A novel method of HPV genotyping using Hybrid Capture sample preparation method combined with GP5+/6+ PCR and multiplex detection on Luminex XMAP.
A novel DNA detection assay comprising Hybrid Capture sample preparation, GP5+/6+ PCR with modifications and Luminex 100 detection was developed and applied to genotyping of human papillomavirus (HPV) in cervical samples. Target-specific sample preparation was performed using magnetic beads conjugated with Hybrid Capture (HC) antibody. DNA-RNA hybrids were formed between DNA target and RNA probes and captured on HC-beads. DNA on magnetic beads was amplified without elution using consensus GP5+/6+ PCR and then genotyped on Luminex beads using hybridization probes for the 17 high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 and an internal control. This new sequence-specific Hybrid Capture sample preparation is fast, efficient and allows direct HPV genotyping by PCR. Compared to traditional non-sequence-specific sample preparation methods, HC sample preparation demonstrated slightly better detection of multiple HPV infections. The clinical utility of this method was demonstrated on cervical samples positive for HR HPV by the Hybrid Capture 2 (hc2) screening assay.
QIAGEN Gaithersburg, Inc., 1201 Clopper Road, Gaithersburg, MD 20878, USA. irina.nazarenko@qiagen.com
I
I
Nazarenko
L
L
Kobayashi
J
J
Giles
C
C
Fishman
G
G
Chen
A
A
Lorincz
D002584:Cervix Uteri / Q000821:virology; D005260:Female; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015347:RNA Probes / Q000235:genetics
D015345:Oligonucleotide Probes; D015347:RNA Probes