PCR-SSP/PCR-SSO两种KIR基因分型方法的比较

Comparison of KIR genotyping between PCR-SSP and PCR-SSO

目的杀伤细胞免疫蛋白样受体(KIR)基因在自然杀伤细胞活性调节、机体抗感染、移植免疫过程中发挥重要作用。在脐带血移植中,KIR基因分型的效率和准确关系到移植的成败。为了满足临床上对KIR基因分型的要求,对比分析聚合酶链反应-序列特异性引物(PCR-SSP)和聚合酶链反应-序列特异性寡核苷酸探针(PCR-SSO)两种方法,试图建立一个高效便捷检测KIR基因分型的方法学体系。方法从2018年到2019年已外送到第三方检测实验室(北京博富瑞医学检验实验室有限公司)做过KIR基因分型检测的脐带血样本中随机取出12份,分别用PCR-SSP及PCR-SSO商品化试剂盒进行KIR基因分型检测,并通过凝胶电泳分析或MATCH IT!DNA软件分析,获取KIR基因分型结果。结果12例脐带血样本中,PCR-SSO方法学检测的结果与原PCR-SSO方法检测结果完全一致,PCR-SSP方法学检测的结果与原PCR-SSO方法检测结果9例相同,有2例因出现漏孔而无法判读,有1例出现了假阳性带。结论PCR-SSO方法学检测KIR基因效果优于PCR-SSP,但是PCR-SSO方法学也存在当微珠的荧光校准值接近于临界值时,结果出现错判的现象;为保证KIR基因分型的准确度,建议PCR-SSP方法出现漏孔或者PCR-SSO方法荧光校准值接近于临界值时两种方法相互验证。

Objective To compare polymerase chain reaction-sequence specific primers(PCR-SSP)and polymerase chain reaction-sequence specific oligonucleotide probes(PCR-SSO),and establish an efficient and convenient methodological system for detecting killer cell immune protein-like receptor(KIR)genotyping and meet clinical requirements for KIR genotyping,under the consideration that KIR gene plays an important role in regulation of natural killer(NK)cell activity,anti-infection and transplantation immunity,and the efficiency and accuracy of KIR genotyping are related to the success of cord blood transplantation.Method Twelve umbilical cord blood samples were randomly selected from the samples analyzed by the third-party laboratory(Beijing Bo Fu Rui Medical Laboratory Co.,Ltd.)from 2018 to 2019,for KIR genotyping using PCR-SSP and PCRSSO commercial kits,and agarose gel electrophoresis or MATCH IT!DNA software was used to obtain KIR genotyping outcomes.Results Of the 12 umbilical cord blood samples,results of PCR-SSO method were completely consistent with that of original PCR-SSO method,and PCR-SSP method results of 9 samples were the same as the original PCR-SSO method results,2 samples were undetected due to leaks,and 1 showed false positive band.Conclusion It is demonstrated that PCR-SSO method is better than PCR-SSP in detecting KIR genes,but wrong outcome occurs at fluorescence calibration value closed to cut-off value.The 2 methods should be mutually verified at leak in PCR-SSP method or fluorescence adjusted value closed to cut-off value in PCR-SSO method for KIR genotyping accuracy.