Microbial community structure in different wastewater treatment processes characterized by single-strand conformation polymorphism (SSCP) technique

In order to investigate microbial community structures in different wastewater treatment processes and understand the relationship between the structures and the status of processes,the microbial community diversity,variety and distribution in five wastewater treatment pro cesses were studied by a culture-independent genetic fingerprinting technique single-strand conformation poly-morphism(SSCP).The five processes included denitrifying and phosphate-removal system(diminished N),Chinese traditional medicine wastewater treatment system(P),beer wastewater treatment system(W),fermentative biohydrogen-producing system(H),and sulfate-reduction system(S).The results indicated that the microbial community profiles in the wastewater bioreactors with the uniform status were very similar.The diversity of microbial populations was correlated with the complexity of organic contaminants in wastewater.Chinese traditional medicine wastewater contained more complex organic components;hence,the population diversity was higher than that of simple nutrient bioreactors fed with molasses wastewater.Compared with the strain bands in a simulated community,the relative proportion of some functional microbial populations in bioreactors was not dom-inant.Fermentative biohydrogen producer Ethanoligenens harbinense in the better condition bioreactor had only a 5% band density,and the Desulfovibrio sp.in the sulfate-reducing bioreactor had less than 1.5%band density.The SSCP profiles could identify the difference in microbial community structures in wastewater treatment processes,monitor some of the functional microbes in these processes,and consequently provide useful guidance for improving their efficiency.

Fluorescence-based Multiplex PCR-Single Strand Conformation Polymorphism (SSCP) Analysis of 16S Ribosomal DNA Using Capillary Electrophoresis

The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tool has been reported widely. This study, aimed at 16S rRNA gene (16S rDNA) and with the help of biomolecular methods, attempted to achieve the goal of rapid identification of common pathogens. In this study, 333 clinical isolated pathogenic bacteria were collected. Two pairs of primers were chosen and labeled with different fluorescent dyes and then used to amplify the genomic DNA extracted from bacteria. The PCR products were then detected by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP). In order to pursue higher resolution and peak-separation effect, a high efficient separating medium, liner polyacrylamidedel (LPA), was put to use in this study. Finally, every bacteria colony generated distinct patterns from each other, which were easily to be used for identification. These results indicated that PCR-CE-SSCP was a rapid identification method for bacterial identification, with the aspects of high efficiency and high precision. Compared with traditional method, this technology is of great utility for clinical use especially for its high sensitivity.

Poly(ADP-ribose) polymerase-1 gene polymorphism in various Chinese nationalities

Poly （ADP-ribose） polymerase-1 （PARP-1） can exacerbate ischemic brain injury and lessen ischemic neuronal death, which may be associated with PARP-1 polymorphisms. The present study investigated human PARP-1 gene polymorphisms in various Chinese nationalities, the results of which could potentially help in the treatment and prevention of neurologic diseases. Genetic polymorphisms of seven exons in the PARP-1 gene, in 898 Chinese Han, Buyi, Shui, Miao, and Zhuang subjects, were investigated by PCR-single-strand conformation polymorphism. A single-strand conformation polymorphism variant in exons 12, 13, 16, and 17 of the PARP-1 gene was identified in 148 people, with two stationary bands showing three degenerative single strands. Results showed that the PARP-1 gene polymorphisms exist in various nationalities, and may act as a biomarker for susceptibility to disease.

Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population

BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.

