单管双向等位基因专一性扩增的单核苷酸多态分型的新方法

目的：建立一种基于等位基因专一性PCR原理的单核苷酸多态（single nucleotidepolymorphism,SNP)分型新方法：单管双向等位基因专一性扩增（single-tube bi-directional allele specificamplification,SB－ASA）,并考察专一性引物的3’端第3位碱基不配对对特异延伸的影响。方法：一个PCR反应体系包含两个3’末端分别与SNP两个等位基因特异结合的引物,它们延伸方向相反,产生长度不同的等位基因专一扩增产物,同时在两个等位基因特异性引物的3’端第3位碱基引入不配对以增加特异性。PCR产物经琼脂糖凝胶电泳后分析确定样本的基因型。观察在不同的温度条件下,近3’末端引入与不引入碱基不配对时两种引物特异延伸的情况,比较两种引物能特异延伸的退火温度（annealingtemperature,Ta)范围。结果：对于4个不同类型的SNP位点,SB－ASA都成功地分型了36个样本,与直接测序的结果完全一致。两条专一性引物3’物第3位碱基引入不配对后,能特异延伸的退火温度Ta范围分别从64℃～69℃、60℃～62℃扩大到46℃～66℃、56℃～61℃。结论：SB－ASA是一种简单快速而有效的SNP分型新方法;在等位基因专一性PCR体系中,专一性引物3’端第3位碱基引入不配对能增加引物对两个等位基因的区分能力。     

AXIN2基因三个位点与先天性巨结肠的关联研究

目的 探讨AXIN2基因单核苷酸多态性(single nucleotide polymorphisms,SNPs)rs2240308、rs8081536和rs9913621 3个位点与先天性巨结肠(Hirschspnmg disease,HSCR)的关联性.方法 对120例HSCR患者和120名正常人群外周血进行基因组DNA抽提,用PCR技术对AXe2基因3个位点(rs2240308、rs8081536和rs9913621)进行PCR扩增,PCR产物用内切酶CviJ I、Dde I和BaN I消化,将SNPs位点进行分型与分析,应用X2检验统计分析病例组和对照组等位基因频率、等位基因型频率及其患病风险;同时将PCR产物进行测序,以进一步确定基因突变位点.结果 HSCR组与对照组AXIN2 rs8081536 CC和CT基因型频率差异无统计学意义(P＞0.05);而HSCR组与对照组AXIN2 rs2240308 GG、AG和从基因型频率及A和G等位基因频率差异有统计学意义(P＜0.05),GC和从基因型及G等位基因的患病风险分别为2.091、0.846和1.703;HSCR组与对照组AXIN2 rs9913621 CC、CT和TT基因型频率及C和T等位基因频率差异有统计学意义(P＜0.05),CC和TT基因型及T等位基因的患病风险分别为0.535、1.113和1.569.测序rs2240308第301位密码子核苷酸GCA→CCA杂合突变;rs913621第199位密码子核苷酸CAC→CAG杂合突变.结论 AXIN2 rs8081536等位基因变异与HSCR的易感性无关;AXIN2 rs2240308和rs9913621与HSCR的发生可能有关联,具有GG基因型与CC基因型患HSCR的危险性相对较高.     

消化后片段长度差异等位基因特异性PCR技术--印记SNP rs220028多态性和甲基化模式研究

目的建立一种能同时分析甲基化和单核苷酸多态性(single nucleotide polymorphism，SNP)的新方法。方法以印记SNP rs220028(A／G)为例，基因组DNA经甲基化敏感限制酶消化后用片段长度差异等位基因特异性PCR(fragment length discrepant allele—specific PCR，FLDAS—PCR)分型；用扩增产物酶消化法来排除酶切位点序列变异，证实检出的是甲基化等位基因。结果消化后FLDAS—PCR可选择性检出甲基化等位基因，家系分析表明其来自母亲。等位基因频率A=0．5085．G=0．4915，多态信息含量为0．3749。结论消化后FLDAS—PCR技术可以实现甲基化和SNP的复合分析；印记SNP rs220028在Prader—Willi／Angelman综合征诊断中有潜在意义。     

