使用PCR-SSP方法检测HLA-DR抗原

目的采用顺序特异引物聚合酶链反应(PCR-SSP)建立人类白细胞抗原DR位点的DNA分型方法.方法合成29个特异性引物和1对阳性对照引物,组成20个PCR反应用于DR位点,建立一步法PCR-SSP.结果所有样本PCR-SSP基因分型获得成功,分型结果经标准DNA,限制性核酸内切酶分析证实符合,特异性和重复性100%.结论 PCR-SSP检测HLA-DR的方法具有快速、准确、特异性高等优点,适合临床应用.     

顺序特异性引物聚合酶链反应技术对HLA-DR DNA的分型

目的：应用顺序特异性引物聚合酶链反应技术(PCR—SSP)进行临床肾移植供受者HLA—DR位点DNA的分型。方法：设计并合成HLA—DR位点16对特异性引物和1对阳性对照引物，建立PCR—SSP法，对52份临床肾移植供受者的外周血淋巴细胞样本进行DR位点基因的分型。结果与微量PCR—SSP基因分型试剂盒分型方法比较。结果：所有临床样本的PCR—SSP基因分型均获得成功，结果与微量PCR—SSP基因分型试剂盒分型方法完全相同，分型时间3h，特异性和重复性100％。结论：应用合成引物为临床肾移植供受者进行HLA—DR位点PCR—SSP基因分型简便快捷，重复性好，适合于临床应用。     

HLA-DRB1基因多态性与急性淋巴细胞白血病和慢性髓性白血病易感性关联的研究

目的探讨急性淋巴细胞白血病（ALL）和慢性髓性白血病（CML）易感性与HLA-DRB1基因多态性之间的关联性,找出急性淋巴细胞白血病和慢性髓性白血病的易感基因。方法采用序列特异性引物聚合酶链反应（PCR-SSP）DNA分型技术对56例ALL患者、48例CML患者和105例健康对照进行了HLA-DRB1基因分型。结果ALL患者组与HLA-DR7基因关联,基因频率为24.1%,RR=2.56,χ     

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。     

使用PCR—SSP方法检测HLA—DR抗原

目的　采用顺序特异引物聚合酶链反应 (PCR -SSP)建立人类白细胞抗原DR位点的DNA分型方法 .方法　合成 2 9个特异性引物和 1对阳性对照引物 ,组成 2 0个PCR反应用于DR位点 ,建立一步法PCR -SSP .结果　所有样本PCR -SSP基因分型获得成功 ,分型结果经标准DNA ,限制性核酸内切酶分析证实符合 ,特异性和重复性 10 0 % .结论　PCR -SSP检测HLA -DR的方法具有快速、准确、特异性高等优点 ,适合临床应用 .     

HLA-DR位点的基因芯片分型与临床应用

目的：建立一种新型的HLA－DR位点的基因分型方法，为HLA－DR的基因分型提供一个较新的思路。方法：利用基因芯片技术HLA不同基因亚型的独特序列设计探讨，制成分型芯片；待检测样品经PCR反应标记上荧光之后，与芯片进行杂交，根据杂交产生的荧光信号值分析确定样品DR位点的基因亚型，将这一方法应用于70份标准DNA和200份医院移植供受者的HLA－DR基因分型并将部分样品进行基因测序。结果：检测结果表明HLA－DR基因分型芯片可准确分辨出DR位点等位基因30大类，耗时3h 。结论：基因芯片用HLA－DR的基因分型，分辨率高，特异性强，重复性好，操作简便，对比常规的PCR－SSP和PCR－SSO方法，HLA－DR基因芯片方法更为直观，并具有集成化优势。可以在一张芯片上同时检测HLAⅠ类的A、B位点，并实现一张芯片多人份，适合于临床应用和骨髓库，脐血库的建立。     

血清学指定HLA—B座位纯合型误差分析及HL—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。     

新疆维吾尔族人自然长寿与HLA-DRB、ACE基因的关联分析

目的探讨人类白细胞抗原(HLA)-DRB基因及血管紧张素转换酶(ACE)基因多态性与新疆维吾尔族人自然长寿的关系.方法研究样本分为4组第1组年龄≥100岁;第2组年龄为90～99岁;第3组年龄为70～90岁;第4组为65-70岁自然死亡者.分别应用聚合酶链反应-序列特异引物法(PCR-SSP)、单链构象多态性(SSCP)分析和直接测序技术对各组进行基因分型.结果百岁组HLA-DR1的频率明显高于对照组(分别为9.5%和1.9%,P＜0.05);HLA-DR6(14)的频率也明显高于对照组(分别为9.5%和0.9%,P＜0.05);HLA-DR6的频率明显高于其余3组;HLA-DR4的频率明显低于对照组(分别为9.5%和23.6%,P＜0.05);HLA-DR9的频率明显低于对照组(分别为1.2%和9.4%,P＜0.05).相关分析显示HLA-DR1及ACE基因的D等位基因与长寿呈正相关,而HLA-DR9呈负相关.结论HLA-DRB基因的DR1等位基因和ACE基因的等位基因D对长寿可能有保护作用;HLA-DRB基因的DR9等位基因可能是不利于长寿的危险因素,HLA-DRB基因多态性和ACE基因多态性相互作用对长寿有协同作用.     

