目视LAMP技术对人乳头瘤病毒18亚型检测的应用价值研究

Application value study of visual loop-mediated isothermal amplification in human papillomavirus type 18 diagnosis

目的探讨环介导等温扩增技术用于检测临床标本乳头瘤病毒HPV18亚型的应用价值。方法设计针对HPV18-L1基因序列中6个区段的4条特异引物，在等温条件下（65℃）进行核酸扩增反应1h，在扩增前加入钙黄绿素作为反应指示剂；利用建立的环介导等温扩增方法进行敏感型检测以及检测正常体检妇女和患者子宫颈分泌物的HPV18亚型。结果LAMP检测HPV18L1基因的敏感度为1000拷贝每微升；阴道炎患者HPV18阳性检出率为5．26％（1／19）；慢性子宫颈炎组HPV18亚型检出率为10％（2／20）；宫颈上皮内瘤变（CINI～Ⅱ级）阳性检出率33．3％（3／9）；子宫颈癌I级阳性检出率为64％（16／25）。结论基于目视的环介导等温扩增方法是一种具有经济、快速筛查临床标本中HPV18亚型的诊断工具，可具有在基层医院推广应用的潜能。

Objective To investigate the application of loop-mediated isothermal amplification（LAMP）in detecting Human papillomavirus type 18（HPV18） from clinical specimens. Methods It is characterized by employing a set of four specially designed primers spanning six distinct sequences of a target gene HPV18-L1. The reaction was performed at 65C for 1 hour with the addition of calcein dye prior to amplification. LAMP was used for sensitivity assay and detecting HPV18 mono infected in cervical secretions from women of physical examination and patients. Results The sensitivity of LAMP for detection of HPV18 L1 is 10-3 per microliter; the positive rate of HPV18 in patients with vaginitis is 5.26%（1/19）; the positive rate of HPV18 in patients with chronic cervicitis is 10%（2/20）; and the positive rate of HPV18 in patients With cervical intraepithelial neoplasia （CIN I- ]I ）and cervical cancer-I is 33.3%（3/9）, 64% （16/25）, respectively. Conclusion LAMP assay is a high efficiency, low cost diagnostic tool, useful for rapid, accurate, direct detection of HPV for clinical diagnosis and might be oooular at the primary hospital in future.