基于二维PCR技术的高危型人乳头瘤病毒及相关肿瘤抑制基因 p53和 RB1的分型检测方法

Development of a typing detection method for high-risk human papillomavirus and related tumor suppressor genes p53 and RB1 based on two-dimensional PCR technology

目的应用高通量二维PCR技术(2D-PCR), 建立一种单管一步法检测和鉴定宫颈细胞中16种高危型人乳头瘤病毒(HR-HPV)亚型(HPV16、18、31、33、35、39、45、51、52、56、58、59、66、68、73、82)、p53(rs1042522)和RB1(rs3092905)基因型的方法。方法根据16种不同亚型HR-HPV、p53及RB1基因DNA序列设计特异性引物, 同时将待测靶基因p53及RB1基因作为评判标本取样及PCR扩增成功与否的内参基因。在3个荧光检测通道中, 采用相应标签标记不同亚型HR-HPV、p53及RB1的上游引物, 并构建完善的2D-PCR检测体系。应用该方法检测804份来源于常州市第一人民医院2022年12月至2023年8月妇科门诊的宫颈刷样本, 将检测结果与PCR-反向点杂交法、流式荧光杂交法、单重实时荧光定量PCR法分别进行一致性比较;同时采用Sanger测序法检测p53和RB1的基因型。采用Kappa检验评价2D-PCR法和其他方法间的一致性。结果 2D-PCR可通过FAM、HEX和Alexa Fluor568通道的特征性熔解谷对16种HR-HPV型别和p53、RB1的基因型进行准确区分和鉴定。2D-PCR法与PCR-反向点杂交法具有较高的一致性(Kappa=0.699), 与流式荧光杂交法具有更高的一致性(Kappa=0.793), 和单重荧光定量PCR法的一致性Kappa=0.880(95%CI 0.862~0.907)。以Sanger测序法为金标准, 2D-PCR法检测p53、RB1基因型的准确度为100%。p53 rs1042522 位点3种基因型(G/G型、G/C型和C/C型)的分布频率为32.09%(258/804)、49.88%(401/804)和18.03%(145/804), RB1 rs3092905位点检出基因型均为A/A型。结论成功开发了一种2D-PCR方法, 用于高危型HR-HPV型别鉴定与宫颈癌相关肿瘤抑制基因p53和RB1的基因分型。

Objective To establish a single-tube,one-step method for detecting and identifying 16 high-risk human papillomavirus(HR-HPV)subtypes(HPV16,18,31,33,35,39,45,51,52,56,58,59,66,68,73,82)and genotyping p53(rs1042522)and RB1(rs3092905)in cervical cells,using high-throughput two-dimensional PCR(2D-PCR)technology.Methods Applied Research.Specific primers were designed according to the DNA sequences of the 16 different HR-HPV subtypes,p53,and RB1 genes,with the target genes p53 and RB1 serving as internal references to assess the success of sample collection and PCR amplification.In three fluorescent detection channels,upstream primers labeled with corresponding tags were used for different HR-HPV subtypes,p53,and RB1,constructing a comprehensive 2D-PCR detection system.Using this method,804 cervical brush samples collected from the gynecology outpatient department of Changzhou First People′s Hospital from December 2022 to August 2023 were tested.The test results were compared for consistency with PCR-reverse dot blot assay,flow cytometric fluorescence hybridization assay,and single-plex real-time quantitative PCR assay,respectively.Meanwhile,the genotypes of p53 and RB1 were detected using Sanger sequencing.The Kappa test was applied to determine the consistency between 2D-PCR method and other methods.Results 2D-PCR accurately discriminated and identified the genotypes of 16 HR-HPV types and p53,RB1 through characteristic melting valleys in the FAM,HEX,and Alexa Fluor568 channels.2D-PCR showed high consistency with PCR-reverse dot blot assay,with a Kappa value of 0.699,even higher consistency with flow cytometric fluorescence hybridization assay,with a Kappa value of 0.793,and the highest consistency with single-plex quantitative PCR,with a Kappa value of 0.880(95%CI 0.862-0.907).Using Sanger sequencing as the gold standard,the accuracy of 2D-PCR method in detecting p53 and RB1 genotypes is 100%.The distribution frequencies of the three genotypes(G/G,G/C,and C/C)at the p53 rs1042522 locus were 32.09%(258/804),49.88%(401/804)and 18.03%(145/804),respectively,while all detected genotypes at the RB1 rs3092905 locus were A/A.Conclusion This study successfully developed a 2D-PCR method for the identification and genotyping of high-risk human papillomavirus types and related tumor suppressor genes p53 and RB1 for cervical cancer.