Single nucleotide polymorphisms in the CDH17 gene of colorectal carcinoma

AIM:To investigate the relationship between c.343A>G and c.2216A>C polymorphism sites in the CDH17 gene and colorectal carcinoma.METHODS:Ninety-three non-consanguineous colorectal carcinoma patients admitted to the Department of Oncology at the First Affiliated Hospital of Zhengzhou University were included in this study.Ninety-three peripheral venous blood samples,of approximately one milliliter from each patient,were collected betweenDecember 2009 and August 2010.The genomic DNA of these peripheral venous blood samples were extracted and purified using a Fermentas Genomic DNA Purification Kit(Fermentas,CA) according to the manufacturer' s protocol.The single nucleotide polymorphisms(SNPs) of the liver-intestine cadherin(CDH17) gene c.343A>G and c.2216A>C were determined by the polymerase chain reaction-single strand conformation polymorphism method(PCR-SSCP) in 93 peripheral venous blood samples from patients suffering with colorectal carcinoma.Typical samples that showed different migration bands in SSCP were confirmed by sequencing.Directed DNA sequencing was used to check the correctness of the genotype results from the PCR-SSCP method.RESULTS:There was a significant association between the c.2216 A>C SNPs of the CDH17 gene and the tumor-node-metastasis(TNM) grade,as well as with lymph node status,in 93 peripheral venous blood samples from colorectal carcinoma patients.The genotype frequencies of A/C,A/A,and C/C were 12.90%,33.33% and 53.76%,respectively.There was a significant correlation between lymph node metastasis,TNM grade,and the genotype distribution(P < 0.05).The C/C genotype raised the risk of lymph node metastasis and the TNM grade.There was a significant difference in the TNM grade and lymph node metastasis between the A/A and C/C genotypes(P = 0.003 and P = 0.013,respectively).Patients with colorectal carcinoma carrying the C allele tended to have a higher risk of lymph node metastasis and have a higher TNM grade.The difference between the TNM grades,as well as the lymph node metastasis of the two alleles,was statistically significant(P < 0.01).CONCLUSION:The SNPs of the CDH17 gene c.2216 A>C might be clinically important in the prognosis of colorectal carcinoma.

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.

Detection of the polymorphism at codon 72 in exon 4 of p53 gene by PCR－SSCP

By employing different gel components and electrophoresis conditions, the PCR-SSCP(polymerase chain reaction-single-stranded conformation polymorphism) method was used to detect the polymorphism(CCC or CGC ) of codon 72 in exon 4 of p53 gene. The results

中国家驴TYRP1基因第二内含子多态性分析

为了探究TYRP1基因对中国家驴毛色的影响,试验采集410只不同毛色中国家驴的血液样本,采用聚合酶链式反应及单链构象多态性(polymerase chain reaction and single-strand conformation polymorphism, PCR-SSCP)方法检测中国家驴TYRP1基因第二内含子中的单核苷酸多态性(single nucleotide polymorphism, SNP)位点,对不同毛色中国家驴个体进行基因分型,并计算基因型频率和等位基因频率,分析TYRP1基因第二内含子多态性与中国家驴不同毛色之间的关系。结果表明:在中国家驴TYRP1基因第二内含子中检测到1个SNP位点(g.1 702A→G),该位点存在AA、AG、GG三种基因型;乌头、黑三粉、青三粉、灰三粉、白色和白化家驴的优势等位基因均为A。乌头、黑三粉和青三粉均为AA基因型频率最高,GG基因型频率最低;灰三粉为AA基因型频率最高,AG基因型频率最低(0)。在乌头、黑三粉、青三粉、灰三粉家驴中,AA基因型频率为灰三粉最高,然后依次为青三粉、黑三粉和乌头;以黑毛色为主色调的家驴(乌头、黑三粉、青三粉)GG基因型频率低于以灰毛色为主色调的家驴(灰三粉),有白毛混杂的黑三粉和青三粉的GG基因型频率低于纯黑的乌头。样本中的白色家驴和白化家驴各有2头,其基因型均为AA纯合子。说明随着中国家驴黑色被毛比例的增加,TYRP1基因g.1 702A→G位点的AA基因型频率逐渐降低,推测该位点可能与中国家驴毛色性状有关。