中国汉族白血病患者及其相关人群罕见的HLA-DR/DQ连锁不平衡研究

 目的 研究中国汉族白血病患者及其相关人群罕见的HLA-DR/DQ连锁不平衡单倍型。方法 对2000－2005年在我院进行异基因造血干细胞移植前HLA配型的白血病患者及与患者有血缘关系的家系供者共1500例的血液标本，采用低分辨序列特异性引物聚合酶链反应（PCR-SSP）方法进行HLA-DR/DQ基因分型，并对两位点间连锁不平衡参数进行统计学分析。1500例中患者650例，平均年龄25岁；家系供者850例，平均年龄42岁。结果 在41例的血液标本中发现13种罕见的连锁不平衡单倍型，主要为HLA-DQ8 或HLA-DQ9与不同DR位点的连锁。其中DR14/DQ4、DR4/DQ5、DR9/DQ6、DR9/DQ7、DR8/DQ8、DR9/DQ8、DR12/DQ8、DR13/DQ8和DR14/DQ9共9种单倍型尚未见报道。650例白血病患者中有20例存在12种罕见的连锁不平衡单倍型，850例家系供者中有21例存在8种罕见的连锁不平衡单倍型。DR8/DQ8单倍型只见于家系供者，而DR14/DQ4、DR12/DQ6、DR11/DQ8、DR13/DQ8和DR14/DQ9单倍型则只见于白血病患者。41例HLA-DR/DQ基因分型结果显示，连锁不平衡单倍型与DR52（DRB3）宽抗原相关联者占58.5%（24/41），与DR53（DRB4）宽抗原相关联者占36.6%（15/41），而与DR51（DRB4）宽抗原相关联者仅占4.9%（2/41）。所发现单倍型频率最高的为DR12/DQ8（0.0023）和DR9/DQ8（0.0023），其次为DR11/DQ9（0.0020）和DR12/DQ9（0.0017）。13种连锁不平衡单倍型的绝对及相对连锁不平衡参数均为负值，说明它们在中国汉族人群中较为罕见，并处于连锁不稳定状态。结论 发现了罕见的DR/DQ连锁不平衡单倍型，对补充中国汉族人群HLA-DR/DQ基因的连锁不平衡类型，提高HLA分型结果的准确性具有一定意义；同时，DR/DQ连锁不平衡单倍型在不同人群中的差异为疾病关联研究提供了思路。     

肾移植患者尿液中供者细胞DNA变化在急性排斥反应诊断中的意义

目的：探讨肾移植受者尿液中供者细胞的出现与急性排斥反应的相关关系及其临床意义。方法：以供者为男性、受者为女性或HLA-DR抗原有错配的80例肾移植受者为研究对象，定期收集尿液标本，从中提取DNA，利用特异性引物聚合酶链反应分别检测Y染色体上特异的基因片段DYZ-1和HLA-DR抗原的基因DRB-1。结果：手术当天受者的尿液中即有供者细胞出现，随着时间的推移，尿液中供者细胞DNA的基因表达强度逐渐减弱，直至术后30天，仍有90．0％受者的尿液中有供者细胞DNA的基因表达，其中8例(29．6％)发生了急性排斥反应；出院后发生急性排斥反应者，90．0％的尿液标本中能检测到供者细胞DNA，抗排斥治疗结束后2周，83．3％转为阴性；肾功能良好的稳定期受者，仅6．7％的受者尿液中DYZ-1或HLA-DRB基因阳性。结论肾移植受者尿中供者细胞DNA的检测可以作为诊断急性排斥反应的一种方法，其基因表达强度变化为定量评价排斥反应提供了可能性。     

