Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Differentiation of Helicobacter pylori isolates by polymerase chain reaction-restriction fragment length polymorphism

Objective: To investigate the association between the diversity of urease gene and urease activity of clinical isolates of Helicobacter pylori (H. pylori). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of urease gene and rapid urease activity test were used to study the urease activity of different clinical isolates of H. pylori. Results: H. pylori clinical isolates were divided into 4 types according to their PCR-RFLP results of urease gene and urease activity. Type Ⅰ , possessing strong urease activity (0. 11) and presented 1 fragment of 1. 7 kb by PCR-RFLP, had close relations with gastric ulcer; type Ⅱ , with the weakest urease activity (0. 07) and 2 fragments (1. 3 and 0. 4 kb respectively) , was associated with duodenal bulb ulcer; type Ⅲ , with the strongest urease activity (0. 12) and 2 fragments (0. 4 and 0. 17 kb) with or without 1 fragment (0. 23 or 0. 37 kb) , was responsible for gastritis; type Ⅳ , with weak urease activity (0. 09) and 2 fragments (1. 5 and 0. 2 kb), was shown to be related to both gastric and duodenal bulb ulcers. Conclusion: The diversity of urease gene decides different urease activities of different clinical isolates of H. pylori, hence the different possibilities of pathogenesis due to this bacteria.

Research on the Correlation Between rs2110385 Polymorphisms of the Visfatin Gene and Nonproliferative Diabetic Retinopathy

Objective:To investigate the association between rs2110385 polymorphisms of the visfatin gene and the risk of type 2 diabetic retinopathy(DR).Methods:172 Han subjects were selected from Xi’an Shaanxi Province;140 patients with type 2 diabetes mellitus(T2DM)and 32 normal controls(NC)were selected from our hospital.Patients with diabetes were divided into a non-DR group(T2DM)(n=69)and a nonproliferative diabetic retinopathy Group(DR)(n=71)after dilated fundus photography and fundus fluorescein angiography.rs2110385/AluⅠgenotypes were detected by standardized polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),and the differences in the detection rates of different genotypes in the above populations were compared.Results:1)The visfatin level in the DR Group was significantly higher than that in the NC and T2DM groups(P<0.05).2)The frequency of GG genotype and G allele of rs2110385 in the DR Group were higher than those in the T2DM and NC groups(80.3,69.6,50.0,86.6,79,65.6,P<0.05).3)There were significant differences in allele frequency and genotype frequency distribution of rs2110385 between the DR Group and the NC group(P<0.01).Conclusion:Visfatin increased in the nonproliferative diabetic retinopathy group and could be a potential indicator for the clinical prediction of DR.The G allele of the rs2110385 polymorphic site may be related to the risk of DR.

Rapid Identification of Mycobacterium Leprae by Polymerase Chain Reaction-restriction Fragment Length Polymorphism Analysis of the Heat Shock Protein 65 Gene from Skin Specimens

INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or inadequate treatment patients. Diagnosis of leprosy depends on the clinical signs and symptoms and slit skin smear positivity. However, it＇s sometimes similar with other granulomatous disease caused by mycobacterial infection. Early stage leprosy is difficult to diagnose by clinical criterion alone because the sensitivity of acid-fast bacilli staining is quite low. The polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） shows the great advantage in rapid identification and diagnosis for early cases and has a differentiation between leprosy and nonleprosy cases.

The Effects of Apolipoprotein E Polymorphism on Serum Lipids, Lipoproteins and Apolipoproteins Variation

Objective: To study the effects of Apolipoprotein E (ApoE) polymorphism onserum levels of lipids, lipoproteins and apolipoproteins. Methods: Fragments of ApoE gene forthex-on containing codon 112 and 158 polymorphic locus were amplified by PCR, and then digested untilCfo I endonuclease. Genotypes and alleles frequencies of 168 healthy persons in Jiangsu area werecalculated. The effects of ApoE genotypes and alleles on serum lipids, lipoproteins andapolipoproteins variation were analyzed. Results: The effects of ApoE alleles on total cholesterol(TC), law density lipoprotein-cholesterol (LDL-C), ApoB was: along a decreasing gradientε_4>ε_3>ε_2. The effect of ε_4 allele was to increase serum levels of TC, LDL-C and ApoB, andthe ε_2 allele had an effect opposite to that of ε_4 allele. Conclusion: ApoE polymorphism is anindependent genetic factor on individual serum levels of lipids and apolipoproteins.

