Materials and methods for detection of enterovirus and norovirus
The invention provides polynucleotides and methods for detecting and quantifying RNA viruses, such as enteroviruses and noroviruses. In one aspect, the invention provides amplification primers and labeled molecular beacons for amplification of viral nucleic acid sequences. In another aspect, the invention provides a synthetic RNA internal control. In another aspect, the invention provides a kit for detecting the presence of enterovirus and/or norovirus in a sample.
1. An isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 5.
2. A method for detecting the presence of an Enterovirus in a sample, comprising contacting a polynucleotide region of the 5′-untranslated region of the Enterovirus genome with amplification primers; amplifying the polynucleotide region of the 5′-untranslated region and detecting the presence of an amplified polynucleotide using a labeled molecular beacon comprising the nucleic acid sequence of SEQ ID NO: 5, where the presence of the amplified product is indicative of the presence of the Enterovirus in the sample.
3. The method of claim 2 , wherein the amplification primers are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4, or a fragment of any of the foregoing.
4. The method of claim 2 , wherein said amplifying comprises nucleic acid sequence based amplification (NASBA).
5. The method of claim 2 , wherein said amplifying comprises RT-PCR.
6. The method of claim 2 , wherein said detecting comprises hybridization of the amplified product with a probe.
7. The method of claim 6 , wherein said detecting comprising serotyping the Enterovirus with a probe specific for an Enterovirus serotype.
8. The method of claim 2 , wherein the sample is a biological sample.
9. The method of claim 2 , wherein the sample is an environmental sample.
10. The method of claim 2 , wherein the Enterovirus is selected from the group consisting of Polio 1, 2, 3; Coxsackie A 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24; Coxsackie B 1, 2, 3, 4, 5, 6; ECHO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34; Entero 68, 69, 70, 71; and Entero 72 (Hepatitis A).
11. The method of claim 2 , wherein said method further comprises determining the quantity of Enterovirus within the sample.
12. The polynucleotide of claim 1 wherein the polynucleotide is labeled.
13. The polynucleotide of claim 1 wherein the polynucleotide is labeled with 6-carboxyfluorescein and 4-(4′-dimethylaminophenylazo)-benzoic acid.
14. The method of claim 2 wherein the molecular beacon is labeled with 6-carboxyfluorescein and 4-(4′-dimethylaminophenylazo)-benzoic acid.