REPORT FROM THE HLA CLASS II TYPING BY PCR-SSP MULTICENTRE STUDY

Results from 360 HLA-DR and -DQ ‘low-resolution’ typings with polymerase chain reaction sequence-specific primers (PCR-SSP), performed by nine laboratories, were analysed for their overall utility in routinely defining the HLA-DR1–DR18, DR51–DR53 and DQ1–DQ9 specificities in less than 2.5 h. Thirty EDTA blood samples and 10 DNA samples were distributed and analysed by each laboratory. DNA was extracted using a rapid bromide salt extraction protocol. Complete HLA-DR and -DQ typings were performed, three by three, on pre-aliquoted 96-tube PCR trays. When compared with reference typing, 351/360 (98%) correct DR typings were obtained, whereas 320/360 (89%) of the DQ phenotypes were correctly assigned. The time for three complete HLA-DR and -DQ ‘low-resolution’ typings, including DNA extraction, ranged from 2.0 h to 2.3 h. Unfortunately, an unusually high level of PCR amplification failures was observed (3%), probably due to diffusion and a significant volume loss from some of the pre-aliquoted primer mixes. Consequently, only 52% of the typings were without any amplification failure, and 0–2 amplification failures where found in 88% of the PCR-SSP typings performed. The number of HLA-DR–DQ retypings needed was 7 and 8%, respectively, reflecting the low number of typings where allelic identification was directly affected by the relatively high level of amplification failures in this study. Thus, a 91–98% success rate of correctly identified HLA-DR and -DQ alleles could be maintained, even under suboptimal typing conditions.     

Rapid DNA typing for HLA-C using sequence-specific primers (PCR-SSP): Identification of serological and non-serologically defined HLA-C alleles including several new alleles

Abstract: Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undectectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.     

A rapid semi automated method for DNA extraction from dried-blood spots: application to the HLA-DR shared epitope analysis in rheumatoid arthritis

Genomic DNA extraction for genotyping analysis is performed from blood samples and is time consuming. We describe a more rapid DNA extraction method, "DBS-miniMAG", that combines filter paper dried blood spots (DBS) with the NucliSens miniMAG semi-automated instrument (bioMérieux). To assess the performance of this method, a post-PCR HLA-DR shared epitope (SE) oligotyping assay was used as a read-out in a cohort of 72 arthritis patients. This new method was compared to the standard manual DBS extraction protocol using FTA reagents (Whatmann Bio-Science), and to a reference phenol-chloroform-based method using EDTA whole blood samples. Higher yield of PCR amplicons was observed with DNA extracts obtained using "DBS-miniMAG" method. The intra- and inter-assay variability of the "DBS-miniMAG" method was similar to that obtained with "DBS-FTA" washing process. Concerning the HLA-DR SE genotyping, "DBS-miniMAG" and "DBS-FTA" methods gave 100% concordance compared to the reference phenol-chloroform method. More importantly, the hands-on time and the turnaround time for "DBS-miniMAG" were both two-times shorter than for "DBS-FTA" protocol. Therefore, the "DBS-miniMAG" combination could facilitate polymorphism analysis in routine clinical practice and the creation of large DNA banks using very small amounts of blood.     

HLA-DR typing for kidney transplants: advantage of polymerase chain reaction with sequence specific primers in a routine hospital laboratory.

AIMS--To determine whether the polymerase chain reaction with sequence specific primers (PCR-SSP) can assign HLA-DR type more accurately than serology in a routine hospital laboratory. METHODS--The 93 patients currently awaiting kidney transplants have been DR typed by serology over the past 14 years, 82% within the past five years. They have now been retyped using the PCR-SSP method described by Bein et al. Where the two results differed, PCR-SSP was repeated, once by the same method and once using the primer set of Olerup and Zetterquist. RESULTS--There were 13 (14%) discrepancies between the results. Of these, two were PCR-SSP failures, later overcome: three were failure to detect DRB1*0103 by serology; five assignment of other alleles by PCR-SSP to serological "blanks"; and three alleles were differently assigned by serology and PCR. The serological typing of the final patient when repeated for this study was at variance with the original findings (14 years ago), but in agreement with PCR. In the remaining patients, serology had not determined the split of 36 DR3 alleles (all DR17 by PCR-SSP) or 13 DR6 alleles (12 DR13 and one DR14 by PCR-SSP). One patient in each case had their antigen splits of DR2 and DR5 assigned by PCR-SSP (DR15 and DR11, respectively) but not by serology. CONCLUSIONS--PCR-SSP provides more reliable and detailed information on HLA-DR polymorphism than serology, and does so within a routine tissue typing laboratory.     

