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.

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.

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

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。

应用PCR-RFLP和芯片生物分析系统鉴别河豚鱼品种

应用聚合酶链式反应-限制性片段长度多态分析和芯片生物分析系统建立5种河豚的分子生物学品种鉴定新方法。首先提取鱼的DNA进行细胞色素b基因的PCR扩增,然后用DdeⅠ、HaeⅢ和NlaⅢ三种限制性内切酶进行酶切,在Agilent DNA1000芯片上对酶切片段进行分离。该方法可成功区分5个河豚鱼品种,是一种快速、简便、有效的鱼类品种鉴定分析手段。

常见食源性感染致病菌属、种用PCR-RFLP及PCR-SSCP技术鉴定探讨

【目的】探讨运用聚合酶链反应邛艮制性片段长度多态性分析(PCR-RFLP)和聚合酶链反应-单链构象多态性分析(PCR-SSCP)进行食源性感染常见致病菌属种鉴定的可行性。【方法】选择细菌23S rDNA基因作为鉴别细菌属的靶基因,通过设计合适的通用引物进行PCR扩增,并对13属食源性感染常见致病菌和5种不同血清型大肠杆菌扩增产物的酶切片段进行RFLP分析和SSCP分析。【结果】运用通用引物可以扩增出13 属食源性感染常见致病菌的23S rDNA基因HpaⅡ酶切产物的RFLP分析表明,不同种属的致病菌表现出不同的RFLP图谱,利于进行细菌属的鉴定。不同种属食源性感染常见致病菌SSCP图谱变异性更大,不利于进行种属间鉴别。对5种不同血清型大肠杆菌的23S rDNA基因扩增产物的RFLP和SSCP分析表明,不同血清型大肠杆菌表现出相似的RFLP图谱。SSCP图谱的变异性较大,这有利于进行血清型间鉴别甚至株间的鉴别。【结论】PCR-RFLP和PCR-SSCP具有应用于食源性感染常见致病菌种、属鉴别诊断的潜力。

PCR-RFLP检测载脂蛋白CⅢ基因T-455C多态性方法的建立及应用

目的建立聚合酶链反应-限制性片段长度多态性技术(PCR-RFLP)检测载脂蛋白CⅢ(apoCⅢ)T-455C多态性的方法,探讨南通汉族人群中apoCⅢ多态性与高三酰甘油血症(HTG)的相关性。方法用PCR扩增apoCⅢ基因启动子区包含T-455C多态性在内的DNA片段,扩增产物用限制性内切酶BseGⅠ酶切后电泳,分析apoCⅢ基因型,同时采用生化方法检测研究对象的血脂和载脂蛋白水平。结果检测到3种基因型,分别为-455TT、-455TC和-455CC。HTG患者组和健康对照组apoCⅢ-455C等位基因频率(50.44%比33.11%)和apoCⅢ-455CC基因型频率(26.55%比12.16%)均有显著差异(P<0.01);C等位基因携带者患HTG的风险是非携带者的2.06倍(95%CI:1.31～3.24);-455C等位基因携带者有较高的三酰甘油(TG)和apoCⅢ蛋白水平(P<0.05)。结论PCR-RFLP是一种快速、敏感的分析apoCⅢ基因启动子区T-455C多态性的方法;apoCⅢ-455C等位基因与HTG的发生相关。

应用PCR-RFLP分析结核分枝杆菌rpsL基因突变

目的 了解结核分枝杆菌链霉素(SM)耐药基因突变情况,建立快速检测耐药突变株的方法。方法 通过PCR-RFLP分析62株结核分枝杆菌临床分离株的rpsL基因突变的部位和性质。结果 以H37RV标准株为对照,13株敏感株的rpsL基因有MboⅡ酶切位点;37株耐SM分离株中,31株(838%)无MboⅡ酶切位点;12株耐其它抗结核药物株中,也有2株无MboⅡ酶切位点。结论 大多数结核分枝杆菌SM耐药分离株的核糖体S12蛋白编码基因(rpsL)第43位密码子有点突变。PCR-RFLP可能成为快速检测结核分枝杆菌耐药突变株的一种新方法。