利格列汀多晶型研究

研究了利格列汀复杂的晶型现象及其晶格中的构象变化.通过培养利格列汀的单晶并进行X射线单晶衍射分析,首次得到了包括晶型A在内的3种晶体结构;结合文献已报道的2种晶体结构,分析了各晶体结构的特点及其晶格中的分子构象.研究结果表明,5种晶体里存在晶型A和晶型F两种晶型,其中晶型F为准多晶型,可以包含多种溶剂,形成通道型溶剂合物,其晶格参数随包含的溶剂略有变化.晶型A的构象与晶型F的构象存在较高能垒,导致2种晶型难以互相转化.晶型F不同溶剂合物之间的分子构象并不完全相同,在粉末X射线衍射(PXRD)谱图上也有显著差异.最后,通过混悬转晶和热分析等方法研究了晶型之间的转化关系.

An automated fluorescent single strand conformation polymorphism technique for high throughput mutation screening

Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1,2 and 7 bp)and 2 deletions(1 and 2 bp)in the hepatocyte nuclear factor(HNF)-4α,glucokinase and HNF-1α genes were tested.During nested PCR,amplified fragments were labeled with three fluorescent dyes.PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in nondenaturing gel conditions.Results Twenty-five of 27 variants(93%)could be detected by combining 5% glycerol and 10% sucrosegel matrix conditions.Twenty-two of 27(82%)and 18 of 27(67%)variants were identified using 5%glycerol and 10% sucrose conditions,respectively.Conclusion This fluorescence-based PCR single strand conformation polymorphism technique represents a simple,non-hazardous,time-saving and sensitive method for high throughput mutation detection.

Single strand conformation polymorphism for analysis of genomic variability of hepatitis C virus nonstructure 5A region

Abstract Objective To establish a convenient method to detect the genomic population with hepatitis C virus (HCV) at nonstructure 5A (NS5A) region and to determine the correlation between the genomic population complexity at NS5A region and disease stage. Methods The sera from 52 patients with chronic hepatitis C virus infections were analysed using single strand conformation polymorphism (SSCP). In the SSCP, an asymmetric polymerase chain reaction (PCR) was carried out on the 455 bp products of the first round PCR at the NS5A region and the number of band of single strand deoxyribonucleic acid (DNA) which reacted with complemental DNA probe specific for the NS5A region after gel electrophoresis was analyzed. Results In 90% patients with chronic persistent hepatitis, the bands of single strand DNA was limited to one, and in those with chronic active hepatitis or liver cirrhosis, two or more bands of DNA were frequently detected. In about half of patients with hepatocellularcarcinoma, three or more bands were found. The number of bands increased with the progression of liver disease. The multivariate analysis showed that the progression of liver disease was the independent factor of viral diversity (P<0.025) and was not related to the age, sex, the route of infection and the titer of hepatitis C virus ribonucleic acid (HCV RNA). Conclusion These results suggest that the genomic variability of HCV at NS5A region increases with the progression of liver disease, and this may be closely related to the clinical features of type C liver disease.

猪LPL基因多态性及其部分DNA片段的测序

脂蛋白脂肪酶（Ｌｉｐｏｐｒｏｔｅｉｎｌｉｐａｓｅ，ＬＰＬ） 是影响动物脂肪沉积的关键酶之一。本文利用ＰＣＲＳＳＣＰ法，对猪的ＬＰＬ基因进行了多态性分析，发现其５’端区域存在ＤＮＡ 多态性。脂肪型猪种梅山猪和通城猪表现出３种等位基因型（ＡＡ、ＡＢ、ＢＢ） ，其等位基因Ａ和Ｂ的频率分别为０．７６、０ ．２４ 和０．７７、０ ．２３ ；而瘦肉型猪种长白猪和大白猪表现出另外不同的３ 种等位基因型（ ＣＣ、ＣＤ、ＤＤ），其等位基因Ｃ和Ｄ 的频率分别为０．７６ 、０ ．２４ 和０．７２、０．２８ 。测序结果表明具四种等位基因纯合子的ＤＮＡ 片段之间存在５ 处碱基突变，分别表示为等位基因ＡＡＴＣＣＴ，ＢＡＴＴＣＴ，ＣＧＣＣＣＴ和ＤＧＣＣＧＣ，其中等位基因Ｄ 第＋ ３２ 处发生Ｔ／Ｃ 的突变导致亮氨酸到脯氨酸的改变（Ｌｅｕ→Ｐｒｏ） 。

