wherein the positive strand RNA virus comprises Hepatitcomprises 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 9 , 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 11 , 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 13 , 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 15 , 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 I;    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 control oligonucleotide probe; and    e) a universal internal control primers.    
     
     
         21 . The RT-PCR mixture of  claim 17 , wherein said oligonucleotide probe includes a detectable label.  
     
     
         22 . The RT-PCR mixture of  claim 17 , wherein the primers specific for Norovirus Genogroup I comprise SEQ ID NO: 1 and SEQ ID NO:2.  
     
     
         23 . The RT-PCR mixture of  claim 17 , wherein the primers specific for Norovirus Genogroup II comprise SEQ ID NO: 5 and SEQ ID NO:6.  
     
     
         24 . The RT-PCR mixture of  claim 17 , wherein the oligonucleotide probe specific for Norovirus Genogroup I comprise SEQ ID NO: 3 and SEQ ID NO:4.  
     
     
         25 . The RT-PCR mixture of  claim 17 , wherein the oligonucleotide probe specific for Norovirus Genogroup II comprises SEQ ID NO:7.  
     
     
         26 . The RT-PCR mixture of  claim 17 , wherein the universal internal RNA control primers comprise SEQ ID NO:11 and SEQ ID NO:12.  
     
     
         27 . The RT-PCR mixture of  claim 17 , wherein the universal internal RNA control oligonucleotide probe comprises SEQ ID NO: 13.  
     
     
         28 . The RT-PCR mixture of  claim 17 , wherein the RT-PCR mixture further comprises primers and oligonucleotide probes specific for detection of a RNA virus.  
     
     
         29 . The RT-PCR mixture of  claim 28 , wherein the RNA virus comprises rotavirus.  
     
     
         30 . The RT-PCR mixture of  claim 28 , wherein the RNA virus comprises a positive strand RNA virus.  
     
     
         31 . The RT-PCR mixture of  claim 30 , wherein the positive strand RNA virus comprises Hepatitis A virus or an Enterovirus group virus.  
     
     
         32 . The RT-PCR mixture of  claim 31 , wherein the Enterovirus group comprises poliovirus, coxsackievirus, echovirus, or enterovirus.  
     
     
         33 . The RT-PCR mixture of  claim 32 , wherein the coxsackievirus comprises coxsackievirus Group A or coxsackievirus Group B.  
     
     
         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 I;    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 primers in a package.    
     
     
         35 . The kit of  claim 28  wherein the kit further comprises instructions for use.