Peng Xiao-Mou, Chen Xue-Juan, Li Jian-Guo, Gu Lin, Huang Yang-Su, Gao Zhi-Liang
Department of Infectious Diseases, the Third Affiliated Hospital, Zhongshan University, Guangzhou 510630, China.
World J Gastroenterol. 2003 Aug;9(8):1743-6. doi: 10.3748/wjg.v9.i8.1743.
Point mutation, one of the commonest gene mutations, is the most important molecular pathogenesis of cancer and chronic infection. The commonest methods for detection of point mutation are based on polymerase chain reaction (PCR). These techniques, however, cannot be used in large scale screening since they are neither accurate nor simple. For this reason, this study established a novel method of competitively differentiated PCR (CD-PCR) for screening point mutation in clinical practice.
Two competitively differentiated primers for mutant-type and wild-type templates respectively with an identically complemented region in 3' end except for last 2 base pairs and a different non-complemented region in 5' end were designed. Thus, competitive amplification might be carried out at a lower annealing temperature at first, and then differentiated amplification at a higher annealing temperature when primers could not combine with initial templates. The amplification was performed in one-tube. The products of CD-PCR were detected using microplate hybridization assay. CD-PCR was evaluated by detecting G1896A variant of hepatitis B virus (HBV) in form of recombinant plasmids and in sera from patients with hepatitis B, and compared with allele-specific PCR (AS-PCR) and competitive AS-PCR.
CD-PCR was successfully established. It could clearly distinguish wild-type and mutant-type plasmid DNA of G1896A variant when the amount of plasmid DNA was between 10(2)-10(8)copies/reaction, while for AS-PCR and competitive AS-PCR, the DNA amount was between 10(2)-10(4)copies/reaction. CD-PCR could detect one copy of G1896A variant among 10-100 copies of wild-type plasmid DNA. The specificity of CD-PCR was higher than those of AS-PCR and competitive AS-PCR in the detection of HBV G1896A variant in sera from patients with hepatitis B. CD-PCR was independent of the amount of HBV DNA in serum. HBV G1896A variant was more often found in HBeAg (-) patients with a lower level of detectable viremia than that with a higher level of detectable viremia (P=0.0192).
CD-PCR is more specific since it is less influenced by the amount of initial templates and the cross amplification between mutant- and wild-type amplified products. It is also simple and time-saving. Thus, CD-PCR might be useful in routine gene typing and point mutation screening. HBV G1896A or other more important mutations have to be routinely detected in patients with a detectable level of viremia after HBeAg/antibody conversion in clinical practice.
点突变是最常见的基因突变之一，是癌症和慢性感染最重要的分子发病机制。检测点突变最常用的方法基于聚合酶链反应（PCR）。然而，这些技术既不准确也不简单，无法用于大规模筛查。因此，本研究建立了一种新型的竞争性差异PCR（CD-PCR）方法用于临床实践中的点突变筛查。
设计了两条分别针对突变型和野生型模板的竞争性差异引物，其3'端除最后2个碱基对外具有相同的互补区域，5'端具有不同的非互补区域。这样，首先可以在较低的退火温度下进行竞争性扩增，然后当引物不能与初始模板结合时，在较高的退火温度下进行差异扩增。扩增在一管中进行。使用微孔板杂交试验检测CD-PCR产物。通过检测重组质粒形式的和乙型肝炎患者血清中的乙型肝炎病毒（HBV）G1896A变异体对CD-PCR进行评估，并与等位基因特异性PCR（AS-PCR）和竞争性AS-PCR进行比较。
成功建立了CD-PCR。当质粒DNA量在10（2）-10（8）拷贝/反应之间时，它可以清楚地区分G1896A变异体的野生型和突变型质粒DNA，而对于AS-PCR和竞争性AS-PCR，DNA量在10（2）-10（4）拷贝/反应之间。CD-PCR可以在10-100拷贝的野生型质粒DNA中检测到一份G1896A变异体。在检测乙型肝炎患者血清中的HBV G1896A变异体时，CD-PCR的特异性高于AS-PCR和竞争性AS-PCR。CD-PCR与血清中HBV DNA的量无关。在HBeAg（-）且可检测病毒血症水平较低的患者中比在可检测病毒血症水平较高的患者中更常发现HBV G1896A变异体（P = 0.0192）。
CD-PCR更具特异性，因为它受初始模板量以及突变型和野生型扩增产物之间交叉扩增的影响较小。它也简单省时。因此，CD-PCR可能有助于常规基因分型和点突变筛查。在临床实践中，对于HBeAg/抗体转换后可检测到病毒血症水平的患者，必须常规检测HBV G1896A或其他更重要的突变。