藏绵羊DQA1基因多态性分析

【目的】研究藏绵羊DQA1基因多态性,确定其等位基因数、核苷酸多态位点、氨基酸多态位点及各等位基因间的遗传关系,同时分析其进化意义。【方法】采用PCR-SSCP方法检测了900只藏绵羊DQA1基因第2外显子多态性;克隆、测序群体内变异产生的各等位基因序列,并分析序列数据。【结果】发现了17个DQA1的等位基因,包括缺失的1种基因,其中5个为发现的新等位基因。16个单倍型序列中发现56个核苷酸多态位点,27个氨基酸多态位点。【结论】藏绵羊DQA1基因第2外显子具有丰富的多态性,群体中可能蕴藏着更多的遗传资源;藏绵羊DQA1基因最初可能是由2个等位基因突变分化成两大类等位基因的;藏绵羊DQA1基因第2外显子序列与牛的DQA1基因第2外显子序列具有较高的同源性,预示绵羊和牛的DQA1基因最早可能来源于它们分歧以前的共同祖先原始序列;DQA1基因在与其相关的特定抗原刺激下发生的免疫应答反应在绵羊和牛上具有相似性;新等位基因C的139位发现了1个新的核苷酸突变位点(A/G),属同义突变;5个新发现的DQA1等位基因遗传关系较近,可能由同一等位基因突变产生。

荷斯坦牛HSP70-1基因遗传多态性与乳腺炎抗性关系

以253头中国荷斯坦奶牛为研究对象,检测HSP70-1基因的多态性,并分析其多态性与中国荷斯坦牛体细胞评分(Somatic cell score,SCS)的相关性。首先以PCR-SSCP法寻找HSP70-1基因编码区的突变,并通过测序确定突变的类型,根据突变类型寻找合适的内切酶,最终采用PCR-RFLP方法鉴定实验牛基因型;然后分析基因多态性与中国荷斯坦牛SCS的相关性。结果表明HSP70-1基因的1623bp处产生G→A→C突变,2409bp处产生G→A突变,两位点都是沉默突变,未引起氨基酸序列的改变;经χ2适合性检验,中国荷斯坦牛在两个位点均未达到Hardy-Weinberg平衡状态;同时,群体基因座不同基因型与SCS相关分析的结果表明,2409位点基因型与SCS相关性不显著(P>0.05),1623位点基因型与SCS相关性显著(P<0.05),CC型SCS显著低于AG、GG型(P<0.05),CC基因型为乳腺炎抗性基因型。在中国荷斯坦奶牛群体中,HSP70-1基因CC基因型可作为改良奶牛乳腺炎抗性性状的分子遗传标记。

脂蛋白脂肪酶基因突变的研究

目的:筛查国人脂蛋白脂肪酶(LPL)基因的突变,并探讨其与高甘油三酯血症的关系。方法:对高甘油三酯血症组(48例)和正常甘油三酯对照组(121例)的各扩增片段进行分析,电泳图谱异常者进行PCR产物测序。对于频率较高的多态性位点,引入限制性核酸内切酶位点利用PCR-RFLP进行鉴定。结果:在高甘油三酯血症人群中,检出2例内含子3受位剪接点上游6bp的C→T转换的突变杂合子,1例外显子5Pro207→Leu突变杂合子,在全部样品中检出1例Ser447→stop突变纯合子,50例Ser447→stop杂合子。结论:天津地区人群中存在着LPL基因突变,除Ser447→stop外,内含子3和外显子5的突变多与高甘油三酯血症相关,可能是高甘油三酯血症的遗传易感因子。

