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.

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.

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.

四川省脊髓灰质炎Ⅰ型野毒株的分子病毒学检测

１９９１年安岳县和１９９２年会东县各１例临床诊断为脊髓灰质炎（脊灰）的患者，从其粪便标本中各分离出１株病毒，血清学定型均为脊灰Ⅰ型病毒，经ＰＣＲ－ＲＦＬＰ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ－ＲｅｓｔｒｉｃｔｉｏｎＦｒａｇｍｅｎｔＬｅｎｇｔｈＰｏｌｙｍｏｒｐｈｉｓｍ ａｓｓａｙ）法检测，２株病毒与Ｓａｂｉｎ Ⅰ有明显差异定为Ⅰ型野毒株。又经核酸序列测定，其与Ｓａｂｉｎ Ⅰ型疫苗株亦有明显的差异，证实为脊灰互型野毒株。结果显示，不同年份、不同地方流行株间的序列有差异。四川省脊灰流行与毒株间核酸序列的差异有关，但我国现行疫苗ＯＰＶ（ＯｒａｌＰｏｌｉｏＶａｃｃｉｎｅ）能有效预防我省脊灰，基础兔疫和强化免疫是预防和消灭脊灰的有力武器。

广西肝癌人群IL－23Rrsl1805303位点单核苷酸多态性相关性分析

目的分析白介素23受体（interleukin-23receptor，IL-23R）基因rsl1805303位点单核苷酸多态性（singlenueleo—tidepolymorphisms，SNPs）与广西肝癌人群的相关性。方法将来自广西的研究对象分为肝癌组（270例）和健康对照组（251例），采用聚合酶链式反应-限制性片段长度多态性（polymerasechainreactiomrestrictionfragmentlengthpolymor—phism，PCR—RFLP）技术检测IL-23Rrsl1805303位点sNPs并分析其与肝癌的关系。结果发现IL-23R基因rsl1805303位点存在三种基因型：CC型、XT型和TT型；在肝癌组与对照组比较中发现IL-23Rrsl1805303位点基因型频率分布差异无统计学意义（χ^2=2．578，P=0．276）；该位点等位基因频率分布差异无统计学意义[（χ^2=2．578，P=0．276，OR（95％CI）：1.164（0．913～1．485）]。结论IL-23Rrsl1805303位点sNPs可能不是广西肝癌人群发病的危险因素。

Research on Hepatitis B virus Genotypes and Subgenotypes among Bai Nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B+C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B+C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B+C) (P=0.077/P=0.663). In Dali, genotypes B, B+C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B+C) in patients.

Aerobic biodegradation of di-n-butyl phthalate by Xiangjiang River sediment and microflora analysis

Di-n-butyl phthalate (DBP),one of phthalate acid esters (PAEs),was investigated to determine its biodegradation rate using Xiangjiang River sediment and find potential DBP degraders in the enrichment culture of the sediment. The sediment sample was incubated with an initial concentration of DBP of 100 mg/L for 5 d. The biodegradation rate of DBP was detected using HPLC and the degraded products were analyzed by GC/MS. Subsequently,the microbial diversity of the enrichment culture was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results reveal that almost 100% of DBP is degraded after merely 3 d,generating two main degraded products:mono-butyl phthalate (MBP) and 9-octadecenoic acid. After a six-month enrichment period under the pressure of DBP,the dominant family in the final enrichment culture is clustered with the Comamonas sp.,the remaining are affiliated with Sphingomonas sp.,Hydrogenophaga sp.,Rhizobium sp.,and Acidovorax sp. The results show the potential of these bacteria to be used in the bioremediation of DBP in the environment.

