用顺序特异引物聚合酶链反应技术对HLA-B基因分型

目的采用顺序特异引物聚合酶链反应技术（ＰＣＲ－ＳＳＰ）建立汉族人群ＨＬＡ－Ｂ基因分型方法。方法肾移植供受者临床样本ＤＮＡ３１９份，Ｂ基因标准系列ＤＮＡ６２份。设计合成Ｂ座位５２个特异性引物和１对阳性对照引物，组成４１个ＰＣＲ反应，建立ＰＣＲ－ＳＳＰ方法。一步法完成对Ｂ座位的基因分型。结果经双盲验证，并与血清学方法比较。结果所有临床样本和标准ＤＮＡ，ＰＣＲ－ＳＳＰ基因分型均获得成功。可准确分辨ＨＬＡ－Ｂ座位等位基因４１个，实际检出汉族人群Ｂ抗原特异性３２个。无假阳性和假阴性，４０份样本的重复率１００％，总耗时５小时。分型结果经双盲验证完全符合。与血清学分型结果比较显示：７８份血清学空白中１７例存在第二个基因，２６份血清学分型结果错误。总误差率１３．５％。结论用ＰＣＲ－ＳＳＰ技术行ＨＬＡ－Ｂ基因分型，分辨率高、特异性强、重复性好、相对简捷快速，分型结果较血清学方法更加精确可靠，适合于临床应用。     

用PCR-SSP分析HLA-B位点血清学分型困难标本

对血清学ＨＬＡ－Ｂ位点分型困难标本用ＰＣＲ－ＳＳＰ方法分析,并寻找血清分型效果不佳原因。方法：用本室建立的ＰＣＲ－ＳＳＰ方法对１００株标准细胞及９１份血清学分型困难标本分别作ＨＬＡ－Ｂ位点分析,前者为作ＰＣＲ－ＳＳＰ方法的参考对照品。结果：１０００株标准细胞分析结果与已知结果一致;９１例疑问标本均顺利地区ＰＣＲ－ＳＳＰ分型,结果共有７０种等位基因格局,其中３６种与血学 同,占５２％,ＰＣＲ－ＳＳＰ     

用PCR—SSP作HAL—B位点分型及B22亚型分析

目的：用ＤＮＡ分型弥补血清学ＨＬＡ－Ｂ位点分型的欠缺并了解南方汉族Ｂ２２亚型分布情况。方法；采用标准细胞作参照，建立Ｂ位点的ＰＣＲ－ＳＳＰ分型方法地血清学Ｂ２２阳性标本进行Ｂ２２亚型分析。结果：１０４株标准细胞ＰＣＲ－ＳＳＰ分型结果与已知结果完全一致，１７例白血病人及同胞分型与血清学一致，１例不符；Ｂ２２亚型以３＾＊５４０１最多，Ｂ＾＊５６０１较少，１例分型格局异常，可能为新Ｂ２２等位基因或错配的     

HLA-A位点PCR-SSP基因分型和血清学分型的比较

目的：建立优化ＨＬＡ－Ａ位点ＰＣＲ－ＳＰ分型方法。方法：采用普通扩增仪和Ｅｐｐｅｎｄｏｒｆ管对细胞系ＤＮＡ和３７个健康正常人进行基因分型,血清学检测采用标准淋巴细胞毒试验方法。结果：发现引物浓度和浓度比、ＤＮＡ的纯度及酶的选用,是影响ＨＬＡ－Ａ位点ＰＣＲ－ＳＳＰ分型准确性的重要因素。该方法对３７个标本的基因分型结果与血清学结果相符合。结论：ＰＣＲ－ＳＳＰ方法具有简便准确的优点,可以作为ＨＬＡ－Ａ位     

HLA-B27基因检定技术

用聚合酶链反应和血清学方法检测ＨＬＡ－Ｂ２７，并进行比较。方法：采用ＰＣＲ技术７８例可疑为强直性脊椎炎患者样本的ＨＬＡ－Ｂ２７基因，并与血清学比较。结果：３６例具有Ｂ２７基因者中２４例同时作血清学试验，５例血清学难以判定结果，并与ＰＣＲ结果不一致。     

