深圳地区结核分枝杆菌耐利福平株rpoB基因突变分布

目的:调查深圳地区结核分枝杆菌耐利福平(RFP)株rpoB基因突变的分布情况,建立结核分枝杆菌耐药基因快速检测的方法.方法:对55株结核分枝杆菌临床分离株的rpoB基因280个碱基(包括其核心区75个碱基)应用PCR-直接测序法(PCR-DS)测定序列,其中耐利福平株51株,敏感株4株.结果:4株敏感株无突变,92.2%(47/51)耐利福平临床分离株存在rpoB基因突变.基因突变导致531位氨基酸突变率为41.2%(21/51);导致526位氨基酸突变率为29.4%(15/51);导致516位氨基酸突变率为13.7%(7/51).联合突变发生率为2.0%(1/51).未检测到发生缺失或插入碱基突变的菌株.结论:深圳地区结核分枝杆菌耐利福平株发生rpoB基因突变最常见的是531位丝氨酸、526位组氨酸和516位天冬氨酸的基因突变,三者突变率之和为84.3%(43/51).PCR-DS方法可快速测定结核分枝杆菌RFP耐药基因突变.     

中国耐多药结核分枝杆菌临床分离株rpoB基因突变特点

为阐明中国耐多药结核分枝杆菌临床分离株rpoB基因的突变特征,对86株结核分枝杆菌临床分离株rpoB基因两个区域,包括81个碱基利福平抗药性决定区(rifampin resistance determining region,RRDR)和V176F区进行序列测定。其中72株耐多药分离株中的65株rpoB基因的RRDR区存在22种不同类型突变、21种点突变和一个插入突变。最常见的突变部位分别位于密码子531(41％)、526(40％)和516(4％),10％耐药分离株未检测到突变。鉴定了RRDR内6个新的等位基因,以及RRDR外部区域5个新的突变。所有分离菌株V176均无突变。     

基于PARMS技术的结核分枝杆菌利福平耐药基因rpoB分子标记的开发

文中旨在建立结核分枝杆菌利福平耐药基因rpoB的荧光分子标记,为分子药物敏感性试验提供简便、可靠的基因分型检测方法。对比分析利福平耐药菌株rpoB基因531、526、516、511、513等氨基酸位点的基因突变与敏感菌株中等位基因的序列差异,结合PARMS技术 (Penta-primer amplification refractory mutation system),建立rpoB基因的荧光分子标记。利用其对104例结核分枝杆菌临床分离株进行检测,经Sanger测序验证,正确率100%。并采用比例法药敏试验对这104份样本进行了利福平耐药性鉴定,与分子标记结果相符率为94.23%。结果表明,分子标记有较强可靠性,能检出表型药敏无法测出的低浓度耐药样本 (511/533单位点突变)。建立的11组荧光分子标记能覆盖92%–96%的利福平耐药菌株rpoB基因突变类型,为快速检测结核分枝杆菌利福平耐药提供新思路。     

PCR-SSCP检测结核分枝杆菌利福平耐药相关基因rpoB突变

目的 建立聚合酶链反应-单链构象多态性(PCR-SSCP)技术快速检测结核分枝杆菌利福平(RFP)耐药相关基因rpoB突变.方法 设计结核分枝杆菌RFP耐药相关rpoB基因PCR引物,建立PCR-SSCP技术检测临床菌株rpoB基因的突变导致的运动变位,同时采用PCR直接测序(PCR-DS)技术检测rpoB基因突变,并对上述方法检测结果进行分析和比较.结果 84株临床菌株均含有rpoB基因;PCR-SSCP和PCR-DS检测结果显示,56株RFP敏感菌株中rpoB基因分别有3株和2株检测出突变,检测特异性分别为94.6％ (53/56)和96.4％(54/56);28株RFP耐药菌株中rpoB基因分别有27株和28株发生突变,检测灵敏度分别为96.4％和100％.结论 本研究建立的PCR-SSCP技术能快速、简便、特异、敏感地检测结核分枝杆菌利福平耐药基因rpoB突变,具有临床应用前景.     

耐药结核分枝杆菌感染动物模型所用菌株的筛选

本研究通过小鼠体内实验检测我国部分地区结核分枝杆菌耐药菌株的毒力,以筛选耐药结核分枝杆菌感染动物模型所用菌株。收集从我国部分省份98例结核病患者痰培养液中分离出的结核分枝杆菌,用比例法药敏试验进行结核分枝杆菌一线和二线药物的药敏试验,筛选出对二线药物敏感而对一线药物利福平或异烟肼耐药或敏感的菌株,然后进行小鼠体内毒力实验,对异烟肼耐药相关基因katG和利福平耐药相关基因rpoB测序并进行基因突变分析。从98株菌中筛选出药物敏感谱清晰的40株,进行小鼠体内毒力实验。结果显示,共35株半数死亡时间≤H37Rv的半数死亡时间,其中18株耐利福平合并耐异烟肼、5株单耐利福平,7株单耐异烟肼、5株对利福平和异烟肼均敏感。通过小鼠毒力研究,分别筛选出基因背景清晰,半数死亡时间≤7d的耐利福平合并耐异烟肼的菌株1株、半数死亡时间≤7d单耐利福平和异烟肼的菌株各1株,作为耐药结核分枝杆菌感染小鼠模型所用菌株及进一步进行豚鼠等其他动物模型感染用候选菌株。     