劳盆地热液喷口沉积物微生物多样性初步研究

采用聚合酶链式反应限制性片段长度多态性分析(PCR-RFLP)技术对劳盆地热液区TVG9站位沉积物中细菌、古菌多样性进行了调查,并构建其细菌、古菌种群的16S rRNA基因文库。研究结果表明,该站位细菌包含变形杆菌(Proteobacteria)、硝化螺旋菌(Nitrospira),疣微菌(Verrucomicrobia)等3个类群,其中变形杆菌占据优势地位,由Alphaproteobactria,Gamaproteobactria,Deltaproteobac-tria等3个亚群组成。古菌包含泉古菌(Crenarchaeota)和广古菌(Euryarchaeota),其中泉古菌占优势,由MGⅠ(Marine GroupⅠ)和MCG(Miscellaneous Crenarchaeotal Group)两类群组成,而广古菌主要由MBGE(Marine Benthic Group E)组成。旨在揭示深海热液区的微生物多样性,为生态环境的研究提供理论支持。

Is type Ⅰ alpha 2 collagen gene responsible for intracranial aneurysm in Northeast China?

In this study, we investigated whether a single nucleotide polymorphism （rs42524 G 〉 C） in the type I alpha 2 collagen gene was associated with sporadic ruptured intracranial aneurysm or its clinical characteristics in patients from Northeast China. Genotyping of the rs42524 G 〉 C polymorphism was carried out using a polymerase chain reaction-restriction fragment length polymorphism assay. The data showed that the frequency of the rs42524 GC ＋ CC genotype was significantly higher than the GG genotype among intracranial aneurysm patients whose Hunt and Hess grading scale was 〉 3. In addition, the rs42524 G 〉 C genotype was found to have a statistically significant association with intracranial aneurysm risk. These findings indicate that the type I alpha 2 collagen gene gene may be involved in a predisposition to intracranial aneurysm in the Northeast Chinese population. Crucially, the rs42524 C allele may be an important risk factor for increased severity of the condition in patients with ruptured intracranial aneurysms.

Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder

BACKGROUND Mutational activation of Ras genes is established as a prognostic factor for the genesis of a constitutively active RAS-mitogen activated protein kinase pathway that leads to cancer.Heterogeneity among the distribution of the most frequent mutations in Ras isoforms is reported in different patient populations with urothelial carcinoma of the bladder(UCB).AIM To determine the presence/absence of mutations in Ras isoforms in patients with UCB in order to predict disease outcome.METHODS This study was performed to determine the mutational spectrum at the hotspot regions of H-Ras,K-Ras and N-Ras genes by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and DNA sequencing followed by their clinical impact(if any)by examining the relationship of mutational spectrum with clinical histopathological variables in 87 UCB patients.RESULTS None of the 87 UCB patients showed point mutations in codon 12 of H-Ras gene;codon 61 of N-Ras gene and codons 12,13 of K-Ras gene by PCR-RFLP.Direct DNA sequencing of tumor and normal control bladder mucosal specimens followed by Blastn alignment with the reference wild-type sequences failed to identify even one nucleotide difference in the coding exons 1 and 2 of H-Ras,NRas and K-Ras genes in the tumor and control bladder mucosal specimens.CONCLUSION Our findings on the lack of mutations in H-Ras,K-Ras and N-Ras genes could be explained on the basis of different etiological mechanisms involved in tumor development/progression,inherent genetic susceptibility,tissue specificity or alternative Ras dysfunction such as gene amplification and/or overexpression in a given cohort of patients.

Detection of genotypic clarithromycin-resistant Helicobacter pylori by string tests

AIM: To evaluate the utility of the string test to detect genotypic clarithromycin-resistant Helicobacter pylori (H. pylori) by polymerase chain reaction (PCR)-restriction fragment length polymorphism.

食管癌不同染色体位点上等位基因杂合缺失的研究

应用聚合酶链反应（ＰＣＲ）技术，配合限制片段长短多态现象（ＲＦＬＰ）分析，对２２例食管癌病人３号染色体短臂３ｐ１４～２４，１７号染色体短臂１７ｐ１３．１杂合缺失（ＬＯＨ）进行了测定。结果显示：３ｐ２４、３ｐ２１、３ｐ１４和１７ｐ１３．１位点ＬＯＨ检出情况分别为８／１１、２／４、１／９和６／６，其中３ｐ２４和１７ｐ１３．１位点的ＬＯＨ较高，提示这一位点ＬＯＨ的测定有可能成为食管癌早期诊断的分子标志。实验发现，在３ｐ２４ＥＡｐＭＤ位点的ＬＯＨ检测中，哈萨克族杂合缺失为５／５，汉族为２／４，维吾尔族为１／２，这是否与种族遗传有关，有必要扩大病例进一步研究。