等位基因3′末端碱基特异性引物定量PCR检测人脂联素基因单核苷酸多态性

目的建立非探针标记荧光定量PCR的SNP检测技术,分析人脂联素(APM1)基因单核苷酸多态性(SNP)与Ⅱ型糖尿病(T2DM)的关系。方法设计3′末端碱基特异性引物,进行定量PCR反应,通过比较各对引物的扩增效率判断基因型,并进行测序验证。利用该方法确定20例T2DM、24例肥胖及28例同年龄组正常人APM1基因的45位和276位碱基的SNP。结果该方法能有效地区分不同的基因型,与测序结果符合率100%。APM1基因45位SNP与T2DM相关(P<0.05),基因型为TT纯合子者发生T2DM的危险性是G/T和GG者的3倍;而肥胖与APM1基因45和276位点SNP无关。结论等位基因3′末端碱基特异性引物定量PCR可以用于基因SNP检测。APM1基因45位碱基的SNP与T2DM发生有关。     

P53(rs1042522)基因多态性与子宫内膜异位症易感性的相关研究

目的 探讨肿瘤抑制基因P53 (tumor suppressor gene,P53)多态性与子宫内膜异位症(endometriosis,EM)遗传易感性的相关性.方法 应用等位基因特异性PCR技术结合DNA测序的方法对460例EM患者、650例无EM妇女(对照组)及113例子宫内膜癌患者的P53基因rs1042522位点(G/C)多态性进行分析.结果 各组均存在P53 (rs1042522) G/C多态性,且P53 (rs1042522)位点等位基因及基因型的分布在EM组与对照组之间差异有统计学意义(P值均小于0.01),其中等位基因C使EM发病风险提高1.179倍,等位基因G使其风险降低0.854倍;GC与GG基因型相比患EM的危险度增高1.548倍(95％CI为1.153～2.081),CC与GG基因型相比患EM的危险度增高1.865倍(95％CI为1.326～2.625).P53 (rs1042522)位点等位基因及基因型的分布在子宫内膜癌组与对照组之间差异有统计学意义(P值均小于0.01),且等位基因C使内膜癌发病风险提高1.278倍,而等位基因G使其风险降低0.772倍;GC与GG基因型相比患内膜癌的危险度增高2.074倍(95％CI为1.197～3.599),CC与GG基因型相比患内膜癌的危险度增高2.864倍(95％CI为1.557～5.263).P53 (rs1042522)位点等位基因及基因型的分布在EM组与子宫内膜癌组之间差异无统计学意义.结论 P53基因rs1042522位点(G/C)的单核苷酸多态性与EM遗传易感性存在相关性,且从遗传学角度分析,EM的发病机制可能更类似于肿瘤.     

等位基因3'末端碱基特异性引物定量PCR检测人脂联素基因单核苷酸多态性

目的 建立非探针标记荧光定量PCR的SNP检测技术,分析人脂联素(APM1)基因单核苷酸多态性(SNP)与Ⅱ型糖尿病(T2DM)的关系.方法 设计3'末端碱基特异性引物,进行定量PCR反应,通过比较各对引物的扩增效率判断基因型,并进行测序验证.利用该方法确定20例T2DM、24例肥胖及28例同年龄组正常人APM1基因的45位和276位碱基的SNP.结果 该方法能有效地区分不同的基因型,与测序结果符合率100%.APM1基因45位SNP与T2DM相关(P＜0.05),基因型为TT纯合子者发生T2DM的危险性是G/T和GG者的3倍;而肥胖与APM1基因45和276位点SNP无关.结论 等位基因3'末端碱基特异性引物定量PCR可以用于基因SNP检测.APM1基因45位碱基的SNP与T2DM发生有关.     

