Chen Qian, Belmonte Irene, Buti Maria, Nieto Leonardo, Garcia-Cehic Damir, Gregori Josep, Perales Celia, Ordeig Laura, Llorens Meritxell, Soria Maria Eugenia, Esteban Rafael, Esteban Juan Ignacio, Rodriguez-Frias Francisco, Quer Josep
Qian Chen, Maria Buti, Damir Garcia-Cehic, Josep Gregori, Celia Perales, Laura Ordeig, Meritxell Llorens, Maria Eugenia Soria, Rafael Esteban, Juan Ignacio Esteban, Josep Quer, Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, 08035 Barcelona, Spain.
World J Gastroenterol. 2016 Nov 21;22(43):9604-9612. doi: 10.3748/wjg.v22.i43.9604.
To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus (HCV).
In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. LightCycler methods, based on real-time PCR with sequence-specific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest.
Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10% (mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80K was detected in 14.6% of G1a patients and 0% of G1b in our setting.
A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.
开发一种快速、低成本的诊断策略，以识别高度可变基因组（如丙型肝炎病毒（HCV））中的单点突变。
在HCV感染患者中，治疗前病毒准种内与耐药相关的氨基酸替代可导致对直接作用抗病毒药物的敏感性降低，并导致治疗失败和病毒学复发。一种这样的自然发生的突变是HCV-NS3蛋白酶基因中的Q80K替代，其赋予对PI抑制剂（特别是simeprevir）的耐药性。能够常规检测这些单点突变的低成本、高灵敏度技术将有助于识别有治疗失败风险的患者。基于实时PCR和序列特异性探针杂交的LightCycler方法已在大多数诊断实验室中实施。然而，该技术无法识别高度可变遗传环境（如HCV基因组）中的单点突变。为了解决这个问题，我们开发了一种新方法，使感兴趣的点突变以外区域中存在的所有核苷酸均匀化。
使用核苷酸特异性探针，在第80位的Q、K和R替代被清晰识别，灵敏度为1０%（检测到频率至少为1０%的突变）。该技术成功应用于识别240份HCV G1血清样本中的Q80K替代，其性能与直接桑格测序（目前用于此目的的标准程序）相当。然后在202名感染HCV G1的加泰罗尼亚人群中验证了该新方法。在我们的研究中，14.6%的G1a患者检测到Q80K，G1b患者中未检测到。
基于实时PCR和荧光共振能量转移探针熔解曲线分析的快速、低成本诊断策略已成功开发，以识别高度可变基因组（如丙型肝炎病毒）中的单点突变。该技术可适用于检测高度可变基因组中的任何单点突变。
