基于环介导等温扩增技术对人乳头瘤病毒的分型检测

Typing and Detection of Human Papillomavirus Based on Loop-Mediated Isothermal Amplification Method

目的:利用环介导等温扩增技术(LAMP)对人乳头瘤病毒(HPV)进行快速分型检测。方法:以HPV59、HPV66和HPV68等3种病毒亚型为实验对象,选择合适的特异性核酸序列作为检测的靶序列并构建质粒,针对每个待检靶序列设计若干组扩增引物,并利用构建的质粒筛选出特异性强、灵敏度高的引物组合;将筛选成功的引物加至微流控芯片上,实现人宫颈脱落上皮细胞样本内HPV的快速核酸分型检测。结果:针对HPV59、HPV66和HPV68这3种病毒亚型,设计筛选出具有一定灵敏度的特异性引物,结合微流控芯片,无需对样本进行复杂的核酸提取纯化操作,实现了对临床样本的直接快速检测,整个检测过程在1.5 h内即可完成。结论:LAMP技术可用于临床样本中HPV的分型检测,与微流控芯片结合后操作简单、检测时间短,在临床上有较好的应用前景。

Objective： To provide a method for rapid typing and detecting human papilloma virus（HPV） based on loop-mediated isothermal amplification（LAMP）. Methods： Three virus subtypes, HPV59, HPV66 and HPV68, were selected as the experimental objects, and from their full sequences, suitable specific nucleic acid sequences were determined as target sequences for detection and recombinant plasmids construction. Several pairs of primers were designed for each target sequence. LAMP primers, with high sensitivity and specificity, were added on microfluidic chip to detect different HPV genotypes rapidly in cervical epithelial cells samples. Results： Primers with high sensitivity and specificity were screened for HPV59, HPV66 and HPV68 subtype. Combing with microfluidic chip, the direct virus detection of clinical samples was realized, which can be finished within 1.5 hours without nucleic acid extraction or purification. Conclusion： LAMP can detect different HPV genotypes rapidly in clinical samples combining with microfluidic chip, suggesting the application of the technology in the field of clinical diagnostics has a bright future.