Carmody M W, Jones M, Tarraza H, Vary C P
Maine Medical Center Research Institute, South Portland 04106, USA.
Mol Cell Probes. 1996 Apr;10(2):107-16. doi: 10.1006/mcpr.1996.0015.
A polymerase chain reaction (PCR) based technique that combines a virus specific primer and a human interspersed repetitive sequence (IRS) specific primer in order to detect integration of human papilloma virus type 16 (HPV-16) is described. Amplification of viral-host DNA junctions occurs when viral integration results in placement of the virus specific primer binding site near (less that 3-4 kb) the primer binding site within a repetitive sequence element. The method relies on enzyme labeled oligonucleotide probes to achieve rapid, specific, and nonradioisotopic detection of viral integration related PCR products since episomal forms of the viral DNA do not lead to exponential accumulation of hybridizable PCR products. The technique is demonstrated for human genomic DNA derived from clinical cervical swab specimens and archival paraffin embedded blocks. Viral integration was detected in 41% of the HPV-16 positive samples (n = 34). In this positive subset, 64% were classified as invasive neoplasias, 29% CIN III and 7% CIN II. Analyzing the positive invasive neoplasias, 6 of 9 (66%) of the fingerprint results were obtained when an HPV primer was paired with an Alu primer. Interestingly, 100% of Alu primed fingerprint results obtained were derived from samples presenting invasive neoplasia (P < 0.025 by chi square).
本文描述了一种基于聚合酶链反应（PCR）的技术，该技术结合了病毒特异性引物和人类散布重复序列（IRS）特异性引物，用于检测16型人乳头瘤病毒（HPV-16）的整合情况。当病毒整合导致病毒特异性引物结合位点位于重复序列元件内引物结合位点附近（小于3-4 kb）时，会发生病毒-宿主DNA连接区的扩增。该方法依赖于酶标记的寡核苷酸探针，以实现对病毒整合相关PCR产物的快速、特异性和非放射性检测，因为病毒DNA的游离形式不会导致可杂交PCR产物的指数积累。该技术已在来自临床宫颈拭子标本和存档石蜡包埋块的人类基因组DNA中得到验证。在41%的HPV-16阳性样本（n = 34）中检测到病毒整合。在这个阳性子集中，64%被归类为浸润性肿瘤，29%为CIN III，7%为CIN II。分析阳性浸润性肿瘤时，当HPV引物与Alu引物配对时，9个指纹结果中有6个（66%）获得。有趣的是，获得的100%的Alu引物指纹结果均来自呈现浸润性肿瘤的样本（卡方检验，P < 0.025）。