建立单管双向荧光PCR方法检测NQO1基因609C/T多态性

目的 建立单管双向荧光PCR方法快速测定人NAD(P)H:醌氧化还原酶1[NAD(P)H:quinone oxidoreductase 1,NQO1]基因609C/T多态性.方法 以人NQO1基因中的609C/T位点,设计双向引物,优化反应条件,应用SYBR GreenⅠ双向荧光PCR扩增191份人基因组DNA标本,并通过对产物进行熔解曲线分析,根据产物Tm值进行等位基因单核苷酸多态性分型.对其中62份标本用经典的PCR-限制性片段长度多态方法进行基因型分型,验证结果准确性.结果 62份样本的单管双向荧光PCR方法的基因型结果与PCR-限制性片段长度多态法分型结果符合率100%.191份样本中,纯合野生型(CC)占28%,杂合型(CT)占50%,纯合突变型(TT)占22%.结论 单管双向荧光PCR方法检测NQO1基因609C/T多态性,操作简便、反应过程快速,结果直观,敏感性、准确性和稳定性好,适用于临床样本检测及流行病学调查研究.     

β地中海贫血杂合子基因突变及Gγ珠蛋白基因-158位点SNP与Hb F的关系

目的　探讨 β地中海贫血 (简称 β地贫 )杂合子基因突变类型和 Gγ珠蛋白基因启动子 - 15 8位点 (Gγ- 15 8)单核苷酸多态性与胎儿血红蛋白 (fetal hemoglobin,Hb F)水平的关系。方法　抗碱 -比色法测定 Hb F水平 ;PCR-寡核苷酸斑点杂交法检测β地贫基因型 ;限制性内切酶 Xmn 消化经 PCR扩增的Gγ基因启动子 DNA片段 ,分析Gγ- 15 8位点的单核苷酸多态性。结果　 6 3例受检的轻型β地贫中 15例 Hb F≥ 2 % (2 .0 6 %～ 10 .4 4 % )。共检出 6种β地贫基因突变 ,分别是 :CD4 1/42 (- TTCT)、CD17(A→T)、nt- 2 8(A→ G)、CD71/72 ( A)、IVS- II- 6 5 4 (C→ T)、IVS- I- 1(G→ T)。 CD4 1/42、CD17、CD71/72、IVS- II-6 5 4的杂合子在 15例 Hb F升高组和 4 8例 Hb F正常组各自所占比例相同。 6 3例个体中有 10例为Gγ-15 8(C→T)突变的杂合子 ,总检出率为 15 .9% ;其中 15例高 Hb F个体中检出 8例 (检出率 5 3.33% ) ,HbF正常的 4 8例检出 2例 (检出率 4 .17% ) ,两组检出率差异有显著性 (P<0 .0 0 1)。结论　 β地贫基因突变CD4 1/42、CD17、CD71/72、IVS- II- 6 5 4与 β地贫杂合子的 Hb F水平无关 ;而 Gγ- 15 8(C→ T)突变与广西地区 β地贫杂合子 Hb F升高密切相关。     