猪食道口线虫ITS-1和ITS-2rDNA的PCR-SSCP分析

以采自我国不同地区猪体的食道口线虫虫株为研究对象,PCR扩增出ITS-1和ITS-2序列片段,然后采用单链构象多态性(SSCP)方法分析PCR产物,对不同地区食道口线虫进行分子鉴定。所有样品经SSCP分析显示两种带型,第一种为有齿食道口线虫带型,另一种为未定种食道口线虫带型。代表性样品的测序结果表明,未定种食道口线虫带型的样品为四棘食道口线虫。本研究在国际上首次报道了中国猪四棘食道口线虫的ITS序列,并建立了区分有齿食道口线虫和四棘食道口线虫的PCR-SSCP方法,从而为食道口线虫的分子生物学的进一步研究奠定了基础。

牛FSHR基因第10外显子单核苷酸多态性及其与双胎性状的关系

以秦川牛和荷斯坦奶牛的双胎母牛和单胎母牛为实验材料 ,以牛的FSHR基因的第 10个外显子作为标记牛双胎性状的候选基因 ,用SNP法进行了多态检测 .结果发现 ,在秦川牛的双胎母牛中突变率 6 0 % (6 10 ) ,而在单胎母牛中突变率为 2 0 % (2 10 ) ;在荷斯坦奶牛中 ,双胎母牛突变率为31 2 5 % (5 16 ) ,单胎母牛突变率为 6 6 7% (1 15 ) ;由此可见双胎牛和单胎牛二者之间FSHR基因的第 10个外显子的突变率差异明显 .这表明 ,选择FSHR基因的第 10个外显子有可能作为双胎性状的候选基因 .序列分析发现 ,在FSHR基因的第 15 0 6位碱基发生了突变 (T→C) ,但氨基酸没有发生变化 .

苯丙氨酸羟化酶(PAH)基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症(PKU)人群中苯丙氨酸羟化酶(PAH)基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR 单链构象多态性(SSCP)分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因:G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7种突变基因在中国PKU人群首次发现;G239D、R241fs、G247S、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库(www.pahdb.mcgill.ca)。突变基因的总频率为30.61%(90/294)。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。

肝癌DKK1基因的表达及突变分析

目的:探讨中国地区人肝癌发生发展过程中有无DKK1基因突变。方法:Northernblot方法分析12例肝癌患者癌和癌旁肝组织DKK1mRNA表达水平。采用PCRSSCP方法,检测20例肝癌患者(包括癌和癌旁肝组织)及8种人肝癌细胞株中有无DKK1基因突变,并采用DNA测序对PCRSSCP突变分析结果加以验证。结果:Northernblot显示12例肝癌患者中7例存在癌组织DKK1mRNA高表达而癌旁肝组织微弱表达或不表达。突变检测发现20例肝癌患者及8种人肝癌细胞株中仅1例肝癌患者存在DKK1基因的同义突变(单核苷酸多态现象)。结论:DKK1在中国人肝癌中存在高表达,但未发现突变发生,提示DKK1基因参与肝癌的癌变过程可能并不依赖于该基因的突变。

ADD1基因PCR-SSCPs标记与猪肌内脂肪含量及背膘厚的关系

采用PCR SSCPs方法在苏太猪脂肪细胞定向分化因子 1(ADD1)基因第 347和 374位点发现单核苷酸多态性(SNP) ,分别为ADD1第 90和 99位氨基酸密码子的第 3位碱基 ,但这两个位点碱基的替换均没有引起氨基酸序列的变化。通过对 10 0头苏太猪肥育猪背最长肌中肌内脂肪含量的相关分析 ,发现杂合子AB肌内脂肪含量最高 ,极显著地高于AA纯合子 (P <0 0 1) ,BB纯合子肌内脂肪含量介于AB和AA之间 ,并有高于AA纯合子的趋势 (P =0 11)。各基因型间背膘厚没有显著差异。提示ADD1基因该位点上的PCR SSCPs可能作为选择肌内脂肪含量 。