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目的通过汇集标本的方法,建立高效筛查稀有血型的试验体系。方法根据Vel阴性(Vel-)稀有血型产生的分子机制,分别设计可特异扩增Vel血型基因SMIM1野生型序列和缺失突变序列的2组引物;合成Vel-对照质粒。将对照质粒与不同数量的随机标本进行汇集,根据扩增结果确定合适的汇集标本数;用缺失突变序列的特异性引物扩增汇集标本,如出现特异性目的条带,则用2组引物进行拆分检测参与汇集的标本,确认基因型。限制性片段长度多态性聚合酶链反应(PCR-RFLP)和DNA测序用于验证筛查结果。结果结合单个随机标本的浓度,最终确定4个血样汇集的筛查体系,可将试验所用时间及检测费用降低4倍。我们在9 122例标本中筛查到1例Vel血型基因SMIM1 c.64_80杂合缺失突变的个体。结论汇集方法的使用大大降低了筛查工作的时间和费用,可推广应用于具有类似分子机制的稀有血型的大规模筛查。
Objective To establish a test system for efficient screening of rare blood groups by pooling specimens. Methods Based on the molecular mechanism of Vel-negative rare blood type, two sets of primers were designed to amplify the wild-type and short mutant sequences of Vel-type gene SMIM1 respectively. The Vel-control plasmid was synthesized. The control plasmid and a different number of random samples were pooled, according to the amplification results to determine the appropriate collection of specimens; collection of pooled specimens with specific primers lacking the mutation sequence, such as the presence of specific bands of interest, with two sets of primers Splitting detection of specimens collected to confirm the genotype. Restriction Fragment Length Polymorphism Polymerase Chain Reaction (PCR-RFLP) and DNA sequencing were used to validate the screening results. Results Combined with the concentration of a single random sample, the screening system of 4 blood samples was finally determined, which could reduce the time and cost of testing by 4 times. We screened 9 122 individuals with one heterozygous deletion mutation of the SMIM1 c.64_80 gene of the Vel gene. Conclusion The use of pooled methods greatly reduces the time and cost of screening work and can be widely applied to large-scale screening of rare blood groups with similar molecular mechanisms.