Association of TNF-α-238G/A and 308 G/A Gene Polymorphisms with Pulmonary Tuberculosis among Patients with Coal Worker’s Pneumoconiosis

Objectives Tumor necrosis factor-α （TNF-α） may play an important role in host＇s immune response to mycobacterium tuberculosis （M. tuberculosis） infection. This study was to investigate the association of TNF-α gene polymorphism with pulmonary tuberculosis （TB） among patients with coal worker＇s pneumoconiosis （CWP）. Methods A case-control study was conducted in 113 patients with confirmed CWP complicated with pulmonary TB and 113 non-TB controls with CWP. They were matched in gender, age, job, and stage of pneumoconiosis. All participants were interviewed with questionnaires and their blood specimens were collected for genetic determination with informed consent. The TNF-α gene polymorphism was determined with polymerase chain reaction of restriction fragment length polymorphism （PCR-RFLP）. Frequency of genotypes was assessed for Hardy-Weinberg equilibrium by chi-square test or Fisher＇s exact probability. Factors influencing the association of individual susceptibility with pulmonary TB were evaluated with logistic regression analysis. Gene-environment interaction was evaluated by a multiplieative model with combined OR. All data were analyzed using SAS version 8.2 software. Results No significant difference in frequency of the TNF-α-308 genotype was found between CWP complicated with pulmonary TB and non-TB controls （2,2=5.44, P=-0.07）. But difference in frequency of the TNF-α-308 A allele was identified between them （2,2-5.14, P=0.02）. No significant difference in frequencies of the TNF-α-238 genotype and allele （P=0.23 and P=0.09, respectively） was found between cases and controls either, with combined （GG and AA） OR of 3.96 （95% confidence interval of 1.30-12.09） at the -308 locus of the TNF-α gene, as compared to combination of the TNF-α-238 GG and TNF-α-308 GG genotypes. Multivariate-adjusted odds ratio of the TNF-α-238 GG and TNF-α-308 GA genotypes was 1.98 （95% CI of 1.06-3.71） for risk for pulmonary TB in patients with CWP. There was a synergic interaction between the TNF-a-308 GG genotype and body mass index （OR=4.92）, as well as an interaction between the TNF-α-308 GG genotype and history of BCG immunization or history of TB exposure. And, the interaction of the TNF-α-238 GG genotype and history of BCG immunization or TB exposure with risk for pulmonary TB in them was also indicated. Conclusions TNF-α-308 A allele is associated with an elevated risk for pulmonary TB, whereas TNF-α-238 A allele was otherwise.

Association of G+1688A Polymorphism of Platelet Endothelial Cell Adhesion Molecule-1 Gene with Myocardial Infarction in the Chinese Han Population

In order to investigate the association of G＋1688A （Ser563Asn） polymorphism of platelet endothelial cell adhesion molecule-1 （PECAM-1） gene with myocardial infarction （MI） in the Chinese Han population, the G＋1688A polymorphism in PECAM-1 gene was detected by polymerase chain reaction-restriction fragment-length polymorphism （PCR-RFLP） method among 502 subjects, including 218 patients with MI and 284 controls. The results showed that there was significant difference in AA frequencies of genotype G＋1688A polymorphism between case and control groups （39% vs 24%, P〈0.001）. A similar trend was observed on the allele frequencies （A/G： 62% vs 49%, P〈0.001）. Among the subjects with high serum total cholesterol level or high systolic blood pressure level, the variant AA genotype was associated with high risk of MI （adjusted OR, 2.13; 95% CI, 1.08 -4.41 and adjusted OR, 2.53; 95%CI, 1.63-3.63）. The single nucleotide polymorphism （SNP） at position ＋1688 in the exon 8 of PECAM-1 gene was associated with MI and the allele A might be a risk factor for MI in the Chinese Han population.

