Detection and quantification of human herpes viruses

A method for detecting infection by HHV1 in a sample from an individual suspected of having HHV1 infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV1 tk gene comprising SEQ ID NO:
• 48, using primers and a probe having sequences set forth in SEQ ID NOS:
• 1, 2, and 3;
• and
• (b) interpolating the number of HHV1 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV2 in a sample from an individual suspected of having HHV2 infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV2 tk gene comprising SEQ ID NO:
• 49, using primers and a probe having sequences set forth in SEQ ID NOS:
• 4, 5, and 6;
• and
• (b) interpolating the number of HHV2 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by drug resistant HHV2 in a sample from an individual suspected of having drug resistant HHV2 infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of a drug resistant HHV2 tk gene comprising SEQ ID NO:
• 50, using primers and a probe having sequences set forth in SEQ ID NOS:
• 7, 8, and 9;
• and
• (b) interpolating the number of drug resistant HHV2 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV3 in a sample from an individual suspected of having HHV3 infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV3 tk gene comprising SEQ ID NO:
• 52, using primers and a probe having sequences set forth in SEQ ID NOS:
• 13, 14, and 15;
• and
• (b) interpolating the number of HHV3 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV5 in a sample from an individual suspected of having HHV5 infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV5 intermediate early gene comprising SEQ ID NO:
• 56, using primers and a probe having sequences set forth in SEQ ID NOS:
• 25, 26, and 27;
• and
• (b) interpolating the number of HHV5 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV7 in a sample from an individual suspected of having HHV7 infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV7 glycoprotein B gene comprising SEQ ID NO:
• 60, using primers and a probe having sequences set forth in SEQ ID NOS:
• 42, 43, and 44;
• and
• (b) interpolating the number of HHV7 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV8 in a sample from an individual suspected of having HHV8 infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV8 K1 gene comprising SEQ ID NO:
• 61, using primers and a probe having sequences set forth in SEQ ID NOS:
• 45, 46, and 47;
• and
• (b) interpolating the number of HHV8 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV4a in a sample from an individual suspected of having HHV4a infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV4a EBNA gene comprising SEQ ID NO:
• 54, using primers and a probe having sequences set forth in SEQ ID NOS:
• 19, 20, and 21;
• and
• (b) interpolating the number of HHV4a viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV4b in a sample from an individual suspected of having HHV4b infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV4b EBNA gene comprising SEQ ID NO:
• 55, using primers and a probe having sequences set forth in SEQ ID NOS:
• 22, 23, and 24;
• and
• (b) interpolating the number of HHV4b viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV6a in a sample from an individual suspected of having HHV6a infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV6a intermediate early gene comprising SEQ ID NO:
• 59, using primers and a probe having sequences set forth in SEQ ID NOS:
• 36, 37, and 38;
• and
• (b) interpolating the number of HHV6a viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by HHV6b in a sample from an individual suspected of having HHV6b infection, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV6b intermediate early gene comprising SEQ ID NO:
• 58, using primers and a probe having sequences set forth in SEQ ID NOS:
• 39, 40, and 41;
• and
• (b) interpolating the number of HHV6b viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by either drug resistant HHV1 or drug resistant HHV2 in a sample from an individual suspected of being infected with either drug resistant HHV1 or drug resistant HHV2, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of a drug resistant HHV1 or a drug resistant HHV2 tk gene comprising SEQ ID NO:
• 51, using primers and a probe having sequences set forth in SEQ ID NOS:
• 10, 11, and 12;
• and
• (b) interpolating the number of either drug resistant HHV1 or drug resistant HHV2 viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by either HHV4a or HHV4b in a sample from an individual suspected of being infected with either HHV4a or HHV4b, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV4 LMP-1 gene comprising SEQ ID NO:
• 53, using primers and a probe having sequences set forth in SEQ ID NOS:
• 16, 17, and 18;
• and
• (b) interpolating the number of either HHV4a or HHV4b viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for detecting infection by either HHV6a or HHV6b in a sample from an individual suspected of being infected with either HHV6a or HHV6b, comprising:

• (a) amplifying, in the course of a single amplification reaction, a target segment of an HHV6 glycoprotein B gene comprising SEQ ID NO:
• 57, using primers and a probe having sequences set forth in SEQ ID NOS:
• 33, 34, and 35;
• and
• (b) interpolating the number of either HHV6a or HHV6b viruses in the sample by comparing the number of amplification cycles required for detection of the target segment to the number of amplification cycles required to detect a known quantity of the target segment.

A method for cloning a segment of genomic HHV viral DNA, comprising:

• (a) selecting a candidate HHV gene, wherein expression of the candidate gene is predictive of active HHV infection;
• (b) performing an informatics analysis of existing gene sequences from different HHV types or strains to identify a target segment within the gene;
• (c) selecting a probe oligonucleotide and at least two primer oligonucleotides capable of selectively directing the amplification, in the course of a single amplification reaction, of the target segment of the particular HHV type or strain;
• (d) using said probe oligonucleotide and at least two primer oligonucleotides to amplify the target segment from isolated genomic DNA from the particular HHV type or strain;
• and
• (e) inserting the amplified target segment into a vector.

A fluorogenic probe that comprises:

• (i) a sequence selected from the group consisting of SEQ ID NOS:
• 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 31, 32, 35, 38, 41, 44, and 47;
• (ii) a fluorescent reporter moiety covalently attached to the probe;
• and (iii) a fluorescence quencher moiety covalently attached to the probe.