单管PCR扩增鉴定ABO血型基因型

本研究目的是建立单管PCR扩增鉴定ABO血型基因型的方法.采用盐析法抽提DNA,应用单管PCR扩增结合基因扫描技术对ABO血型的基因型进行鉴定.结果表明：用本方法鉴定出的132例样本的ABO基因型结果与其血清学结果完全符合,A、B、O基因频率分别为0.205、0.159、0.636,其中AA基因型8例（6.1%）,AO基因型31例（23.5%）,AB基因型7例（5.3%）,BB基因型6例（4.5%）,BO基因型23例（17.4%）,OO基因型57例（43.2%）.结论：单管PCR扩增结合基因扫描技术能鉴定ABO血型基因型.     

应用PCR—SSP法分析中国人血小板抗原基因型频率

本研究建立了在同一PCR反应条件下同时检测人血小板抗原（human platelet antigen,HPA）系统HPA-1到HPA-5的PCR-SSP检测法。应用该方法分析了110例健康献血员的HPA-1到HPA-5的基因型,并以此为依据推算了中国人HPA-1到HPA-5各亚型的基因频率。结果表明HPA-1a和HPA-1b的基因频率分别为0.91和0.09,HPA-2a和HPA-2的基因频率分别为0.86和0.14,HPA-3a和HPA-3b的基因频率分别为0.60和0.40,HPA-4a和HPA-4b的基因频率分别为0.92和0.08,HPA-5a和HPA-5b的基因频率分别为0.85和0.15。结论：基因组DNA的血小板抗原PCR-SSP分型法切实可行,可广泛应用于临床血小板抗原的分型。     

序列特异性引物PCR法检测MN血型基因型

目的　建立MN血型系统基因分型的方法 ,检测人群中MN基因的频率。方法　用快速盐析法抽提样本DNA ,序列特异性引物PCR法检测MN基因。结果　 115例汉族人群中MM基因型为 2 5例 ,MN基因型为 6 0例 ,NN基因型为 30例。M基因频率为 0 .4 78,N基因频率为 0 .5 2 2。结论　该方法可以检测MN血型基因型。浙江汉族中N基因频率大于M基因频率     

HLA—Ⅰ、Ⅱ类PCR—SSP基因分型在移植配型及亲子鉴定中的应用

目的：建立快速、准确的HLA基因分型方法,满足临床移植配型的需要。方法：对25对已进行HLA抗原血清学分型的骨髓移植的供、受者,12例亲子鉴定应用聚合酶链反应－序列特异引物（PCR－SSP）进行HLA－Ⅰ、Ⅱ类基因A、B、DRB1、DQB1位点扩增,琼脂糖凝胶电泳分析PCR产物并确定其基因型。结果：基因分型结果与血清学分型一致者有22对,基因分型能明显判断而血清学分型无法判断者有3对;排除亲子关系的有2例。结论;PCR－SSP方法具有操作简单、快速、结果可靠的特点,不仅适用于移植配型、法医学亲子鉴定和个人识别,也可用于相关疾病及人类遗传学的研究。     

婴儿ABO血型的鉴定及应用于临床输血

为了研究6个月内婴儿ABO血型正确定型方法及影响因素，采用常规血清学法、凝胶柱法及PCR-SSP3种方法对6个月以内的婴儿进行血型鉴定。结果表明：在使用不同方法检测婴儿ABO血型结果不符的33例中，常规血清学方法32例红细胞定型与血清定型不符，1例是由细菌感染产生类B抗原所致的假相合。凝胶柱法可正确定型27例，正确率84.4%。PCR-SSP法可对所有标本做出正确定型。凝胶柱法与PCR-SSP法有显性差异。结论：对6个月以内的婴儿进行ABO血型鉴定，推荐采用凝胶柱技术。凝胶柱法红细胞定型与血清定型相符的婴儿输血采用同型输注，但若婴儿患有感染性疾病时，应考虑到类B抗原引起的假相合。凝胶柱法红细胞定型与血清定型不符的婴儿输血时使用O型洗涤红细胞等成分血，待PCR-SSP法分型结果做出后，改为同型输血。     

序列特异性引物PCR法检测Rh血型E/e基因型

目的　建立Rh血型E/e基因分型的方法，以检测人群中E/e基因频率。方法　采用快速盐析法抽提样本DNA，采用PCR—SSP方法检测E/e基因。结果　在本研究的RhD阳性人群中E基因频率为0.223，e基因频率为0.777。RhD阴性人群中E基因频率为0.040，e基因频率为0.960。结论　人群中以e基因为主，Rh阳性与阴性人群中E和e基因频率存在明显的差异。     

PCR—SSP分析视网膜色素膜炎易感基因

采用聚合酶链式反应和顺序特异性引物（ＰＣＲ－ＳＳＰ）基因分析方法，对视网膜色素膜炎患者２９例及３０例无血缘关系的健康人的人类主要组织相容性复合体（ＭＨＣ）ＤＱ各等位基因及亚基因进行了检测分析。实验结果显示：ＭＨＣ－ＤＱＢ１＊０３０１基因可能是视网膜色素膜炎的相关基因之一；ＰＣＲ－ＳＳＰ具有快速、简便、敏感、准确和可靠等优点，值得推广应用。     