HLA-B40交叉反应组高分辨度DNA分型

目的　采用顺序特异引物聚合酶链反应技术（ＰＣＲＳＳＰ） ， 建立汉族人群ＨＬＡＢ４０ 交叉反应组高分辨度ＤＮＡ 分型方法。方法　Ｂ４０ 组标准ＤＮＡ１０ 份，血清学Ｂ４０ 阳性临床样本１６４ 份，快速盐析法提取模板ＤＮＡ。设计合成１３ 个特异引物和１ 对阳性对照引物，组成８ 个ＰＣＲ 反应，建立一步法ＰＣＲＳＳＰ 快速ＨＬＡＢ４０ 组高分辨度ＤＮＡ 分型方法。结果　所有样本和标准ＤＮＡ 采用ＰＣＲＳＳＰ 分型均获得成功，可准确分辨出Ｂ４０ 组所有等位基因。无假阳性和假阴性出现，重复率１００ ％ ，总耗时４ ～５ 小时。分型结果经双盲验证符合。临床应用结果显示： 三分之一的汉族人群存在Ｂ４０组抗原，Ｂ６０ 占绝大多数（６７ ．３ ％ ） ，血清学对Ｂ４０ 组的误差率达１０ ％ 。本方法比血清学更加准确， 对血清学分型存在困难、亚型分辨不清的纯合子或杂合子均能作出准确的分型。结论　本研究建立的ＨＬＡＢ４０ 组ＰＣＲＳＳＰ 高分辨度ＤＮＡ 分型方法， 分辨度高、特异性强、结果精确可靠、明显优于血清学方法，适合于临床应用。     

应用微量SSP法进行器官移植供受者HLA-Ⅱ类基因分型

目的：探讨应用于临床器官移植的组织配型新技术。方法：采用快速ＤＮＡ抽提技术，建立微量ＰＣＲ－ＳＳＰ－ＨＬＡ－ＤＲＢ／ＤＱＢ基因分型方法，并与单克隆抗体一步法分型结果进行对比研究。结果：应用２种方法对７３份标本进行ＨＬＡ－Ⅱ类抗原／基因分型，结果完全相符，但微量ＰＣＲ－ＳＳＰ法不但快速，需血量少，而且分辨率高。结论：微量ＰＣＲ－ＳＳＰ法具有快速，准确，省血等优点，适合在临床器官移植配型中推广应用。     

HLA—A,B血清学分型与DNA分型的比较

目的：比较１３６例样本ＨＬＡ－Ａ，Ｂ血清学分型与ＤＮＡ分型结果。方法：应用聚合酶链反应－序列特异性引物（ＰＣＲＳＳＰ）技术，设计３８对引物，检测常见的ＨＬＡ－Ａ，Ｂ基因。结果：１３６例样本中有６０例血清学分型与ＤＮＡ分型结构不符合（４４．１％）；ＨＬＡ－Ａ特异性血清学分型错误率在纯合子中分别占２３．３％与９．４％（共１１．２％），在ＨＬＡ－Ｂ中分别占４７．４％与１８．４％（共２０．６％）；ＨＬＡ－Ａ特异性血清学分型假阴性６．６％，假阳性２．５％，错误指认特异性２．１％，ＨＬＡ－Ｂ错异性则分别为６．７％，３．６％，１０．３％。结论：ＩＬＡ－Ａ纯合子血清学分型错误率明显高于杂合子（Ｐ〈０．０５）；ＨＬＡ－Ｂ纯合子血清学分型错误率也明显高于杂合子（Ｐ〈０．００５）；ＨＬＡ－Ｂ特异性血清学分型错误率显著高于ＨＬＡ－Ａ特     

HLA－DPB131个等位基因的PCR－RFLP高分辨分型

本文报道建立了一种基于ＰＣＲ－ＲＦＬＰ技术的ＨＬＡ－ＤＰＢ１分型方法。经计算机分析而选出１０个限制性内切酶，在３１个ＤＰＢ１等位基因组成的４９６个ＤＰＢ１基因型中，可唯一性分辨４８４个。用于确定ＤＰＢ１基因型的３３个多态性酶切片段有２９个在８种纯合／杂合子分型细胞ＤＮＡ的ＤＰＢ１分型中得到验证。经研究来自十个家系２３人的ＤＰＢ１等位基因分布，证实在等位基因和杂合性多态片段的分布上均符合孟德尔分离律。与目前其他ＰＣＲ－ＲＦＬＰ和ＰＣＲ－ＳＳＯＤＰＢ１分型系统比较，本分型系统在分辨的ＤＰＢ１等位基因数目和唯一性反应格局率方面居于领先，特别适用于异基因骨髓移植供者的选择。     

