Quantitative epstein barr virus PCR rapid assay
First Claim
1. An isolated and purified oligonucleotide primer pair for enzymatic amplification of Epstein-Barr virus DNA, consisting essentially of a pair of nucleic acid sequences which complement and specifically hybridize to a nucleic acid sequence of an Epstein-Barr virus under stringent conditions, which encodes a conserved EBNA2 region of the Epstein-Barr virus genome, wherein the pair of nucleic acid sequences is at least 95% homologous to sequences of SEQ ID NO:
- 1 and SEQ ID NO;
2, respectively.
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Abstract
The present invention provides novel compositions comprising Epstein-Barr virus-specific oligonucleotides that are useful as primers to amplify particular regions of the genome during enzymatic nucleic acid amplification. The invention also provides a rapid, sensitive and specific method for the detection and quantitation of the virus which may be present in a clinical specimen, using the virus-specific primers and enzymatic nucleic acid amplification; hybridization of amplified target sequences, if present, with one or more Epstein-Barr virus-specific oligonucleotide probes which are labeled with a detectable moiety; and detection of the detectable moiety of labeled oligonucleotide probe hybridized to amplified target sequences of Epstein-Barr virus DNA.
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Citations
25 Claims
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1. An isolated and purified oligonucleotide primer pair for enzymatic amplification of Epstein-Barr virus DNA, consisting essentially of a pair of nucleic acid sequences which complement and specifically hybridize to a nucleic acid sequence of an Epstein-Barr virus under stringent conditions, which encodes a conserved EBNA2 region of the Epstein-Barr virus genome, wherein the pair of nucleic acid sequences is at least 95% homologous to sequences of SEQ ID NO:
- 1 and SEQ ID NO;
2, respectively.
- 1 and SEQ ID NO;
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2. An isolated and purified oligonucleotide primer pair for enzymatic amplification of Epstein-Barr virus DNA, consisting essentially of a pair of nucleic acid sequences which complement and specifically hybridize to a chromosomal gene of an Epstein-Barr virus under stringent conditions, and which encodes a conserved EBNA2 region of the Epstein-Barr virus genome, wherein the pair of nucleic acid sequences is selected from the group consisting of (a) the oligonucleotide pair of SEQ ID NO:
- 1 and SEQ ID NO;
2, and (b) a primer pair, which differs from SEQ ID NO;
1 and SEQ ID NO;
2 by a one base change or substitution therein, and (d).
- 1 and SEQ ID NO;
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3. A method of detecting the presence of Epstein Barr virus DNA in a sample comprising:
- a) contacting the sample with oligonucleotide primer pair of SEQ ID NO. 1 and SEQ ID NO;
2 under suitable conditions permitting hybridization of the oligonucleotides to the Epstein Barr virus DNA, b) enzymatically amplifying a region of the Epstein Barr virus DNA using the oligonucleotide pair SEQ ID NO;
1 and SEQ ID NO;
2 to form nucleic acid amplification products, c) contacting the amplified Epstein Barr virus DNA sequences from step (b), if present, with hybridization probes comprising the oligonucleotide pair of SEQ. ID. NO. 3 and SEQ. ID. NO. 4 or the oligonucleotide pair of SEQ. ID. NO. 7 and SEQ. ID. NO. 8, labeled with a detectable moiety under suitable conditions permitting hybridization of the labeled oligonucleotide probe to amplified Epstein Barr virus DNA sequences, and d) detecting the presence of amplified Epstein Barr virus DNA sequences by detecting the detectable moiety of the labeled oligonucleotide probe hybridized to the amplified Epstein Barr virus DNA sequences. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20)
- a) contacting the sample with oligonucleotide primer pair of SEQ ID NO. 1 and SEQ ID NO;
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16. A method of selecting an appropriate dosage or type of antiviral agent for treating an infection caused by Epstein Barr virus comprising the steps of:
- a) obtaining a sample from a patient to be treated;
b) preparing the sample for PCR amplification;
c) adding PCR reagents to the prepared sample, including one primer pair SEQ ID NO;
1 and SEQ ID NO;
2 and complementary sequences thereof;
d) maintaining the prepared sample of step c under conditions suitable for amplification;
e) adding at least one probe labeled with a detectable moiety corresponding to the primer pair, selected from the group consisting of the oligonucleotide pair of SEQ. ID. NO. 3 and SEQ. ID. NO. 4, the oligonucleotide pair of SEQ. ID. NO. 7 and SEQ. ID. NO. 8, and complementary sequences thereof, under suitable conditions permitting hybridization;
f) measuring quantitatively one or more of the Epstein Barr virus species contained in the sample; and
vii) selecting the type or adjusting the dosage of the antiviral agent based on the quantitative measurement. - View Dependent Claims (17, 18, 19)
- a) obtaining a sample from a patient to be treated;
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21. A method for the simultaneous amplification and detection of Epstein Barr Virus DNA in a sample comprising:
- a) processing the sample to produce denatured opposing strands of DNA;
b) simultaneously subjecting the denatured opposing strands of DNA to polymerase chain reaction in the presence of;
i) an aqueous solution buffered to a pH of about 6 to about 9; and
ii) first and second primers which are specific to and hybridizable with the denatured opposing strands of DNA, wherein the sequences of the first and second primers are SEQ ID NO;
1 and SEQ ID NO;
2 and c) simultaneously detecting the amplified DNA using hybridization probes comprising the oligonucleotide pair of SEQ. ID. NO. 3 and SEQ. ID. NO. 4 or the oligonucleotide pair of SEQ. ID. NO. 7 and SEQ. ID. NO. 8. - View Dependent Claims (22)
- a) processing the sample to produce denatured opposing strands of DNA;
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23. A diagnostic test kit for detection of Epstein Barr virus comprising:
- (a) at least one oligonucleotide primer pair SEQ ID NO;
1 and SEQ ID NO;
2 and both the oligonucleotide pair and (b) at least one oligonucleotide probe labeled with a detectable moiety selected from the group consisting SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 7 and SEQ. ID. NO. 8. - View Dependent Claims (24, 25)
- (a) at least one oligonucleotide primer pair SEQ ID NO;
Specification