Flow-rSSO及PCR-SBT方法检测KIR基因有无的对比研究

Concordance of identifying the presence or absence of KIR genes by Flow-rSSO and PCR-SBT methods:a comparative study

目的研究流式磁珠序列特异性寡核苷酸探针(Flow-rSSO)杂交及测序分型(PCR-SBT)方法鉴定KIR基因有无的一致性。方法对131例汉族人群的DNA标本采用Flow-rSSO方法鉴定全部16种KIR基因的有无,并采用本实验室建立的PCR-SBT方法对14种功能性KIR基因进行高分辨水平基因检测;分析Flow-rSSO及PCR-SBT两种方法鉴定14种功能性KIR基因有无的一致性。对初检结果不一致的标本,采用同一厂家、不同批号的Flow-rSSO试剂盒进行复检,并采用序列特异性引物-PCR(PCR-SSP)方法进行检测。结果131例标本中有129例完全一致,2例不一致,占1.5%(2/131)。不一致的两例标本,1例标本3DL1基因、另1例标本2DS3及2DS5基因,其Flow-rSSO初检结果均为阴性,而PCR-SBT结果均为阳性。更换新的批号Flow-rSSO试剂盒复检,两例标本的结果均为阳性;采用PCR-SSP方法检测的结果亦为阳性。结论经Flow-rSSO试剂盒检测KIR基因有无,出现2例标本初检结果错误,提示检测KIR基因有无时,试剂的质控工作至关重要。

Objective To study the concordance of identifying the presence or absence of KIR genes using flow reverse sequence-specific oligonucleotide probe(Flow-rSSO)hybridization and sequencing based typing-PCR(PCR-SBT)methods.Methods A total number of 131 cases of DNA samples from Han population were subjected to identify the presence or absence of all 16 KIR genes by Flow-rSSO method,and then sequenced at coding sequence for all 14 functional KIR genes using our in-house KIR PCR-SBT assay.The concordance of identifying the presence or absence of all functional KIR genes by Flow-rSSO and PCR-SBT was analyzed.Samples with inconsistent initial results were re-tested using the Flow-rSSO commercial kits with different Lot number,and further tested using the PCR-SSP commercial kit.Results The presence or absence of 14 functional KIR genes for 129 of 131 samples were completely in accordance via the PCR-SBT and Flow-rSSO methods.Two samples,one with 3DL1 negative,the other with both 2DS3 and 2DS5 negative initially-identified by Flow-rSSO,were actually all positive tested by PCR-SBT.Further retest by Flow-rSSO commercial kits with different Lot number and PCR-SSP commercial kit indicated that the two samples were all positive,which agreed well with PCR-SBT results.Conclusion In this paper,the initial test results of the presence or absence of KIR genes identified by Flow-rSSO for 2 samples were wrong,which indicated the importance of carrying out the quality control for reagents in KIR gene testing.