牦牛乳蛋白多态性对奶酪凝乳特性的影响

为了检测青海部分地区7家不同牧场牦牛乳κ-酪蛋白和αs1-酪蛋白基因多态性,作者分析了基因多态性对奶酪凝乳特性的影响。通过提取牦牛乳体细胞,采用限制性片段长度多态性聚合酶链式反应(polymerase chain reaction-restrition fragment length polymophism,PCR-RFLP)分析技术,对提取的DNA进行PCR扩增及酶切,对电泳条带进行基因分型;提取牦牛乳蛋白,采用高效液相色谱法(high performance liquid chromatography,HPLC)分析各样品与标准品色谱图,对出峰时间和出峰形状进行基因分型。利用流变仪测定不同基因型牦牛乳凝乳过程中流变特性,记录奶酪凝乳时间,计算奶酪得率。牦牛乳κ-酪蛋白基因有AA型、AB型和BB型3种基因型,3种基因型与凝乳特性的分析表明,在凝乳时间方面A等位基因为有利等位基因。牦牛乳αs1-酪蛋白存在AA型、AB型和BB型3种类型,3种基因型与凝乳特性的分析表明,在凝乳时间、奶酪得率、最大动力黏度和最大剪切速率方面B等位基因为有利等位基因。以上结果表明,青海7家牧场牦牛乳κ-酪蛋白和αs1-酪蛋白均存在基因多态性,κ-酪蛋白A等位基因和αs1-酪蛋白B等位基因是影响牦牛乳凝乳特性的主效基因。

PEAR1基因多态性与缺血性脑卒中复发易感性的关系研究

目的分析血小板内皮聚集受体1(PEAR1)基因多态性与缺血性脑卒中复发的相关性,为防治缺血性脑卒中复发提供依据。方法选取该院神经内科门急诊和住院确诊为急性缺血性脑卒中的150例患者作为研究对象,根据是否为脑卒中复发分为初发脑卒中组(127例)和复发脑卒中组(23例)。应用聚合酶链反应-限制性片断长度多态性(PCR-RFLP)方法分析PEAR1基因rs12041331位点单核苷酸多态性,并测序验证基因型。结果初发脑卒中组和复发脑卒中组PEAR1基因rs12041331G>A位点GG、GA、AA基因型和G、A等位基因频率比较,差异有统计学意义(P<0.05);复发脑卒中组的年龄较初发脑卒中组高,差异有统计学意义(P<0.05)。Logistic回归分析显示,年龄、PEAR1基因rs12041331位点AA基因型与缺血性脑卒中复发有关,是缺血性脑卒中复发的危险因素(P<0.05)。结论PEAR1基因rs12041331G>A位点多态性与缺血性脑卒中复发相关。PEAR1基因纯合突变可能是缺血性脑卒中复发的危险因素,PEAR1基因可能是缺血性脑卒中复发风险预测的候选基因。

PEAR1基因多态性与缺血性脑卒中的相关性研究

目的分析PEAR1基因多态性与缺血性脑卒中相关性,为缺血性脑卒中发病机制的进一步研究提供科学依据,为疾病防治提供新的思路。方法收集昆明医科大学附属延安医院神经内科门急诊或住院确诊为急性缺血性脑卒中患者150例作为实验组,同期150例健康体检者作为对照组,应用聚合酶链反应-限制性片断长度多态性(PCR-RFLP)方法分析PEAR1基因rs12041331位点单核苷酸多态性(SNP),并测序验证基因型。结果卡方检验显示缺血性脑卒中组和正常对照组PEAR1基因rs12041331G>A位点GG、GA、AA基因型和G、A等位基因频率间的差异有统计学意义(P<0.05);缺血性脑卒中组的糖尿病、高血压比例较正常对照组多,同型半胱氨酸(HCY)水平较正常对照组高,差异有统计学意义(P<0.05);Logistic回归分析显示PEAR1基因rs12041331G>A位点突变可能是缺血性脑卒中发生的危险因素。结论PEAR1基因rs12041331G>A位点基因多态性与缺血性脑卒中发生相关。rs12041331G>A位点基因可作为缺血性脑卒中风险预测的候选基因。