基因扫描技术在ABO血型基因型鉴定中的应用

目的 建立基因扫描（GeneScan）技术鉴定ABO血型基因型的方法。方法根据O1基因在261位G缺失而非O1基因无缺失，B基因第803位核苷酸与A基因不同的特点，设计4对引物，分别在上游引物5’端标记荧光基团，PCR-SSP扩增后，用基因扫描技术鉴定ABO血型基因型。结果129例样本用Genescan技术鉴定的ABO血型结果与血清学结果完全符合，A、B、O基因频率分别为0．198、0．159、0．643。其中AA基因型8例（6．2％），AO基因型29例（22．5％），AB基因型6例（4．7％），BB基因型6例（4．7％），BO基因型23例（17．8％），OO基因型57例（44．2％）。结论Genescan技术鉴定ABO血型基因型具有可行性，提供了一种可选择的方法。     

聚合酶链式反应-特异性序列引物基因定型在新生儿溶血病ABO血型定型中的应用

目的研究聚合酶链式反应-特异性序列引物(PCR-SSP)基因定型在新生儿溶血病(CHDN)ABO血型定型中的应用。方法收集20例来自广东省肇庆市第一人民医院新生儿溶血病(HDN)的样本,对这些样本进行ABO血型鉴定等一系列血型血清学方法进行检测,然后用PCR-SSP基因定型技术进行样本的基因分型。结果与其血型的血清学分型结果进行比较,新生儿溶血病ABO血型的定型是新生儿输血的必要保障,而PCR-SSP基因分型较血型血清学方法更快捷、准确。结论 PCR-SSP基因正型技术将不再局限于疑难血型的鉴定中,并会越来越多地应用于HDN的鉴定中。     

中国汉族人群ABO血型系统基因分型研究与应用

目的 研究中国汉族人群ABO基因多态性，并将基因分型技术用于解决临床输注中血型血清学难题：方法 快速盐析法提取外周血中的DNA，采用聚合酶链反应—序列特异性引物(PCR—SSP)基因方法对ABO血型定型，并且在吸收放散试验、唾液血型物质凝集抑制等血清学试验基础上，用PCR—SSP法扩增疑难ABO血型的等位基因：结果 在中国汉族符合Hardy—Weeinberg平衡的随机群体(260人)中，检出O1、B、A101(A267c)和A102／103(A467r)四种等位基因，其基因频率分别为、0.5827,0．1．846、0.0096、0.2231：在疑难血型鉴定中6份血清学定为A2的标本中，只有2例基因分型确定为A201O1，其余均为A102／A103O1型，在一家系疑难血型鉴定中，兄弟三人均为类孟买型，ABO基因分型分别为A102B、A102B、A102O1.结论 ABO RCR—SSP基因分型是一种方便、快速、可靠的技术，与血型血清学相比有着显著优势：研究发现中国汉族人群A2等位基因构成与国际报道的基因构成存在差异。     

Human platelet antigen-2 and -3 genotyping by PCR-SSP

SUMMARY. Allele-specific PCR using sequence specific primers (PCR-SSP) is a simple and reliable technique to detect point mutations in genes. We have developed a PCR-SSP to enable the detection of a C-T mutation at position 482 of the GPIb gene and a T-G mutation at position 13962 in exon 26 of the GPIIb gene. These point mutations are at the basis of the HPA alloantigens 2a, 2b and 3a, 3b respectively. One primer of each primer set has a 3' nucleotide complementary to the DNA sequence coding for one allele. PCR product is only produced when the corresponding DNA is present and thus the genotype is determined by the presence or absence of a band in agarose electrophoresis of PCR products. A second set of primers in the same reaction yields a product regardless of the HPA genotype to control the efficiency of the PCR amplification. The HPA-2 and -3 genotypes determined in this way were in strict concordance with those established by conventional genotyping using PCR followed by restriction enzyme digestion (PCR-ASRA). PCR-SSP is a rapid and reliable technique that can be used for the determination of alleles which code for platelet alloantigens.     