HLA-B27基因在强直性脊椎炎中的临床应用

目的：为评价ＨＬＡ－Ｂ２７基因在强直性脊椎炎的临床诊断价值。方法：采用ＰＣＲ－ＳＳＰ地１４１例风湿性病人ＨＬＡ－Ｂ２７工与血沉、影像学Ｘ光片报告开面指标进行比较。结果：强直性脊椎炎病人组ＨＬＡ－Ｂ２７是性率６８．８％与风湿必 人组ＨＬＡ－Ｂ２７基因阳性率１１．４％,比较,Ｐ〈０．００５,有非常显著性划;强直性脊椎炎病人Ｂ２７基因阳性率６８．８％,与血沉结果升高率８７．５％比较,Ｐ〉０．０５,无显著     

血清学指定HLA-B座位纯合型误差分析及HLA-B座位表达变异检测的意义

目的 :用DNA分型法对HLA B座位抗原纯合型进行分型 ,探明是否存在HLA B基因发生变异引起结果差异。方法 :经血清学鉴定HLA B座位纯合型样本 75例 ,用PCR SSP法进行DNA分型和表达变异筛查。结果 :75例血清学鉴定为纯合型后经DNA分型证实有 12例存在第二基因 ,经PCR SSP法进行变异筛查发现有 1例存在第四外显子额外胞嘧啶插入 ,经DNA分型发现的第二基因实为沉默基因。结论 :用PCR SSP法进行HLA B基因分型可弥补血清学方法的不足 ,同时筛查表达变异使分型结果更加准确 ,能有效地指导临床应用。     

HLA-B27 PCR辅助诊断幼年强直性脊椎炎

目的：辅助诊断幼年强直性脊椎炎。方法：用ＰＣＲ技术对２４例临床表现为类风湿关节炎少关节型患儿的ＤＮＡ进行ＨＬＡ－Ｂ２７基因分析。结果：２４份标本中７例ＰＣＲ检测Ｂ２７基因阳性,占分析的２９．２％（７／２４）。７例Ｂ２７基因阳性病例中男５例,女２例,平均年龄１０．４岁。结论：Ｂ２７检测可作为诊断或鉴别诊断幼年强直性脊椎炎的重要指标,利于疾病病鉴别和预后估计。     

应用顺序特异性引物聚合酶链反应检测华南人群HLA-B座位第4外显子变异

目的：为探明中国华南人群中因HLA-B座位发生基因突变而引起表达变异存在的可能性。方法：用顺序特异性引物聚合酶链反应（PCR-SSP）对75例经血清血分型为纯合型样本再次分型，对有差异的结果使用PCR-SSP方法检测第四外显子突变情况。结果：75例血清学指定为纯合型标本经DNA分型证实有12例为杂合型。12例结果有差异的样本经PCR-SSP法表达变异的筛查发现有1例存在第四外显子额外胞嘧啶插入，经DNA分型发现的第二基因为沉默基因。结论：应用PCR-SSP方法可检测HLA-B等位基因突变，该突变导致HLA-B基因表达无效，进行HLA基因表达变异的检测可提高HLA分型的准确性，更加有效的指导临床进行器官和骨髓的选配。     

A case of maternal–foetal chimerism identified during routine histocompatibility testing for hematopoietic stem cell transplantation

This report describes a case of maternal‐foetal chimerism identified in a boy diagnosed with SCID, who underwent HLA testing in preparation for HSCT. The first analysis was carried out on DNA from peripheral blood and included HLA‐A, HLA‐B, HLA‐DRB1 typing using PCR‐SSO. The patient's HLA‐B typing results were noninterpretable. All samples were re‐typed for HLA‐B using PCR‐SSP, again resulting in noninterpretable typing of patient's HLA‐B. In both cases, several weak positive probes/reactions interfered with the interpretation when using commercial software. Next round of HLA typing, using PCR‐SSP and PCR‐SSO methods, included the patient's bone marrow sample and HLA‐C locus, but interpretation was again not possible. The PCR‐STR analysis performed on both peripheral blood and bone marrow samples revealed seven STRs for which two maternal and one paternal allele were detected. Retrospective manual interpretation of HLA‐B and HLA‐C typing revealed that weak positive reactions were indeed owed to paternal HLA‐B and HLA‐C alleles and that the patient had both maternal and one paternal allele. Retyping of HLA‐B and HLA‐C loci and STR analysis on the patient's buccal cells sample revealed the expected one maternal/one paternal allele pattern. In summary, the combination of several different typing methods and manual interpretation were necessary to obtain the patient's HLA typing results.     