中国汉族和维吾尔族药物代谢酶CYP3A4、CYP2C9、CYP2C19、CYP2D6基因多态性分析

目的对中国汉族、维吾尔族健康人群CYP3A4、CYP2C9、CYP2C19、CYP2D6进行基因多态性分析,并对汉族和维吾尔族人群等位基因频率和基因型频率进行比较。方法聚合酶链反应一限制性片段长度多态性(PCR-RFLP)法对CYP3A4、CYP2C9、CYP2C19、CYP2D6进行分型。结果汉族、维吾尔族健康人群CYP3A4*5等位基因频率为0,CYP3A4*18等位基因频率分别为0.183 8、0.140 2;CYP2C9*2等位基因频率分别为0.011 0、0.095 8,CYP2C9*13等位基因频率分别为0、0.002 3;CYP2C19*2等位基因频率分别为0.386 0、0.324 8,CYP2C19*3等位基因频率分别为0.051 5、0.021 0;CYP2D6*10等位基因频率分别为0.573 5、0.224 3。结论本研究在汉族、维吾尔族健康人群中未发现CYP3A4*5等位基因。汉族、维吾尔族健康人群CYP3A4*18、CYP2C9*13、CYP2C19*2、CYP2C19*3等位基因频率差异均无统计学意义。维吾尔族健康人群CYP2C9*2等位基因频率远高于汉族(P<0.01);CYP2D6*10等位基因频率远低于汉族(P<0.01),存在民族差异。

冠状动脉粥样硬化性心脏病患者凝血酶激活的纤溶抑制物及其编码区基因多态性的研究

目的探讨凝血酶激活的纤溶抑制物（thrombin actiratable fibrinolysis inhibitor，TAFI）及其编码基因CPB2单核苷酸多态性与冠状动脉粥样硬化性心脏病（冠心病）之间的联系。方法应用聚合酶链反应．限制性片段长度多态性分析技术（polymerase chain reaction-restriction fragmentlengthpo lymorphisin，PCR-RFLP）检测210例冠心病患者和190名正常对照者的似仃基因编码区Thr325Ile、Thrl47Ala多态性分布特点，同时应用发色底物法和ELISA法分别测定TAFI的活性及抗原，并进一步分析基因多态性与TAH的活性及抗原之间的关系。结果冠心病组（心肌梗死组及心绞痛组）血浆中TAFI的活性及抗原水平均较对照组显著增高，差异有统计学意义。CPB2基因C1040T（Thr325Ile）及GSOSA（Thrl47Ala）2个位点的3种基因型在冠心病组和对照组的频率分布分别为C1040C（Thr325Thr）67（31．9％）、64（33．6％）；C1040T（Thr325Ile）109（51．9％）、92（48．4％）；T1040T（lle325Ile）34（16．2％）、34（17．8％）；G505G（Alal47Ala）75（35．7％）、72（37．8％）；G505A（Thrl47Ala）112（53．3％）、96（50．5％）；A505A（Thrl47Thr）23（10．9％）、22（11．7％），经x^2检验，基因型分布符合Hardy-Weinberg平衡，并且两组之间各种基因型频率分布差异无统计学意义（P〉0．05）。在冠心病组和对照组Thr325Ile不同的基因型对TAFI活性没有影响；对TAH抗原含量的影响则以Thr325Thr纯合基因型者血浆TAH抗原浓度最高，较其他两型差异有统计学意义（P〈0．05），Thr325Ile与TIle325Ile型之间则差异无统计学意义（P〉0．05）。而Thrl47Ala基因多态性与血浆中TAFI活性及抗原水平之间的差异均无统计学意义（P〉0．05）。结论TAFI具有抑制纤溶的作用，可能是冠心病发病的危险因子。TAFI编码区基因Thr325Ile的多态性对血浆中TAFI抗原水平有明显影响，但Thr325Ile、Thrl47Ala的多态性与冠心病的发生没有明显的相关性。