结核分枝杆菌eis基因突变与氨基糖苷耐药的研究

目的：探讨结核分枝杆菌eis基因突变与氨基糖苷耐药之间的相互关系。方法：以本室保存的35株已确定耐一线药物（异烟肼、利福平、乙胺丁醇、链霉素）的结核分支杆菌为研究对象,应用BECTEC960测定其二线药物（阿米卡星、卡那霉素）的耐药情况,同时应用基因测序的方法测定结核分枝杆菌eis基因突变情况,分析eis基因突变与氨基糖苷耐药之间的相互关系。结果：氨基糖苷耐药的部分结核分杆杆菌中,eis基因487位碱基出现突变,相应的163位氨基酸密码子由CGT突变为CAT,即由缬氨酸变为异亮氨酸。结论：eis基因V163I突变（缬氨酸变为异亮氨酸）可能与结核分枝杆菌耐氨基糖苷类药物有关。     

结核分枝杆菌eis 基因突变与氨基糖苷耐药的研究

目的:探讨结核分枝杆菌eis基因突变与氨基糖苷耐药之间的相互关系。方法:以本室保存的35株已确定耐一线药物(异烟肼、利福平、乙胺丁醇、链霉素)的结核分支杆菌为研究对象,应用BECTEC960测定其二线药物(阿米卡星、卡那霉素)的耐药情况,同时应用基因测序的方法测定结核分枝杆菌eis基因突变情况,分析eis基因突变与氨基糖苷耐药之间的相互关系。结果:氨基糖苷耐药的部分结核分杆杆菌中,eis基因487位碱基出现突变,相应的163位氨基酸密码子由CGT突变为CAT,即由缬氨酸变为异亮氨酸。结论:eis基因V163I突变(缬氨酸变为异亮氨酸)可能与结核分枝杆菌耐氨基糖苷类药物有关。     

应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型,评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系,比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中,78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸,6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T,14株（14．3％）未发现突变;69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致,RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明,应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性,反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。     

应用专一性寡核苷酸微阵列可靠地检测结核分枝杆菌利福平耐药性

DNA微阵列代表聚合酶链反应产物诊断测序的发展方向 .根据结核分枝杆菌rpoB基因利福平抗药性决定区域内点突变及其它重排的特征 .研制一种快速地鉴定结核分枝杆菌利福平耐药菌株的中等密度微阵列方法 .利福平抗药性通过使荧光标记扩增遗传物质与微阵列杂交测定 .检测5 3株利福平耐药结核分枝杆菌和 15株利福平敏感结核分枝杆菌 .微阵列方法的检测结果与药物敏感性试验和DNA测序结果完全一致 .临床标本PCR扩增后仅 1 5h可检出利福平耐药临床分离株 .表明寡核苷酸微阵列是高效的、专一性的方法 ,可作为检测利福平抗药性的快速方法以弥补传统培养方法的不足     

新疆南疆MTB临床分离株异烟肼耐药情况及耐药相关基因的突变研究

目的:探索新疆南疆部分地区维吾尔族结核病人群中分离到的结核分枝杆菌基因组中的katG、inhA、kasA、ahpC基因突变与耐异烟肼的关联,揭示新疆肺结核病高患病率、高死亡率的可能原因.方法:收集新疆南疆部分地区维吾尔族结核病患者痰液标本,对MTB进行分离培养后,应用比例法检测其对异烟肼耐药性,运用PCR技术对所分离菌株的上述基因相关片段扩增并进行测序及序列分析.结果:筛选出实验菌株99株,其中敏感株63株,耐异烟肼菌株36株,耐药率为36.4%.耐药株中katG基因突变率为63.9%,315位点突变,突变类型AGC→ACC(Ser→Thr)、AGC→AAC(Ser→Asn);inhA基因突变率为47.2%,有缺失、同义突变和错义突变;kasA基因突变率为41.7%,突变类型为缺失和错义突变;ahpC基因突变率为8.3%,属于错义突变.结论:新疆南疆结核病高患病率、高死亡率的可能原因之一是结核分枝杆菌对INH产生了耐药,结核分枝杆菌对INH耐药机制可能与耐药菌株基因组内katG、inhA、kasA和ahpC基因发生了突变有关.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.