青海省汉族妊娠期高血压疾病与维生素D受体基因FokⅠ多态性的关系

目的 探讨维生素D受体(VDR)基因单核苷酸多态性(SNP)FokⅠ(rs2228570/rs10735810)位点多态性与青海省汉族妇女妊娠期高血压疾病(HDCP)的相关性。方法 选择青海省汉族HDCP患者137例(HDCP组),正常汉族孕妇146例(对照组),使用聚合酶链式反应-限制性核酸内切酶片段长度多态性(PCR-RFLP)方法检测HDCP组和对照组FokⅠ位点的基因分型并测序验证,使用SPSS 19.0统计学软件检验两组一般临床资料、基因型及等位基因频率分布差异是否具有统计学意义。结果 HDCP组和对照组FokⅠ位点FF、Ff、ff基因型频率分别为51.82%、37.96%、10.22%和34.93%、43.15%和21.92%(χ^(2)=11.099,P<0.05),HDCP组FF基因型(OR=2.004,95%CI=1.243~3.231)频率高于对照组;HDCP组和对照组FokⅠ位点F和f等位基因频率分布差异有统计学意义(χ^(2)=12.454,P<0.001),HDCP组F等位基因(OR=1.253,95%CI=1.105~1.421)频率高于对照组。结论 VDR基因FokⅠ位点多态性与青海省汉族HDCP相关,其中F等位基因可能是HDCP的易感基因,FF基因型可能是HDCP的易感基因型。

青海汉族妊娠期高血压疾病白细胞介素10基因启动子区-592A/C多态性分子特征

目的探讨白细胞介素10(IL-10)基因启动子区-592A/C(rs1800872)多态性与青海汉族妇女妊娠期高血压疾病(HDP)的相关性,并确定该基因在HDP组和健康对照组中的表达情况。方法选择青海省HDP患者(HDP组)和正常妊娠妇女(对照组)各140例,以血液DNA为模板,应用限制性内切酶片段长度多态性聚合酶链反应(PCR-RFLP)方法检测HDP组和对照组IL-10-592A/C多态性分型并测序验证。应用Real-time PCR检测两组胎盘组织中IL-10 mRNA的表达。应用ELISA检测两组血浆IL-10水平。结果HDP组和对照组IL-10基因AA、AC、CC基因型频率分别为24.29%、44.29%、31.42%和13.57%、41.43%、45.00%,两组基因型分布差异有统计学意义(P<0.05);AA基因型频率HDP组(24.29%)高于对照组(13.57%)(P<0.05),CC基因型频率HDP组(31.42%)低于对照组(45.00%)(P<0.05),而AC基因型频率两组间差异无统计学意义(P>0.05);IL-10-592A/C多态性A、C等位基因频率两组间分布有差异,HDP组A等位基因频率高于对照组(χ^(2)=8.079,P<0.05);HDP组胎盘组织中IL-10 mRNA表达量低于对照组(P<0.01);HDP组血浆IL-10水平(1.53±0.68)ng/L低于对照组(1.79±0.72)ng/L(P<0.01)。结论IL-10-592A/C多态性与青海汉族HDP相关;IL-10-592A/C多态性中A等位基因可能通过调节IL-10的表达参与HDP的遗传易感性。