PCR-RFLP鉴别念珠菌、曲霉和隐球菌的探讨

为快速鉴别 3类医学上重要真菌———念珠菌、曲霉和隐球菌 ,应用聚合酶链反应 限制性片段长度多态(PCR RFLP)的分子生物学方法对白念珠菌、克柔念珠菌、季也蒙念珠菌、近平滑念珠菌、热带念珠菌、乳酒念珠菌、新生隐球菌格梯变种、新生隐球菌新生变种、罗伦隐球菌、烟曲霉、黄曲霉和杂色曲霉进行体外研究。经PCR念珠菌、曲霉和隐球菌均扩增出 3 10bp的DNA片段 ,再行限制性内切酶HaeIII消化后分别呈现 2条、4条和 3条片段。本实验建立的PCR RFLP技术鉴别念珠菌、曲霉和隐球菌感染 ,具有快速和特异的特点 ,是对PCR诊断的重要补充 ,有潜在的临床应用价值。

PCR-RFLP在细粒棘球绦虫种株鉴定中的应用

目的根据细粒棘球绦虫线粒体DNA rrnL区段基因特征,建立一种新的细粒棘球绦虫种株(基因型)鉴定技术。方法应用PCR-RFLP方法,对新疆不同地区44个囊型包虫病(CE)病人分离株标本提取DNA,用特异性引物对mtDNA rrnL基因片段进行PCR扩增,将扩增产物用限制性内切酶SspI和Bg1Ⅱ消化,再用琼脂糖凝胶电泳分析片段大小,进行基因型鉴别。结果CE病人分离株PCR扩增产物均不能被限制性内切酶SspI酶切,为细粒棘球绦虫;其中43个病人分离株的PCR扩增产物不能被限制性内切酶Bg1Ⅱ酶切,为细粒棘球绦虫G1基因型;1个病人分离株的PCR扩增产物被Bg1Ⅱ切成158 bp和403 bp 2条DNA片段,为细粒棘球绦虫G6基因型。结论根据细粒棘球绦虫线粒体DNA rrnL区段基因特征建立的PCR-RFLP技术可用于细粒棘球绦虫的基因型鉴别。

PCR-RFLP法检测生殖道沙眼衣原体感染

目的 建立检测沙眼衣原体的聚合酶链反应 -限制性内切酶切片段长度多态性分析 (PCR -RFLP)方法。方法 在利用PCR扩增沙眼衣原体的基础上 ,引入RFLP方法 ,并与美国ABI公司的金标免疫层析法试剂盒和镜检包涵体法比较。结果 共检测 40例临床确诊病人 ,镜检包涵体法有 15例 (37.5 % )阳性 ,金标免疫层析法有 2 3例 (5 7.5 % )阳性 ,而PCR-RFLP法有 2 9例 (70 .3 % )阳性 ,各组间有显著性差异 (P <0 .0 5 )。结论 我们认为PCR -RFLP法优于镜检包涵体法。

复合PCR-RFLP技术用于尖音库蚊复组抗性相关酯酶基因分子分型

目的应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)检测法,建立尖音库蚊抗性酯酶等位基因分型方法。方法根据尖音库蚊抗性酯酶基因保守区序列设计引物;提取单个蚊虫基因组DNA,采用PCR-RFLP分析的方法,对抗性酯酶等位基因分型。结果酯酶基因扩增片段长度为860 bp,经限制性内切酶酶切后,尖音库蚊抗性Est-β11纯合型有5条带(70、89、147、198和347 bp);Est-β2纯合型出现4条带(60、147、207和437 bp);Est-β11/Est-β2杂合型出现5条带;Est-βN纯合型出现3条带(147、207和506 bp)。结论PCR-RFLP方法能快速、有效地检测尖音库蚊抗性酯酶等位基因类型。

多重PCR-RFLP检测XRCC1基因两位点多态性

目的探讨多重聚合酶链反应-限制性片断长度多态性(多重PCR-RFLP)技术在单核苷酸多态性检测中的应用。方法应用多重聚合酶链反应(Multiplex)(多重PCR)原理设计人类X射线交错互补修复基因1(XRCC1基因)194、399位点引物,通过PCR-RFLP技术判断XRCC1基因2个SNP位点多态性。结果设计的两对引物可同时PCR扩增分别含2个多态位点的DNA片段,MspI限制性内切酶酶切可判断2位点多态性,PCR和酶切效果均较好。结论多重PCR-RFLP技术可应用于单核苷酸多态位点的检测,并节省时间和费用,提高检测效率。

