运用荧光定量多重PCR技术检测RB1基因突变

目的　建立一种半自动、简易、快速和不需放射性同位素技术,用以检测RB1 基因突变的方法。方法　应用包括扩增RB1 基因27 个外显子和启动区在内的荧光定量多重PCR 技术。将全基因分成若干组,每组3～7 对引物,同时含对照C4 引物并使用4 对外对照,分别为RB的缺体、单体、双倍体及三体。在测试标本中,一个片段的拷贝数根据比较对照与标本的荧光强度而获得。利用自动片段处理软件2.1 处理结果。结果　观察到小片段缺失、插入及外显子拷贝数缺失等突变。测序结果和RB瘤细胞杂合性丢失进一步证实荧光定量多重PCR 的筛查结果。结论　此法是筛查患者基因缺失、插入的一种快速、简易的方法。应用此法可查出约50% 的阳性病例。查出的突变不仅有小缺失、插入,也可发现用以往的方法所不能见到的外显子杂合性缺失病例。对临床基因缺陷的诊断有重要意义

Detection of RB1 mutations by using quantitative fluorescent multiplex PCR

Objective To develop a half automatic,simple,non radioactive technique for rapid detection of mutation in the RB gene.Methods Quantitative fluorescent multiplex PCR(QFM PCR) involves amplification of the promoter region and all 27 exons of the RB1 gene with fluorescein labeled primers in multiplex sets.Primers were divided into multiplex sets of three to seven pairs of primers,also contained were internal control primers C4.Four external controls were also tested by using nullisomic,monosomic,diploid,and trisomic for RB1.The number of copies of a fragment in a test sample was calculated by comparing fluorescence intensity of the fragment with these standards.Fragment value detection and subsequent calculations were performed automatically by Fragment Manager 2.1 software.Results Small deletions,insertions and whole exon deletion in the RB1 gene were detected from genomic DNA.Sequencing analysis and RB tumor homozygous mutation(LOH) confirmed the defects detected by QFM PCR.Conclusion The approach is a rapid,cost effective initial method for screening patient samples.It has been used to identify approximately 50% positives of tested samples.The mutations shown were not only small deletion and insertion,but also the single copy exons heterozygous mutations in the RB1 gene which the previously used methods were unable to detect. These features make the technique an attractive approach to clinical diagnosis of gene defects.