HLA-B27 genotyping by fluorescent resonance emission transfer (FRET) probes in real-time PCR

Several polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) genotyping methods are in use, but none is fully satisfactory. The introduction of real-time PCR (rt-PCR) with fluorescence resonance energy transfer (FRET) probes provides a powerful tool to overcome the drawbacks of current methods such as the long processing time and the requirement for post-PCR manual procedures. Here we present evidence that the FRET-fluorotyping principle may resolve HLA-B27 variants, providing a higher resolution in less time than the techniques currently in use. The protocol uses between one and three consecutive amplification reactions depending on the resolution required. The first reaction, aimed at detecting HLA-B27-positive samples, uses beta-globin coamplification as control. The second reaction, aimed at resolving most frequent B27 alleles, uses two hybridization probes whose melting temperatures curves allow the classification of HLA-B27 alleles into eight groups. By adding a third reaction, even the rarest alleles associated and not associated to ankylosing spondylitis (AS) may be discriminated. The technique was blindly tested on 60 samples from individuals previously typed and confirmed by standard PCR sequence-specific oligoprobes-PCR sequence and PCR-based typing PCR-SBT (30 B27+, 30 non-B27). There was a complete concordance rate, thus confirming the potential of this new technique for clinical HLA-B27 typing and for HLA typing in general.     

A modified PCR‐SSP method for the identification of ABO blood group antigens

The ABO blood group antigens are carbohydrate molecules synthesized by the glycosyltransferases encoded by the ABO gene on chromosome 9. Kidney transplantation across the ABO barrier generally leads to rapid humoral graft rejection due to the presence of naturally occurring antibodies to the A and B antigens. We have developed a method for ABO typing our cadaveric organ donors by the polymerase chain reaction using sequence‐specific primers (PCR‐SSP). The method uses 12 primers in eight PCR mixtures and is performed under the same conditions as our routine HLA‐A, B, C PCR‐SSP typing. The PCR‐SSP‐based types of 166 regular blood donors and 148 cadaveric organ donors all showed total concordance with their serologically assigned ABO groups. Six individuals possessing the ABO A subgroups (A3, Ax and Aend) all typed as A1 by PCR‐SSP, as expected. PCR‐SSP is an appropriate method for ABO typing of cadaveric organ donors and, importantly, enables both ABO and HLA typing to be performed on the same DNA material.     

HLA-B27 subtypes determination in patients with ankylosing spondylitis from Zulia,Venezuela

To perform an investigation regarding the distribution of the human leukocyte antigen (HLA)-B27 subtypes in the Zulian population with ankylosing spondylitis (AS), 48 unrelated Mestizos, HLA-B27 positive by serology, were studied using the polymerase chain reaction-specific sequence oligonucleotides probe (PCR-SSOP) and specific sequence primers (SSP) to analyze the polymorphism in exons 2 and 3 of the HLA-B27 gene. Only two of eight HLA-B27 subtypes studied (B*2701-B*2708) were found. The distribution of these alleles in the population of patients was: B*2705, 68.8%, and B*2702, 31.2%. B*2705 subtype showed significant association with patients being male. In the healthy controls, the most common subtype was B*2708. These results were compared with frequencies reported in other Mestizo and Spanish populations and showed significant differences, such as a high frequency of B*2702. Such results show that HLA*B2705 and HLA*B2702 are the subtypes most frequently associated with AS in our Mestizo population and suggest a possible protector role for HLA*B2708, which was found only in the healthy population.     

Functional characterization and exon 2 – intron 2 – exon 3 gene sequence of HLA-B*2712 as found in a British family

The HLA-B*27 group of alleles has been extensively studied due to the association of particular B*27 alleles with ankylosing spondylitis (AS). We describe here an HLA-B*27 allele (B*2712) encoding an antigen that lacks reactivity with B27 monoclonal antibodies (moabs) and alloantisera but reacts with some B40/B60 moabs and alloantisera and expresses the Bw6 public epitope. This allele was discovered by the segregation of an HLA-B allele undetectable by PCR–SSP within a Caucasian family from the British population referred for routine bone marrow transplant HLA typing and found in the haplotype A*29; B*2712 ; Cw*1203; DRB1*13; DQB1*0603. Serological typing showed a lack of reactivity with four B27 moabs and four alloantisera but positive reactivity with moabs and alloantisera specific for B40/B60 and Bw6 public epitopes. Subsequent sequencing showed the closest homology was with B*2708 with three mismatches in exon 2 at positions 204, 209 and 210. The intron 2 sequence was identical with other B*27 lineage alleles including a 2 base pair deletion at positions 95 and 96. The relationship between HLA-B*2712 and reported B60 associations with susceptibility to AS remains to be determined.