利用多重微流控环介导等温扩增和可视化检测方法对 30 种皮肤人乳头瘤病毒进行基因分型。
Genotyping of 30 kinds of cutaneous human papillomaviruses by a multiplex microfluidic loop-mediated isothermal amplification and visual detection method.
机构信息
Department of Dermatology, The First Hospital of China Medical University, No.155 Nanjing Bei Street, Heping District, Shenyang, Liaoning Province, 110001, PR China.
NHC Key Laboratory of Immunodermatology (China Medical University), No.155 Nanjing Bei Street, Heping District, Shenyang, Liaoning Province, 110001, PR China.
出版信息
Virol J. 2020 Jul 9;17(1):99. doi: 10.1186/s12985-020-01373-3.
BACKGROUND
Human papillomaviruses (HPVs), a group of non-enveloped small viruses with double-stranded circular DNA which lead to multiple skin diseases such as benign warts, are commonly seen in clinics. The current HPV detection systems aim mainly at mucosal HPVs, however, an efficient clinical approach for cutaneous HPVs detection is lacking.
OBJECTIVES
To establish a rapid detection system for cutaneous HPVs using a colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue (HNB) dye in combination with microfluidic technology.
METHODS
L1 DNA sequences of the 30 cutaneous HPVs were chemically synthesized, and LAMP primers against L1 DNA were designed with use of an online LAMP designing tool. Isothermal amplification was performed with use of a water bath and the amplification results were inspected with the naked eye. Using PCR sequencing as a control method, the specificity and sensitivity of the new detection system were obtained by detecting clinical samples.
RESULTS
The lower detection limit of the LAMP assay was 10 viral DNA copies/μl when tested on synthesized L1 DNA sequences, which was better than the conventional PCR. Compared to PCR sequencing, the sensitivity of HPV27, HPV2, HPV1, HPV57, HPV3, HPV4, HPV7 and HPV75 genotypes detections were 100%, whereas the specificity was 34.55, 45.12, 95.83, 98.59 and 97.62% respectively, when tested on clinical samples.
CONCLUSIONS
The new cutaneous type HPV detection system is characterized by both a good sensitivity and specificity compared to conventional methods.
背景
人乳头瘤病毒（HPV）是一组无包膜的小型双链环状 DNA 病毒，可导致多种皮肤疾病，如良性疣，临床上较为常见。目前的 HPV 检测系统主要针对黏膜型 HPV，但缺乏有效的皮肤型 HPV 检测方法。
目的
建立一种利用显色环介导等温扩增（LAMP）结合微流控技术，以羟基萘酚蓝（HNB）染料检测皮肤型 HPV 的快速检测系统。
方法
化学合成 30 种皮肤型 HPV 的 L1 DNA 序列，利用在线 LAMP 设计工具设计针对 L1 DNA 的 LAMP 引物。采用水浴进行等温扩增，肉眼观察扩增结果。以 PCR 测序为对照方法，通过检测临床样本，获得新检测系统的特异性和敏感性。
结果
当检测合成的 L1 DNA 序列时，LAMP 检测的下限为 10 个病毒 DNA 拷贝/μl，优于常规 PCR。与 PCR 测序相比，HPV27、HPV2、HPV1、HPV57、HPV3、HPV4、HPV7 和 HPV75 基因型的检测敏感性分别为 100%，特异性分别为 34.55%、45.12%、95.83%、98.59%和 97.62%，检测临床样本时。
结论
与传统方法相比，新型皮肤型 HPV 检测系统具有良好的敏感性和特异性。
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