A simple and rapid genotyping method for beta-2 receptor (beta2 AR) gene using allele specific multiplex PCR

BACKGROUND: Polymorphism of the beta2-adrenergic receptor (beta2 AR) gene is an important determinant of the function of this receptor. It affects receptor down-regulation and beta2-agonist responses. It has also been a focus of interest in attempts to elucidate the genetic basis of asthma, hypertension, obesity and cystic fibrosis. Several different techniques have been established to determine beta2 AR genotypes but none of these methods are simple enough to detect simultaneously all the five alleles of our research interest (Arg16/Gly16, -20T/C, Gln27/Glu27, -47T/C and Thr164/Ile164). OBJECTIVES: To develop a simple and rapid PCR based method for the simultaneous detection of five beta2 AR alleles. METHOD: DNA was extracted from whole blood using standard alkali lysis method. Primers specific at the 3' end for the polymorphic sites were designed. The nested allele specific PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, Taq polymerase, primers and annealing temperature, to produce specific bands of interest. DNA samples were selected at random and submitted for direct PCR sequencing. RESULT: The first PCR produced a fragment of size 710 bp, which was used as template for the subsequent duplex and triplex PCR to detect the mutation sites of the five alleles. The method was found reproducible and specific when used to genotype patients with bronchial asthma. The sequencing results confirmed the specificity of the PCR method. CONCLUSION: The simple and rapid method for the simultaneous detection of the five beta2 AR alleles is suitable for the study of beta2 polymorphism and its clinical consequences.     

PCR-SSP技术对羊水细胞ABO血型的基因鉴定

目的通过PCR-SSP基因技术检测胎儿羊水细胞ABO血型基因型,产前诊断胎儿ABO血型。方法选取了6名孕16 W以上的孕妇,抽取羊水细胞并进行分离,提取羊水细胞DNA,运用PCR-SSP技术分析其ABO血型基因型,并通过出生后的脐带血的血型鉴定进行确认。结果 6例羊水标本均通过PCR-SSP方法检测出了ABO血型的基因型;该6名胎儿的脐带血的ABO血型与羊水细胞的血型一致。结论 PCR-SSP技术可以准确地检测胎儿羊水细胞的ABO血型。     

应用PCR—SSP方法进行人类血小板抗原1～6系统的基因分型

目的研究采用PCR—SSP技术，建立人类血小板抗原1．6系统（HPA—1，2，3，4，5，6）的基因分型方法。方法合成18条序列特异性引物，通过调节引物浓度、Mg^2＋离子浓度和探索最佳PCR扩增条件．建立HPA—1．6系统同步基因分型技术。对第10届及第11届国际输血协会（ISBT）血小板基因定型协作组送检的考核样本进行盲检来验证。并应用该技术对198名深圳地区健康的血小板志愿捐献者进行基因分型。结果应用本研究的方法，对第10届及第11届ISBT送检的考核样本进行基因分型，结果与ISBT公布的结果完全一致，符合率达100％。对198名随机的血小板志愿捐献者观察到的基因频率分别是：HPA—1a和1b为0．9924和0．0076，HPA-2a和2b为0．9545和0．0455，HPA-3a和3b为0．5556和0．4444，HPA-4a和4b为0．9975和0．0025，HPA-5a和5b为0．9848和0．0152，HPA-6a和6b为0．9798和0．0202。结论本研究建立的HPA基因分型技术具有简便、快速、准确的特点，适合于常规HPA基因分型，具有广泛的应用前景。     

Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers

D_el (D-elute) in the Rh blood group system is a variant with very weak D antigen and no agglutination is found by the indirect antiglobulin test. This variant is characterized by the presence of anti-D eluate obtained after an adsorption-elution test using anti-D antibodies. We studied here the molecular genetic status of D_el by using polymerase chain reaction with sequence-specific primers (PCR-SSP).
We screened 306 serologically apparent D-negative Japanese donors comprising 102 D_el types for exons 7, 4 and 10 of the RHD gene. No PCR product was found in all 204 non-D_el samples from the D-seronegative donors. However, PCR products were found in all 102 D_el samples and all 70 D-seropositive samples tested by the three PCR methods for exons 7, 4 and 10 analysis.
D_el was found with CCee, CcEe and Ccee, but not with CCEe, CcEE, ccEE, ccEe or ccee phenotype. The incidences of D_el in the samples with the serological phenotypes CCee, CcEe and Ccee were 80.0% (4/5), 68.2% (45/66) and 61.6% (53/86), respectively.
The results provide evidence that D_el samples have exons 4, 7 and 10 of an RHD gene present in some form. This is consistent with the evidence that D antigen is present on the cells although only detected by antibody adsorption and elution. The PCR-SSP method in the present study is useful to confirm D_el among serologically apparent D-negative samples.     

Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR-SSP method

Genotyping of the human platelet alloantigens (HPA) is useful for the diagnosis and therapy of the patients with alloimmune thrombocytopenic syndromes, such as post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura and foetomaternal alloimmune thrombocytopenia. We have developed, optimized and validated a new method for simultaneous genotyping of HPAs - HPA-1, HPA-2, HPA-3 and HPA-5 - by using the real-time polymerase chain reaction (PCR) based on TaqMan technology. Its performances were compared to those of the standard PCR-sequence-specific primers (SSP) method by testing 120 DNA samples. Several discrepancies between the two methods have been observed, especially in the HPA-3 genotyping. Evidently, the PCR-SSP method produced several false positive results due to its technical drawbacks. Based on our comparison, we believe that the new real-time TaqMan PCR assay for the HPA-1, HPA-2, HPA-3 and HPA-5 genotyping is faster, more reliable and reproducible, compared to the standard PCR-SSP.