PCR-SSP技术在红细胞ABO血型快速基因分型中的应用研究  

Application of PCR-SSP technology in rapid genotyping of ABO blood group

作　　者：梁伟 周建霖 杨亮 许德义 黄丹丹 邓刚 梅传亮 韩新乐 张哲 

机构地区：[1]浙江省宁波市中心血站输血研究所,315041 [2]浙江省宁波市中心血站检验科,315041 [3]江苏省新沂市人民医院输血科,221400 [4]浙江省宁波市卫生局,315010

出　　处：《中华全科医学》2015年第8期1323-1327,F0003,共6页Chinese Journal of General Practice

摘　　要：目的探讨PCR-SSP技术用于临床标本红细胞ABO血型快速定型的可行性。方法利用PRIMER 5软件针对A101等位基因c DNA第261、703 SNP位点不同的碱基设计4对序列特异性引物;通过温度梯度PCR反应确定各个引物对的最佳退火温度;利用基因测序技术来评价PCR-SSP的性能;用100例临床盲样的SSP结果与其相应的血清学结果的比对来观测该方法临床应用的有效性;选择26例血清学定型结果有疑问的临床疑难标本进一步考察该法的性能。结果确定261G、261A、703A以及703G的最佳退火温度分别为70℃、64℃、60℃、64℃;通过随机抽取5例不同反应格局的标本进行第6、7外显子基因测序,测序结果表明该4对引物可以对261位点G缺失情况以及703位点G/A的分布情况进行有效鉴别;100例的临床盲样检测与血清学完全一致;26例血清学定型有疑问的标本通过该法可以为血清学实验提供有效的参考依据。结论 PCR-SSP基因分析技术在红细胞ABO血型快速鉴定方面具有可行性,在临床常规标本血型方面性能可靠并可作为ABO定型临床疑难标本进行进一步的DNA测序的依据。Objective To evaluate the feasibility of rapidgenotyping of ABO blood group using the polymerase chain reaction with sequence-specific primers（ PCR-SSP） typing. Methods The four pairs of special specific primers were designed according to the c DNA nts 261 and 703 of A101 allele using PRIMER 5 software. The optimal annealing temperatures were determined through the temperature gradient PCR reaction;the performance of the PCR-SSP technology was examined by the DNA sequencing technology and the comparison of serological results of the 100 clinical blind samples,furthermore 26 difficult serological typing samples were also tested by the PCR-SSP technology. Results The best annealing temperature was 70 ℃,64 ℃,60 ℃,64 ℃ at the nts 261 G,261A,703 A and 703 G,respectively. The sequencing technology was executed for the 6 and 7 exons of five random specimens which reaction pattern was different using the PCR-SSP technology and the results showed the 4 pairs special specific primers distinguished the SNP at nts 261 and 703 effectively;The results of PCR-SSP and serological typing were consistent perfectly. Conclusion The PCR-SSP technology can discriminate the genotype of ABO blood group gene rapidly. It was reliable to detect clinical routine specimens,moreover,supplied the molecular assistance for the special specimens which were difficult to type the ABO blood group only using serological method. The PCR-SSP can provide the molecular screening evidences which determined whether or not the DNA sequencing technology should be chosen in the next step.

关 键 词：序列特异性引物聚合酶链反应 单核苷酸多态性 ABO血型 基因分型 

分 类 号：R457.11[医药卫生—治疗学] R446.41[医药卫生—临床医学]