Monavari Seyed Hamidreza, Fateh Roohollah, Vaziri Farzam, Rahimi Jamnani Fatemeh, Anvari Enayat, Sadeghi Farzin, Afrough Parviz, Behrouzi Ava, Sakhaee Fatemeh, Meidaninikjeh Sepideh, Mollaie Hamidreza, Hadizadeh Tasbiti Alireza, Yari Shamsi, Sadeghi Maryam, Fateh Abolfazl, Siadat Seyed Davar
a Department of Virology , Iran University of Medical Sciences , Tehran , Iran.
b Department of Microbiology and Immunology, Faculty of Medicine , Qom University of Medical Sciences , Qom , Iran.
Scand J Clin Lab Invest. 2017 Jul;77(4):247-252. doi: 10.1080/00365513.2017.1299207. Epub 2017 Mar 10.
Interleukin-28B (IL28B) single-nucleotide polymorphisms (SNPs) constitute important host-related factors influencing the response rate to Hepatitis C virus (HCV) standard antiviral therapy. In the last few years, several new technologies for SNP detection have been developed. However, the sensitivity and specificity of various methods are different and needs evaluation. Five different methods (resolution melting curve [RMC], polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP], PCR-sequencing analysis, amplification refractory mutation system [ARMS], and zip nucleic acid probe-based real-time PCR [ZNA]) were developed for genotyping rs12979860 associated with IL28B. In this study, limit of detection (LD), costs and turnaround time of these methods were compared in 350 subjects. As for IL28B rs12979860 polymorphisms, 348/350 (99.4%) samples were consistent among the five methods, while results for 2/350 (0.57%) samples were concordant by ZNAs and PCR-sequencing, and discordant by other methods. Without considering the cost of DNA extraction, the price of each reaction for ARMS-PCR, RMC, PCR-RFLP, ZNA and PCR-sequencing were respectively: US$3.10, US$5.0, US$5.50, US$8.50 and US$17.0. RMC was the fastest method, while the ZNA method was easy to use, reliable and effective. Lower LD was determined to be 50-60 copies/μL for the PCR-RFLP, RMC and ARMS-PCR assays; whilst ZNA assay was able to detect 2-3 copies/μL. In conclusion, in the current study, all four methods are suitable for IL28B rs12979860 genotyping, but the ZNA assay can be a reliable tool. Due to its lower LD for SNP identification, this method is better than others for detecting this type of polymorphism.
白细胞介素-28B（IL28B）单核苷酸多态性（SNP）是影响丙型肝炎病毒（HCV）标准抗病毒治疗应答率的重要宿主相关因素。在过去几年中，已开发出几种用于SNP检测的新技术。然而，各种方法的敏感性和特异性各不相同，需要进行评估。针对与IL28B相关的rs12979860进行基因分型，开发了五种不同的方法（熔解曲线分析[RMC]、聚合酶链反应-限制性片段长度多态性[PCR-RFLP]、PCR测序分析、扩增阻滞突变系统[ARMS]和基于拉链核酸探针的实时PCR[ZNA]）。在本研究中，对350名受试者比较了这些方法的检测限（LD）、成本和周转时间。对于IL28B rs12979860多态性，五种方法中有348/350（99.4%）的样本结果一致，而2/350（0.57%）样本的结果在ZNA和PCR测序中一致，但在其他方法中不一致。不考虑DNA提取成本，ARMS-PCR、RMC、PCR-RFLP、ZNA和PCR测序每个反应的价格分别为：3.10美元、5.0美元、5.50美元、8.50美元和17.0美元。RMC是最快的方法，而ZNA方法易于使用、可靠且有效。PCR-RFLP、RMC和ARMS-PCR检测的较低LD确定为50-60拷贝/μL；而ZNA检测能够检测2-3拷贝/μL。总之，在本研究中，所有四种方法都适用于IL28B rs12979860基因分型，但ZNA检测可能是一种可靠的工具。由于其在SNP鉴定中的较低LD，该方法在检测此类多态性方面优于其他方法。
