用地高辛标记的PCR/SSO方法进行HLA-B27等位基因检测

Detection of HLA B27 alleles by using polymerase chain reaction and digoxigen 11 2 3 dideoxyuridinetriphosphate labelled oligonucleotide probes

目的建立检测ＨＬＡ－Ｂ２７等位基因的聚合酶链反应／顺序特异的寡核苷酸探针方法，并分析其实用性。方法采用４对引物Ｅ４０ｓ、Ｅ９０ａｓ；Ｅ９１ａｓ、Ｅ１３６ａｓ；Ｅ９１ｂｓ、Ｅ１８１ａｓ；Ｅ４０ｓ、Ｅ１３０ａｓ分别扩增靶ＤＮＡ，用１０个探针ＣＬ－１～９和ＰＡＮ－Ｂ２７分别进行杂交，探针用地高辛系统标记和检测，可检定Ｂ＊２７０１～２７０８共８个等位基因。结果通过对Ｂ２７等位基因已明确的８个阳性对照ＤＮＡ、５例Ｂ２７阴性标本及７５例阳性标本的检测，表明本方法技术稳定结果可靠，不会与其它ＨＬＡⅠ类基因产生交叉杂交而影响结果的指定。结论本研究为ＨＬＡ－Ｂ２７多态性和强直性脊椎炎关联研究提供了一种有效方法，也为ＨＬＡⅠ类分子基因分型研究积累了经验。

Objective To establish a method of polymerase chain reaction and sequence specific oligonucleotide probes for identification of HLA B27 alleles,and analyse the practicability of the method. Methods Genomic DNA was amplified by using four pairs of primers and amplified DNA was hybridized with ten probes ,respectively.The probes were labelled with Dig 11 dd UTP at the 3end by using terminale transferase.The hybridized probes were detected with antidigoxigenin alkaline phosphatase conjugate Fab fragments and CSPD for visualization.Eight HLA B27 alleles named B*2701,02,03,04,05,06,07 and 08 can be identified. Results A total of seventy five B27(+) and five B27(-) samples from Shanghai were analysed with this method.Eight DNA samples which have been well typed as B*2701,02,03,04,05,06,07 and 08 were used as hybridization controls.The results showed that the method is simple,highly specificitic and sensitive . Conclusions The results suggest that PCR/SSO method will be useful for the study on HLA B27 alleles and Ankylosing spondylitis in Chinese.