慢性HBV感染前C区变异与病毒复制水平关系

探讨HBV前C基因变异与病毒复制水平的关系在慢性HBV感染者中的意义。应用错配聚合酶链反应 (PCR) -限制性片段长度多态性 (RFLP)分析与荧光定量聚合酶链反应检测HBVDNA相结合 ,对 30例HB sAg(+ )、抗 -HBe(+ )及抗 -HBc(+ )慢性HBV感染者 ,其中无症状携带者 (AsC) 9例、慢性乙型肝炎 (CHB) 12例及慢性重症肝炎 (CHF) 9例进行前C区基因变异与HBVDNA水平关系进行分析。AsC组 3例 (33 33 % ) ,CHB组9例 (75 % )及CHF组 8例 (88 89% )有A83(nt 1896 )变异。荧光定量PCR结果表明HBVDNA含量在CHF组中最高。HBV前C变异在HBV不同感染状态中都可见 。

陕西地区乙型肝炎患者血清中病毒的基因分型

目的 了解陕西地区乙肝病毒 (HBV)基因分型情况 .方法 用套式 PCR方法从 10 0份 HBs Ag阳性的乙型肝炎患者血清中扩增 5 85 bp的 S基因 ,用限制性内切酶 Bsr ,Sty ,Dpn 和 H pa 平行酶切鉴别 HBV A～ F基因型 .结果 所有标本用内引物 P1和 P2均扩增获得的 5 85 bp的靶基因 ,继之用上述限制性内切酶消化鉴定 ,证明 10 0份血清标本中的 HBV可分为 B型 36份 (36 % ) ,C型 5 6份 (5 6 % ) ,D型 8份 (8% ) .结论 陕西地区 HBV分型以 B,C型为主 ,D型较少 ,未发现 A,E,F型 .结果同时表明 PCR- RFL P方法灵敏性高 ,特异性强 ,且酶切图谱简明单一 ,易于识别 。

宁夏地区乙型肝炎病毒基因型分布及D基因型的测序鉴定

为确定乙型肝炎病毒 (HBV) D基因型在我国的存在 ,建立我国 HBV D基因型的参照序列 ,作者用聚合酶链式反应 (PCR)结合限制性片段长度多态性分析 (RFL P)技术 ,调查了可能存在 D基因型的我国宁夏地区 HBV基因型分布。对 90例 HBe Ag阳性 HBV感染者的血清进行前 S区 RFL P基因型分型。结果发现 :C基因型 86 .7% (78/ 90 ) ;D基因型 1 0 % (9/ 90 ) ;无 A、B、E和 F型 ;不能明确分型者 3.3% (3/ 90 )。 2例 RFL P分型为 D基因型的病例经测序证实。结果表明 ,宁夏地区 HBV优势基因型为 C型 ,并首次在该地区发现 D基因型的存在。本研究为我国 HBV基因型分布积累了新的资料 ,为进一步建立我国 HBV

DQA1、DQB1和DQB2基因多态性与精神分裂症的关系

目的 :探讨 1 1 6个家系的 6号染色体短臂 ( 6p) MHC区 DQA1、DQB1和 DQB2基因多态性与精神分裂症的关系。方法 :PCR技术和限制性内切酶片段长度多态性方法。结果 :DQA1位点G、A等位基因和 DQB2位点 G、C等位基因在病例组和对照组的频数分布差异无显著性( P>0 .0 5 )。DQB1位点 G、C等位基因在病例组和对照组的频数分布差异有显著性 ( P<0 .0 5 )。结论 :DQB1位点的基因多态性与精神分裂症有关联 ,G等位基因的频数分布病例组高于对照组 ,而DQA1和 DQB2位点的基因多态性与精神分裂症无关联 ;DQA1位点的等位基因在 3种不同程度情感迟钝、淡漠的精神分裂症患者中分布差异有显著性 ;DQB2位点的等位基因分布在精神分裂症不同的诊断分型之间的差异有显著性 ,其中的等位基因

