Establishment Of Consensus-degenerate Hybrid Oligonucleotide Primers (CODEHOP) Method For The Detection Of High-risk Human Papillomaviruses And Development And Application Of Quality Control Materials For Human Papillomavirus Type16, 18 Nucleic Acid Test

1. Development and ap plication of quality control materials for human papillomavirus type16.18 nucleic acid testOBJECTIVETo develop quality control materials for Human papillomavirus (HPV) type 16,18 nucleic acid test and to evaluate the applicability of the materials in external quality assessment (EQA) of HPV- 16,- 18 clinical detection.METHODSThe target gene from HPV-16 was cloned into pEGFP-C1 plasmid, then the recombinant plasmid pEGFP-C1-HPV-16 was transfected into a HPV-18-carrying Hela epithelium cell line. The cultured epithelia cells which carried both HPV-16 target gene and HPV-18 were collected and fixed using methanol. The EQA samples for HPV-16, -18 test survey were prepared from the above prepared cells and distributed to the EQA participants nationwide. The reports from the participants were summarized and evaluated.RESULTSThe quality control materials which was diluted to 1:10, 1:50, 1:100, 1:500 were detected with real-time PCR. The data showed the corresponding CT values were 29.10, 31.19, 32.15 and 32.73 for HPV-16 and 30.32, 32.13, 32.22 and 35.55 for HPV-18, respectively. The stability testing data indicated the quality control materials were stable at least for 40 days when stored at 2-8℃. Meanwhile, for those returned EQA samples which were blindly mailed to Wulumuqi, Huhehaote and Xiamen, no significant changes were detected with real-time PCR, which demonstrated the stability of the quality control materials during the mailing. The EQA data from 44 participants showed that the average corrective ratio for positive samples with concentration more than 1×10~4 copies/ml was 95.13%. However, if the concentration of samples were less than 1×10~3 copies/ml, the average corrective ratio was only 57.4%. The average corrective ratio for negative samples was about 98.3%.CONCLUSIONThe quality control materials for HPV-16,-18 molecular detection was developed, and applicability of the materials in EQA was verified through our HPV-16, -18 EQA survey. From the external quality assessments results, it was suggested that the sensitivity for HPV-16,-18 molecular tests need to be improved in some clinical laboratories.2. Consensus-degenerate hybrid oligonucleotide primers (CODEHOP) for the detection of human papillomaviruses high-risk typesOBJECTIVEWe exploit the recently described Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) design and PCR strategy for targeted isolation of human papillomavirus high-risk types, and to evaluate the applicability of this methods in clinical detection.METHODSCODEHOPs are designed from aminoacid sequence motifs that are highly conserved gene family L1. Complete L1 protein sequences were aligned and put into Block Maker multiple sequence alignment site on the BLOCKS WWW Server hosted at the Fred Hutchinson Cancer Research Center, Seattle, WA, and 11 blocks of conserved protein sequences were identified. Primers were designed in the corresponding nucleotide regions of eleven blocks. One pair of primers derived from blocks E and J appeared promising when compared with established consensus primers. The PCR reaction condition was optimized.RESULTSBroad-range primers (CODEHOP) for detecting papillomaviruses were designed in the corresponding nucleotide regions of eleventblocks. One pair of primers, derived from blocks E and J, appeared promising when compared with established consensus primers. Application of these primers to 59 clinical samples the average corrective ratio for positive samples was 67.8%(40/59), The average corrective ratio for negative samples was 33.2%(19/59).CONCLUSIONCODEHOP PCR primers derived from amino acid sequence motifs which are highly conserved between members of a protein family have proven to be highly effective in the identification and characterization of distantly related family members. Our primer sets were broader in range and more sensitive against most papillomavirus types tested, confirmed a high level of sensitivity for detecting HPV high-risk types from these clinical samples. These primers should facilitate the search for human papillomaviruses high-risk types.