Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.
Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.
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DOI：
10.7754/clin.lab.2016.160906
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发表时间：
2017-03
影响因子：
0.7
通讯作者：
Junxiao Lin;B. Ma;Jiehong Fang;Ye-Fu Wang;Haizhen He;Wei-Yee Lin;Wei Su;Mingzhou Zhang
中科院分区：
文献类型：
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作者：
Junxiao Lin;B. Ma;Jiehong Fang;Ye-Fu Wang;Haizhen He;Wei-Yee Lin;Wei Su;Mingzhou Zhang
背景人乳头瘤病毒(HPV)感染与宫颈癌有关。随着分子生物学和流行病学技术的发展，检测和治疗HPV已成为预防宫颈癌的重要手段。方法建立了一种简便、快速、灵敏的比色环介导等温扩增(LAMP)方法，对23种HPV基因进行了检测。LAMP反应的引物序列定位于HPV基因组的L1基因。由于它是一种荧光染料，所以在反应前加入了钙黄绿素。反应在65℃等温条件下进行40分钟。阳性反应由黄色变为荧光绿色。荧光曲线图代表了实时定量仪器的监控。对2 3种不同HPV基因单发感染患者的450份宫颈拭子标本进行检测，评价其特异性。结果与其他HPV基因型别无交叉反应。对含有23个HPVL1基因序列的克隆载体进行连续稀释，以评价其敏感性。不同的HPV亚型具有不同的检测能力。LAMP法检测不同亚型HPV的敏感性在1.0×10~(-1)-4.0×10~3拷贝/反应之间。采用LAMP法和反向斑点印迹法(RDB)对450例临床标本进行HPV检测和基因分型。LAMP和RDB检测HPV阳性标本分别为385例(85.6%)和375例(83.3%)，HR-HPV阳性标本分别为306例(68.0%)和296例(65.8%)。两种方法的符合率分别为93.3%(κ=0.75)和94.7%(κ=0.88)。结论该方法可用于资源有限的医院或乡村诊所HPV感染的快速筛查。
BACKGROUND Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. METHODS A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. RESULTS The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. CONCLUSIONS It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.