PCR-RFLP技术在布氏菌鉴定与分型中的应用

目的利用PCR-RFLP技术扩增7株布氏菌临床分离株16S RNA基因片段,为该菌的分子诊断学、遗传学和流行病学研究提供实验依据。方法参考GenBank公布的布氏菌ATCC 25840标准株序列,设计针对该菌16S RNA保守区的特异性引物,从临床分离培养7株布氏菌,提取DNA并行16S RNA片段的PCR扩增。将PCR产物克隆至PGEM-T载体进行测序并选用合适的限制性内切酶进行RFLP分析,将分析结果用于临床株的同源性及变异位点的研究,并对感染患者的临床资料进行回顾性分析,研究不同布氏菌基因型与感染患者临床特征的相关性。结果利用PCR扩增出的目的片段与预期一致,大小约为1500bp。测序及同源性分析表明,7株临床分离株之间存在98.88%的同源性和11个变异位点。RFLP结果将7株菌分为4个基因型,临床资料回顾性分析患者不同基因型与临床表现之间无明显联系。结论利用PCR技术扩增布氏菌16SRNA基因片段可以实现对该菌的早期诊断,为布氏菌的分子遗传学和流行病学提供依据。

RT-PCR-RFLP技术在登革病毒感染早期诊断中的应用

目的建立疑似登革病毒感染的病原快速鉴定分型方法,并对2004年我院收治的一例疑似登革感染患者进行确诊。方法用免疫层析法(ICT)检测DV-IgM抗体、免疫斑点法(DIBA)检测DV-IgG抗体对疑似登革病毒感染患者进行动态监测;用登革病毒通用引物通过逆转录-聚合酶链反应(RT-PCR)扩增患者血清中的登革病毒核酸,用限制性内切酶BstnⅠ酶切,聚丙烯酰胺凝胶电泳分析鉴定分型。结果发病第5天患者DV-IgM抗体开始呈弱阳性,第9天DV-IgM抗体滴度达到1∶8,第13天DV-IgM抗体滴度开始下降;发病第9天患者DV-IgG抗体呈弱阳性,第12天DV-IgG抗体滴度达到1∶8。用RT-PCR-RFLP检测疑似登革病毒感染患者早期血标本,确定2004年我院收治的一例患者为登革Ⅰ型病毒感染所致。结论利用RT-PCR-RFLP技术对登革病毒进行分型鉴定具有敏感、特异、快速的优点,与登革热抗体检测结合起来应用可缩短疑似登革感染的确诊时间、降低检测成本,具有很好的社会效益和经济效益。

应用PCR-RFLP技术检测阿德福韦耐药相关性HBV变异

目的建立一种简便、快速、实用的检测阿德福韦(ADV)耐药相关性HBV变异(rtA181V/T/S和rtN236T变异)的方法。方法根据GenBank收录的HBV基因全序设计套式-PCR引物,使野毒株的PCR产物上游末端引入BglI的酶切位点,使rt236变异株的PCR产物下游末端引入BseDI(SecI)的酶切位点,建立PCR-限制性片段长度多态性(RFLP)方法。用此方法分别检测含rt181变异株、rt236变异株及野毒株质粒和3例ADV耐药慢性乙型肝炎患者血清标本。将变异株和野毒株配成不同比例,再用此方法检测,以了解该方法的敏感性。结果本研究建立的PCR—RFLP方法可同时检测rt181变异和rt236变异;用此方法检测血清标本,结果与测序及克隆分析一致;此方法可检测出标本中10%的变异株。结论应用PCR- RFLP方法可同时检测rtA181V/T/S和rtN236T变异,具有较高的敏感性,可用于ADV耐药变异的早期诊断。

PCR-RFLP技术DLA-DRB1基因分型在狗卵巢移植配型中的应用

目的探讨狗组织相容性系统（ＤＬＡ）－ＤＲＢ１基因分型方法及用于狗卵巢移植配型的可行性。方法 选择特定的引物和 ６种限制性内切酶，采用聚合酶链反应－限制性片段长度多态性（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎｂａｓｅｄｏｎｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔ ｌｅｎｇｔｈ ｐｏｌｙｍｏｒｐｈｉｓｍ， ＰＣＲ－ＲＦＬＰ）技术建立了 ＤＬＡ－ＤＲＢ１基因分型的方法。用 ＰＣＲ方法对 ６ 只无亲缘相关的个体纯种比格狗ＤＲＢ１基因扩增，通过选用 ＨａｅⅢ， Ｈｉｎｆ Ⅰ， ＭｓｐⅠ， ＨｈａⅠ， Ｓａｕ９６Ⅰ及 ＲｓａⅠ等６种限制性内切酶对该片段进行酶切分析。结果酶切图谱显示出ＤＬＡ－ＤＲＢ１基因具有高度多态性。应用于３对卵巢移植狗的基因配型获得成功。结论研究表明，该方法在狗器官移植配型中有较好的实用价值。