Evidence for the involvement of two different MHC class II regions in susceptibility or protection in allergic bronchopulmonary aspergillosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a disease with uncertain pathology. Studies have suggested a pathogenic role for T(H)2 cells. Previously, we demonstrated, in a small group of patients, that T(H)2 reactivity to a major Aspergillus fumigatus antigen was restricted by HLA-DR2 or HLA-DR5 alleles. OBJECTIVES: We sought to confirm whether susceptibility to ABPA is exclusively associated with HLA-DR locus and to investigate the involvement of HLA-DQ genes in the development of ABPA. METHODS: Genomic DNA was extracted from patients with ABPA, patients without ABPA but with positive A fumigatus skin test responses and asthma or cystic fibrosis, and healthy control subjects. HLA-DR and HLA-DQ genes were detected by using low-resolution typing; high-resolution typing was done only on HLA-DR2- and HLA-DR5-positive individuals by using sequence-specific primers (PCR-SSP). RESULTS: A significantly higher frequency of HLA-DR2 was observed in patients with ABPA versus those without ABPA (corrected P <.01) or healthy control subjects (corrected P <.01). Genotype analysis revealed that susceptibility to ABPA is associated with HLA-DR2 alleles DRB1*1503 and DRB1*1501 and, to a lesser extent, with the HLA-DR5 allele DRB1*1104. The presence of DR4 or DR7 alleles in non-DR2/5 patients with ABPA suggests that these alleles may also be contributing factors in this disease. Another striking observation was the significantly high frequency of HLA-DQ2 in patients without ABPA (67. 4%) compared with patients with ABPA (20.5%) and normal control subjects (37.7%), suggesting that these alleles may confer protection in the population without ABPA. CONCLUSION: These genetic studies suggest that HLA-DR molecules DR2, DR5, and possibly DR4 or DR7 contribute to susceptibility while HLA-DQ2 contributes to resistance and that a combination of these genetic elements determines the outcome of ABPA in patients with cystic fibrosis and asthma.     

云南彝族2型糖尿病与HLA-DRB1等位基因多态性关联的研究

目的探讨云南彝族2型糖尿病与HLA-DRB1等位基因多态性的关联。方法采用聚合酶链反应-序列特异性引物技术,对云南楚雄地区彝族2型糖尿病患者79例和同地区47名彝族正常对照进行HLA-DRB1等位基因分型,做2型糖尿病与HLA-DRB1等位基因多态性关联分析。结果云南彝族2型糖尿病组与正常对照组相比,DR7、DR11等位基因频率均高于正常对照组,差异有统计学意义(P=0.009,RR=8.329;P=0.029,RR=7.734)。结论DR7、DR11等位基因可能是云南彝族2型糖尿病的易感基因。     

Rapid HLA-DR fluorotyping based on melting curve analysis

Real-time polymerase chain reaction (PCR) has been used in the study of human leukocyte antigen (HLA) genotyping as a potential alternative for routinely used molecular methods such as PCR-sequence specific primers (PCR-SSP) and PCR-sequence specific oligonucleotide probes (PCR-SSO). Combined with fluorescent dye like SYBR GREEN I, it has more advantages such as low cost and consistent background. The aim of this study was to optimize the fluorescent dye-based method and introduce it into the fluorotyping for HLA-DR lotus. 24 pairs of allele-specific primers and 1 pair of internal control were optimized to discriminate HLA-DRB1, -DRB3/B4/B5 alleles. Additionally, conditions of real-time PCR amplifying and melting curve recording had been improved for convenient and clear readout. Forty-two clinical samples previously typed by conventional PCR-SSP or sequence based typing (SBT) were tested and all got identical results. With this technique, 15 DNA samples can be assayed in parallel within 2 hours on the Real-time PCR instrument. These data strongly suggest a rapid HLA-DR fluorotyping method based on melting curve analysis, which could be a more economic and automatic alternative for clinical HLA-DR typing.     

Rapid HLA-DR Fluorotyping Based on Melting Curve Analysis

Real-time polymerase chain reaction (PCR) has been used in the study of human leukocyte antigen (HLA) genotyping as a potential alternative for routinely used molecular methods such as PCR-sequence specific primers (PCR-SSP) and PCR-sequence specific oligonucleotide probes (PCR-SSO). Combined with fluorescent dye like SYBR GREEN I, it has more advantages such as low cost and consistent background. The aim of this study was to optimize the fluorescent dye-based method and introduce it into the fluorotyping for HLA-DR lotus. 24 pairs of allele-specific primers and 1 pair of internal control were optimized to discriminate HLA-DRB1, -DRB3/B4/B5 alleles. Additionally, conditions of real-time PCR amplifying and melting curve recording had been improved for convenient and clear readout. Forty-two clinical samples previously typed by conventional PCR-SSP or sequence based typing (SBT) were tested and all got identical results. With this technique, 15 DNA samples can be assayed in parallel within 2 hours on the Real-time PCR instrument. These data strongly suggest a rapid HLA-DR fluorotyping method based on melting curve analysis, which could be a more economic and automatic alternative for clinical HLA-DR typing.