椎间盘退行性变与HTRA1和HAPLN1基因多态性的相关性

文题释义：HTRA1：在Duchenne型肌营养不良骨骼肌及骨关节炎和和类风湿性关节炎软骨中HTRA1表达上调, HTRA1的上调可能有助于此类疾病的进展。对HTRA1单核苷酸多态性基因型的等位基因频率与高椎间隙狭窄评分之间的关联性进行分析,HTRA1基因启动子区域的单核苷酸多态性基因型与椎间盘退变相关,在携带G等位基因组(GG+GA)和未携带组(AA)的患者中均观察到椎间隙狭窄程度显著增加,因此HTRA1可能在椎间盘病理学中起作用。  HAPLN1：是软骨细胞外基质的关键组分,通过透明质酸链结合蛋白多糖,从而稳定蛋白多糖与透明质酸的聚合体,有助于关节的抗压和减震。试验中HAPLN1基因仅在rs179851单核苷酸多态性受试者中观察到骨赘形成和椎间隙狭窄的显著组间差异,而其余3个单核苷酸多态性(5’侧翼rs975563、内含子1 rs10942332和内含子4 rs4703570)则未见。因此,HAPLN1可能是软骨稳态的重要调节因子,并有助于椎间盘退变引起的骨关节炎发病机制。背景：研究发现,HTRA1基因启动子区域的单核苷酸多态性基因型与椎间盘退变相关,而HAPLN1基因与椎间盘退变引起的骨关节炎相关。目的：探讨分泌型丝氨酸蛋白酶HTRA1和骨细胞外基质的关键组分HAPLN1变异在椎间盘退变发病机制中的作用。方法：选择2015年4月至2018年12月在定州市人民医院接受体检的498名绝经后女性受试者,利用TaqMan PCR方法检测受试者HTRA1基因启动子rs11200638单核苷酸多态性和HAPLN1基因5’侧翼rs975563、内含子1 rs10942332、内含子2 rs179851和内含子4 rs4703570的单核苷酸多态性,分析HTRA1和HAPLN1基因多态性与椎间盘退变影像学特征之间的相关性。试验已通过定州市人民医院伦理道德委员会批准。结果与结论：①在498名受试者HTRA1基因rs11200638单核苷酸多态性中,178名为GG纯合子,222名为GA杂合子,98名为AA纯合子,将具有至少一个G等位基因(GG+GA,n=400)和没有G等位基因(AA,n=98)受试者间的椎间盘退变参数进行比较;②在HTRA1基因rs11200638单核苷酸多态性中,GG+GA等位基因组的椎间隙狭窄评分低于AA等位基因组(P < 0.001);随着椎间隙狭窄评分的升高,受试者中AA等位基因型发生风险增高(P ≤ 0.001);③在498名受试者HAPLN1基因单核苷酸多态性中,137名为TT纯合子,230名为CT杂合子,131名为CC纯合子,将CC+TT等位基因(n=361)、TT等位基因(n=137)的椎间盘退变参数进行比较;④在HAPLN1基因中,仅rs179851单核苷酸多态性的CC+TT等位基因组与TT等位基因组骨赘形成、椎间隙狭窄存在显著差异(P < 0.01);⑤在HAPLN1基因rs179851单核苷酸多态性中,椎间隙狭窄≥6分受试者的TT等位基因型发生风险显著增高(P < 0.05);随着骨赘形成评分的升高,受试者TT等位基因TT等位基因型发生风险显著增高(P < 0.001);⑥结果表明,HTRA1和HAPLN1特定基因位点的遗传变异与椎间盘退变相关。ORCID: 0000-0002-4567-2930(杨金丰)中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

A New Specific Quantitation-in-Gel Method Differentiating Commercial Human Serum Standards Intended for RID Analyses

A rapid, sensitive technique is described which measures small amounts of protein applied on agarose gel containing specific antibody. Alternating current through the gel reduces the influence of diffusion, and specific immunoprecipitate spots are formed. Their density after staining is proportional to applied protein. Quantitation of IgG and IgA in human serum standards was highly reproducible ( P < 0.001, Spearman coefficient of rank correlation test), and as little as about 35 ng Ig could be detected. Results are available within hours. Human sera and some purified preparations of 7S IgG and secretory IgA were grouped according to immune-gel filtration findings. The group of samples containing fragments, aggregates, or unusually small amounts of monomeric IgG or IgA was also differentiated by specific immunoprecipitate spot (SIS) analysis from those mainly monomeric in character (Wilcoxon rank test, P < 0.02). A World Health Organization reference serum (67/97) was used as standard. The study indicates that a human serum pool stored in aliquots at -20°C is a good standard for quantitating serum IgG and IgA and that purified preparations art mi better. It is suggested that the SIS assay could be advantageously applied to screening biologic fluids for unusual amounts or types of IgG and IgA.     

Simple and Accurate PCR-Based System for Typing Vacuolating Cytotoxin Alleles of Helicobacter pylori

Alleles of the vacuolating cytotoxin gene (vacA) of Helicobacter pylori vary between strains, particularly in the region encoding the signal sequence (which may be type s1 or s2) and the midregion (which may be type m1 or m2). Using a PCR-based typing system developed in the United States, we showed that 36 strains from Asia and South America were all vacA signal sequence type s1; 3 were midregion type m1 and 11 were m2, but 22 could not be typed for the vacA midregion. All strains possessed cagA (cytotoxin-associated gene A), another virulence marker. vacA nucleotide sequence analysis showed that midregion typing failure was due to base substitutions at the primer annealing sites. Using the new sequence data, we developed two new PCR-based vacA midregion typing systems, both of which correctly typed 41 U.S. strains previously typed by the old system and successfully typed all 36 of the non-U.S. strains. All previously untypeable strains were vacA m1, other than one m1/m2 hybrid. In summary, we describe and validate a simple PCR-based system for typing vacuolating cytotoxin (vacA) alleles of H. pylori and show that this system correctly identifies the signal and midregion types of vacA in 77 strains from Asia and North and South America.     