鉴别猪瘟强毒和弱毒的反转录-复合套式聚合酶链式反应(RT-nPCR)检测方法的建立

【目的】建立一种能区分猪瘟病毒(classicalswinefever,CSFV)强毒和弱毒的反转录-复合套式聚合酶链式反应(RT-nPCR)检测方法。【方法】根据GenBank上登录的现有CSFV基因组全序列,选择高度保守区设计了一对CSFV通用引物,并在该对引物跨越区域的内部设计了猪瘟兔化弱毒疫苗和强毒特异性引物,建立了一种能区分猪瘟强毒和弱毒的RT-nPCR鉴别诊断方法。【结果】应用该方法从猪瘟兔化弱毒疫苗和石门强毒株基因组中扩增出了大小分别为447和343bp的一条特异性片段,从两种病毒基因组混合物中扩增出了大小为447和343bp的两条特异性片段,但对牛病毒性腹泻病毒和其它常见猪源病毒细胞培养物以及正常细胞基因组进行检测时均为阴性。该方法可以检测出0.04pg的CSFVRNA。对从黑龙江省采集的15份疑似猪瘟病料进行了检测,结果表明,有14份类似猪瘟强毒,1份类似弱毒疫苗。限制性片段长度多态性和种系发生分析证实了RT-nPCR的检测结果。【结论】本研究建立的RT-nPCR可以有效区分猪瘟强毒和弱毒,减少了未感染的免疫猪被误杀的可能性。

肿瘤坏死因子β基因多态性在中国汉族SLE病人中的分布特点及与不同人种的比较研究

目的 :探讨中国江苏地区汉族人群TNFβ基因多态性在SLE病人中分布特点 ,并与不同人种进行比较研究。 方法 :收集江苏地区 16 8名无血缘关系健康个体及 6 6例SLE病人的静脉血提取DNA ,应用聚合酶链反应限制性片段长度多态性(PCR RFLP) ,分析TNFβ基因的多态性。 结果 :中国汉族SLE病人与白种SLE病人TNFβ基因频率差异无统计学意义 ;中国汉族健康个体TNFβ 1等位基因频率明显高于白种健康个体 ;SLE病人TNFβ 2基因频率较正常人明显升高 (SLE病人 6 7 4% ,正常人 5 5 1% ,P <0 0 5 ,R =1 6 8) ;中国汉族SLE病人与白种SLE病人TNFβ基因频率差异无统计学意义。 结论 :TNFβ等位基因频率在正常人分布具有种族差异 ,但在SLE病人分布无种族差异。

猪品种间ESR基因PCR-RFLP的初步研究

利用聚合酶链反应 (PCR)技术 ,检测了不同猪品种的ESR基因PvuⅡ RFLP。结果表明 :品种间基因频率差异极显著 (P <0 .0 1 )。并试分析了PvuⅡ

e抗原阴性重症乙型肝炎患者HBV前C区热点变异研究

目的 研究ＨＢＶ信号肽区热点变异与重症乙型肝炎发生及转归的关系。方法 采用错配聚合酶链反应（ＰＣＲ）和限 制性片段长度多态（ＲＦＬＰ）分析方法检测６８例ＨＢｅＡｇ阴性的重症乙型肝炎病人（其中急性重肝６例、亚急性重肝３８ 例、慢性重肝２４例）及４４例慢性乙型肝炎患者的Ｔ１８６２、Ａ１８９６变异情况。结果 急性重肝的Ａ１８９６、Ｔ１８６２变异分别 为６６．７％（４／６）、０（０／６）；亚急性重肝４２．１％（１６／３８）、１５．８％（６／３８）；慢性重肝 ２５．０％（６／２４）、１６．７％（４／２４）；慢性肝炎 ４５．５％（２０／４４）、２．３％（１／４４）。重症乙型肝炎组与慢性肝炎组比较Ｔ１８６２变异率有显著差异（Ｐ＜０．０１），Ａ１８９６变异率无显 著差异（Ｐ>０．０５）。结论 提示Ｔ１８６２变异与乙型肝炎的重症化密切相关。而Ａ１８９６变异与乙型肝炎重症化进程是否有 关还不能确定。