CYP1A1和CYP1A2基因多态性与汉族女性乳腺癌的关系

目的 探讨CYP1A1基因MspⅠ位点与CYP1A2基因C734A位点多态性与汉族女性乳腺癌的关系.方法 应用聚合酶链反应-限制性片段长度多态性技术和限制性核酸内切酶酶切的方法,检测2011年9月至2012年8月在四川省医学科学院四川省人民医院确诊的144例女性乳腺癌患者（乳腺癌组）和152例同期健康体检正常女性（对照组）CYP1A1基因MspⅠ与CYP1A2基因C734A多态性位点的基因型,用χ2检验比较两组等位基因频率的差异.结果 在乳腺癌组与对照组中,CYP1A1基因MspⅠ位点T等位基因频率分别为0.73和0.65,两者差异有统计学意义（χ2=4.94,P=0.03）,C等位基因与T等位基因相比,乳腺癌发病风险OR为0.67 （95%CI：0.47～0.96）;CYP1A2基因C734A位点C等位基因频率分别为0.26和0.29,两者差异无统计学意义 （χ2=0.63,P=0.43）.将乳腺癌组按照ER、PR表达与否进一步分组后,CYP1A1基因MspⅠ与CYP1A 2基因C734A 2个多态性位点的等位基因频率在ER（＋）与ER（-）组之间以及PR（＋）与PR（-）组之间差异均无统计学意义[ER（＋）组比ER（-）组：χ2=0.34、0.01;PR（＋）组比PR（-）组：χ2=0.60、0.68;P均〉0.05].结论 汉族女性CYP1A1基因MspⅠ位点多态性与乳腺癌相关联.

Glacial Refugia of Ginkgo biloba and Human Impact on Its Genetic Diversity:Evidence from Chloroplast DNA

Variations in the trnK region of chloroplast DNA were investigated in the present study using polymerase chain reactionrestriction fragment length polymorphism to detect the genetic structure and to infer the possible glacial refugia of Ginkgo biloba L. in China. In total, 220 individuals from 12 populations in China and three populations outside China were analyzed, representing the largest number of populations studied by molecular markers to date. Nineteen haplotypes were produced and haplotype A was found in all populations. Populations in south-western China, including WC, JF, PX, and SP, contained 14 of the 19 haplotypes and their genetic diversity ranged from 0.771 4 to 0.867 6. The TM population from China also showed a high genetic diversity （H = 0.848 5）. Most of the genetic variation existed within populations and the differentiation among populations was low （GsT = 0.2）. According to haplotype distribution and the historical record, we suggest that populations of G. biloba have been subjected to extensive human impact, which has compounded our attempt to infer glacial refugia for Ginkgo. Nevertheless, the present results suggest that the center of genetic diversity of Ginkgo is mainly in south-western China and in situ conservation is needed to protect and preserve the genetic resources.

PCR-RFLP and AP-PCR of rbcL and ITS of rDNA Show That × Taxodiomeria peizhongii （ Taxodium × Cryptomeria） Is not an Intergeneric Hybrid

Taxodiorneria peizhongii Z. J. Ye, J. J. Zhang et S. H. Pan was regarded as a new Intergenerlc hybrid between Taxodlum mucronatum Tenore （as the female donor） and Cryptomeria fortunei Hoolbrenk ex Otto et Dletr （as the male donor）. To confirm the authenticity of the intergeneric hybrid, we analyzed the rbcL gene and the Internal transcribed spacer （ITS） of 26S-18S ribosomal RNA gene of the three species using polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） and arbitrarily primed PCR （AP-PCR）, and ob- tained the following results：i） Taxodiomeria peizhongii had the same RFLP maps of the rbcL gene and the ITS as Taxodlum mucronatum, but was different from C. fortunei; II） a 311-bp PCR amplification product was obtained In C. fortunei by AP-PCR of ITS, but was not found in Taxodiomeria peizhongii. Our results have demonstrated that C. fortunei did not provide any genome for Taxodiomeria peizhongii, Implying that T. peizhongii Is not an Intergenerlc hybrid between the two species.