Degenerate and Nested PCR: a Highly Sensitive and Specific Method for Detection of Human Papillomavirus Infection in Cutaneous Warts

The role of human papillomavirus (HPV) in anogenital carcinogenesis is firmly established, but evidence that supports a similar role in skin remains speculative. Immunosuppressed renal transplant recipients have an increased incidence of viral warts and nonmelanoma skin cancer, and the presence of HPV DNA in these lesions, especially types associated with the condition epidermodysplasia verruciformis (EV), has led to suggestions that HPV may play a pathogenic role. However, differences in the specificities and sensitivities of techniques used to detect HPV in skin have led to wide discrepancies in the spectrum of HPV types reported. We describe a degenerate nested PCR technique with the capacity to detect a broad spectrum of cutaneous, mucosal, and EV HPV types. In a series of 51 warts from 23 renal transplant recipients, this method detected HPV DNA in all lesions, representing a significant improvement over many previously published studies. Cutaneous types were found in 84.3% of warts and EV types were found in 80.4% of warts, whereas mucosal types were detected in 27.4% of warts. In addition, the method allowed codetection of two or more distinct HPV types in 94.1% of lesions. In contrast, single HPV types were detected in all but 1 of 20 warts from 15 immunocompetent individuals. In summary, we have established a highly sensitive and comprehensive degenerate PCR methodology for detection and genotyping of HPV from the skin and have demonstrated a diverse spectrum of multiple HPV types in cutaneous warts from transplant recipients. Studies designed to assess the significance of these findings to cutaneous carcinogenesis are under way.     

Performance Characteristics of Updated INNO-LiPA Assays for Molecular Typing of Human Leukocyte Antigen A (HLA-A), HLA-B, and HLA-DQB1 Alleles

We carried out a multicenter performance evaluation of three new DNA-based human leukocyte antigen (HLA) typing assays: INNO-LiPA HLA-A Update, INNO-LiPA HLA-B Update, and INNO-LiPA HLA-DQB1 Update. After optimization, the accuracy rates were all 100%, and the final observed resolutions were 99.4, 92.4, and 85.6%, respectively. These rapid and easy-to-perform assays yielded results fully concordant with other DNA-based tissue typing tests.     

Clinical Usefulness of Multiplex PCR Lateral Flow in MRSA Detection: A Novel, Rapid Genetic Testing Method

Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I-V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3?h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.     

Molecular Genotyping of Staphylococcus aureus Strains: Comparison of Repetitive Element Sequence-Based PCR with Various Typing Methods and Isolation of a Novel Epidemicity Marker

Repetitive sequence-based (Rep)-PCR genotyping as described here is based on the presence of homologues of Mycoplasma pneumoniae repeat-like elements in Staphylococcus. In this study we comparatively evaluated the usefulness of rep-PCR typing with two sets of well-defined collections of Staphylococcus aureus strains. Rep-PCR analysis of the first collection of S. aureus strains (n = 59) and one Staphylococcus intermedius strain showed 14 different rep-PCR patterns, with each pattern harboring 6 to 15 DNA fragments. The discriminatory power of rep-PCR typing compared well to those of arbitrarily primed PCR (average of 20 types) and pulsed-field gel electrophoresis (11 types). S. aureus strain collection I comprised four outbreak-related groups of isolates. The isolates in only one group were found to have identical rep-PCR profiles. However, in an analysis of isolates from three additional independent local outbreaks (n for outbreaks 1 and 2 = 5, n for outbreak 3 = 12), identical rep-PCR types were found among strains isolated during each outbreak. Therefore, we conclude that rep-PCR genotyping may be an easy and fast method for monitoring of the epidemiology of nosocomial Staphylococcus infections. Rep-PCR analysis of strain collection II, which consisted of epidemic and nonepidemic methicillin-resistant S. aureus (MRSA) strains, revealed that a cluster of similar rep-PCR profiles was found among MRSA isolates which were more frequently isolated and which were most often associated with outbreaks.