Compositions and methods for the selective detection of tumor-derived viral DNA
The present disclosure provides methods and compositions of modified oligonucleotide primer and probe combinations, structurally modified with locked nucleic acids, quenchers, and dyes, effective to detect tumor-derived Human Papilloma Virus (HPV) and tumor-derived Epstein-Barr virus (EBV) and, especially, to distinguish viral DNA derived from tumors from viral DNA derived from infectious viral particles.
1 . A composition for detecting tumor-derived Human Papilloma Virus type 31 (HPV31) in a sample from a subject, comprising at least a triad of modified oligonucleotide primer and probe sets selected from the group consisting of:
Set 1: SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136,
Set 2: SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139,
Set 3: SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142,
Set 4: SEQ ID NO: 143, SEQ ID NO: 144, and SEQ ID NO: 145,
Set 5: SEQ ID NO: 146, SEQ ID NO: 147, and SEQ ID NO: 148,
Set 6: SEQ ID NO: 149, SEQ ID NO: 150, and SEQ ID NO: 151,
Set 7: SEQ ID NO: 152, SEQ ID NO: 153, and SEQ ID NO: 154,
Set 8: SEQ ID NO: 155, SEQ ID NO: 156, and SEQ ID NO: 157,
Set 9: SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160,
Set 10: SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163,
Set 11: SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166,
Set 12: SEQ ID NO: 167, SEQ ID NO: 168, and SEQ ID NO: 169,
Set 13: SEQ ID NO: 170, SEQ ID NO: 171, and SEQ ID NO: 172,
Set 14: SEQ ID NO: 173, SEQ ID NO: 174, and SEQ ID NO: 175,
Set 15: SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178,
Set 16: SEQ ID NO: 179, SEQ ID NO: 180, and SEQ ID NO: 181, and
Set 17: SEQ ID NO: 182, SEQ ID NO: 183, and SEQ ID NO: 184,
wherein the first primer and probe set of the triad is configured to produce a first amplicon signal,
wherein the second primer and probe set of the triad is configured to produce a second amplicon signal,
wherein the third primer and probe set of the triad is configured to produce a third amplicon signal, and
wherein the primer and probe set of the triad with the smallest set number corresponds to the first primer and probe set, and the primer and probe set of the triad with the largest set number corresponds to the third primer and probe set.
2 . The composition of claim 1 , wherein the triad contains three primer and probes sets of which no two primer and probe sets have consecutive set numbers.
3 . The composition of claim 1 , wherein the triad contains three non-consecutive numbered primer and probe sets.
4 . The composition of claim 1 , comprising primer and probe sets numbered 4, 7, and 10.
5 . A method for detecting tumor-derived Human Papilloma Virus type 31 (HPV31) in a sample from a subject, the method comprising:
providing at least a triad of modified oligonucleotide primer and probe sets comprising the composition of any one of claim 1 ;
fractionating a plurality of HPV DNA fragments from the sample into droplets at a concentration wherein only 0 or 1 molecule of the DNA fragments is present in each droplet;
amplifying HPV DNA in each droplet with the triad of primer and probe sets to produce amplicon signals;
detecting in each droplet any amplicon signals;
wherein detection within a droplet of the second amplicon, but not the first or third amplicon, indicates that the HPV DNA fragment fractionated into the droplet is a tumor-derived HPV DNA fragment.