Detection of Enterovirus

• US 20110045458A1
• Filed: 08/20/2010
• Published: 02/24/2011
• Est. Priority Date: 08/20/2009
• Status: Abandoned Application

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Inventors: Smith, Thomas F., Espy, Mark J.
• Application Number:
  US12/859,917
• Publication Number:
  US 20110045458A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

This document describes methods and materials relating to viral diagnostics, and more particularly to the detection of enterovirus. For example, primers and probes for the detection of enterovirus are provided. Articles of manufacture containing such primers and probes for detecting enterovirus are further provided.

Claims

1. A method for detecting the presence or absence of enterovirus in a biological sample from an individual, said method comprising:

performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step, wherein said amplifying step comprises contacting said sample with a pair of enterovirus primers to produce an enterovirus amplification product if an enterovirus nucleic acid molecule is present in said sample, wherein said pair of enterovirus primers comprises a first enterovirus primer and a second enterovirus primer, wherein said first enterovirus primer comprises the sequence 5′

-CCG GCC CCT GAA TG-3′

(SEQ ID NO;

1), and wherein said second enterovirus primer comprises the sequence 5′

-CAC CGG ATG GCC AAT-3′

(SEQ ID NO;

2), wherein said hybridizing step comprises contacting said sample with

(a) a pair of enterovirus probes, wherein the members of said pair of enterovirus probes hybridize to said enterovirus amplification product within no more than five nucleotides of each other, wherein a first enterovirus probe of said pair of enterovirus probes is labeled with a donor fluorescent moiety and said second enterovirus probe of said pair of enterovirus probes is labeled with a corresponding acceptor fluorescent moiety, wherein said first enterovirus probe comprises the sequence 5′

-GGG CAA CTC TGC AGC GGA ACC GAC-3′

(SEQ ID NO;

3), and wherein said second enterovirus probe comprises the sequence 5′

-TGG GTG ACC GTG TTT CTT TT-3′

(SEQ ID NO;

4);

or

(b) one enterovirus probe, wherein said enterovirus probe comprises the sequence 5′

-GGG CAA CTC TGC AGC GGA ACC GAC-3′

(SEQ ID NO;

3) or the sequence 5′

-TGG GTG ACC GTG TTT CTT TT-3′

(SEQ ID NO;

4), wherein said enterovirus probe is labeled with a first fluorescent moiety and a second fluorescent moiety, wherein said first and second fluorescent moieties are within no more than 5 nucleotides of each other on said enterovirus probe;

and

detecting the presence or absence of fluorescence resonance energy transfer (FRET) between said donor fluorescent moiety of said first enterovirus probe and said acceptor fluorescent moiety of said second enterovirus probe,

wherein the presence of FRET is indicative of the presence of enterovirus in said sample, and wherein the absence of FRET is indicative of the absence of enterovirus in said sample.

2. The method of claim 1, wherein said donor fluorescent moiety is fluorescein.

3. The method of claim 1, wherein said detecting step comprises exciting said sample at a wavelength absorbed by said donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by said corresponding acceptor fluorescent moiety.

4. The method of claim 1, wherein said detecting comprises quantitating said FRET.

5. The method of claim 1, wherein said detecting step is performed after each cycling step.

6. The method of claim 1, further comprising determining the melting temperature between one or both of said enterovirus probe(s) and said enterovirus amplification product, wherein said melting temperature confirms said presence or said absence of said enterovirus.

7. The method of claim 1, wherein said biological sample is cerebrospinal fluid.

8. The method of claim 1, wherein said amplification employs a polymerase enzyme having 5′

to 3′

exonuclease activity.

9. The method of claim 1, wherein said enterovirus probe comprises a nucleic acid sequence that permits secondary structure formation, wherein said secondary structure formation results in spatial proximity between said first and second fluorescent moiety.

10. A method for detecting the presence or absence of enterovirus in a biological sample from an individual, said method comprising:

performing at least one cycling step, wherein a cycling step comprises an amplifying step and a dye-binding step, wherein said amplifying step comprises contacting said sample with a pair of enterovirus primers to produce an enterovirus amplification product if an enterovirus nucleic acid molecule is present in said sample, wherein said dye-binding step comprises contacting said enterovirus amplification product with a double-stranded DNA binding dye; and

detecting the presence or absence of binding of said double-stranded DNA binding dye into said amplification product,

wherein the presence of binding is indicative of the presence of enterovirus in said sample, and wherein the absence of binding is indicative of the absence of enterovirus in said sample.

11. The method of claim 10, further comprising determining the melting temperature between said enterovirus amplification product and said double-stranded DNA binding dye, wherein said melting temperature confirms said presence or absence of said enterovirus.

12. An oligonucleotide having a sequence selected from the group consisting of 5′

-CCG GCC CCT GAA TG-3′

(SEQ ID NO;

1);

5′

-CAC CGG ATG GCC AAT-3′

(SEQ ID NO;

2);

5′

-GGG CAA CTC TGC AGC GGA ACC GAC-3′

(SEQ ID NO;

3); and

5′

-TGG GTG ACC GTG TTT CTT TT-3′

(SEQ ID NO;

4).

14. The article of manufacture of claim 12, wherein said first enterovirus probe is labeled with said donor fluorescent moiety and wherein said second enterovirus probe is labeled with said corresponding acceptor fluorescent moiety.

13. An article of manufacture, comprising:

a pair of enterovirus primers;

a pair of enterovirus probes; and

a donor fluorescent moiety and a corresponding acceptor fluorescent moiety,

wherein said pair of enterovirus primers comprise a first enterovirus primer and a second enterovirus primer, wherein said first enterovirus primer comprises the sequence 5′

-CCG GCC CCT GAA TG-3′

(SEQ ID NO;

1), wherein said second enterovirus primer comprises the sequence 5′

-CAC CGG ATG GCC AAT-3′

(SEQ ID NO;

2), and

wherein said pair of enterovirus probes comprises a first enterovirus probe and a second enterovirus probe, wherein said first enterovirus probe comprises the sequence 5′

-GGG CAA CTC TGC AGC GGA ACC GAC-3′

(SEQ ID NO;

3), and wherein said second enterovirus probe comprises the sequence 5′

-TGG GTG ACC GTG TTT CTT TT-3′

(SEQ ID NO;

4).