内蒙古地区结核分枝杆菌串联重复序列基因多态性分析

目的 了解内蒙古地区结核分枝杆菌临床分离株的串联重复序列(variable number tandem repeat,VNTR)基因多态性和VNTR基因型构成,及不同VNTR位点在该地区结核分枝杆菌基因分型中的应用。 方法 从内蒙古自治区结核病防治研究所收集临床分离的结核分枝杆菌菌株及其病例背景资料,采用多位点串联重复序列分析(MLVA)对收集的菌株进行22个VNTR 位点分析,计算Hunter-Gaston 指数(HGDI),分析各个位点的分辨率, 同时用BioNumerics 软件对VNTR结果进行聚类分析。且统计学分析民族与主要基因型间的关系。 结果 372株菌共分为308个基因型,47簇,261个独特基因型,成簇率为17.20%。22个VNTR位点的基因多态性存在较大的差异,位点VNTR3820(HGDI 0.838)的分型分辨能力最高,MIRU23(HGDI 0.068)和MIRU27(HGDI 0.083)分型分辨能力较差。随着VNTR 位点的增加,分型的分辨能力也有所提高。Ⅰ群基因型菌株与民族易感性的分析表明,汉族与蒙古族间Ⅰ群基因型菌株分布差异无统计学意义(χ2=0.337, P=0.561>0.05)。 结论 内蒙古地区结核分枝杆菌临床分离株基因具有多态性,不同VNTR位点在内蒙古地区结核分枝杆菌中具有不同的分辨能力。且Ⅰ群基因型为该地区主要流行株,而Ⅰ群基因型菌株与民族易感性间无关联。     

炭疽高发地区土壤样本中常见芽胞杆菌的分离及鉴定

目的 通过分离鉴定炭疽可疑污染土壤样本的芽胞杆菌,评价消毒效果和了解监测地区土壤中的芽胞杆菌分布情况。 方法 采集炭疽监测点土壤样本60份,对炭疽芽胞杆菌和其他芽胞杆菌进行分离培养和聚合酶链反应扩增鉴定。 结果 在可疑污染土壤样本中未分离到炭疽芽胞杆菌;从分离到的48个单克隆菌落中扩增到33个目的片段,经测序和blast比对,确定得到地衣芽胞杆菌13株,枯草芽胞杆菌8株,短小芽胞杆菌扩增11株,蜡样芽胞杆菌扩增到1株,巨大芽胞杆菌引物和环状芽胞杆菌引物特异性不好。 结论 本研究提示该监测点炭疽疫情消毒效果可信,但需要进一步研究验证;几种芽胞杆菌在土壤中广泛存在,在炭疽监测工作中进行病原体分离时需要加以鉴别,可通过特异基因扩增来辅助检验。     

应用多位点VNTR分析进行铜绿假单胞菌院内感染溯源的研究

目的应用多位点可变数目串联重复序列(VNTR)分析方法进行多重耐药铜绿假单胞菌分子分型,为由铜绿假单胞菌引发的院内感染的溯源和流行病学调研提供依据。方法从铜绿假单胞菌基因组中筛选12个VNTR位点,设计合成引物;对98株多重耐药铜绿假单胞菌进行PCR扩增,产物毛细管电泳;根据产物大小计算各VNTR位点的重复数,构建系统发育树,进行数据分析。结果 12个位点的多态性指数(DI)为0.45~0.88,将98株铜绿假单胞菌分成5个群,88个基因型,同病区分离的菌株具有一定的同源性。结论多位点VNTR分析具有很高的分辨率,适用于铜绿假单胞菌基因分型和溯源研究,具有很好的应用前景。     

载脂蛋白A5基因多态性与脂类代谢疾病相关性研究进展

载脂蛋白A5(apoA5)是新近通过蛋白组学技术发现的载脂蛋白家族新成员,与血浆三酰甘油(TG)浓度呈负相关.apoA5基因至少有23个单核苷酸多态性(SNP)位点,其中多个位点的变异影响血浆TG水平,且存在着种族差异.国内外均有针对apoA5基因SNP位点与脂类代谢疾病的报道.现对apoA5基因分子结构、影响TG代谢的机制作简要介绍,并着重讨论其基因多态性与脂类代谢疾病的相关性.     

延边地区健康人群Nelin基因单核苷酸多态性研究

目的分析延边地区健康人群Nelin基因单核苷酸多态性(SNP)的分布特征。方法采用DNA序列测定技术,检测114例延边地区健康人Nelin基因外显子及外显子/内含子连接区的核苷酸序列。利用核酸序列分析软件与Genbank的Nelin基因序列比对,对SNP位点进行序列分析。结果通过对4个外显子SNP位点的序列分析,共发现2个SNP位点。其中1个为NCBI数据库中报道的rs1166698位点,新发现1个位点,NCBI数据库中报道的本研究人群中没有发现的位点3个。rs1166698位点为第8外显子区G/A错义突变(48364365 G/A),其基因型频率为GG:31.6%,GA:52.6%,AA:15.8%,等位基因频率为G:57.9%,A:42.1%。通过对延边地区人群该位点的民族、性别比对分析,基因型、等位基因频率在朝汉族、男女间的分布无明显差异。新发现的位点是第9外显子区A/G错义突变(1195 A/G),基因型AA占88.6%(101/114),AG占11.4%(13/114),未发现突变纯合子GG基因型。结论通过本研究了解了延边地区Nelin基因相关SNP位点遗传分布特征,为研究Nelin基因多态性与疾病的关系奠定了基础。     

蜡样芽胞杆菌快速检测及其毒力基因筛选

目的筛选蜡样芽胞杆菌鉴定基因和快速检测毒力基因。方法选择分离自食品和土壤的代表性蜡样芽胞杆菌共329株,聚合酶链式反应（PCR）方法检测gyrB和groEL基因的种特异性,检测毒力基因在菌株中的分布特征。 基于检测结果,用多重PCR检测方案快速检测蜡样芽胞杆菌鉴定基因及其毒力因子。结果在蜡样芽胞杆菌及其近缘芽胞杆菌中,除1株苏云金芽胞杆菌扩增阳性外,gyrB基因具有蜡样芽胞杆菌种特异性;而groEL基因在4种芽胞杆菌中均有扩增。 6种毒力基因nheA、entFM、bceT、hblC、cytK和ces的携带率分别为84.19%、79.64%、49.24%、47.72%、47.11%和1.52%。 选择nheA、hblC、entFM、ces、cytK和gyrB用于快速鉴定蜡样芽胞杆菌及其毒力基因,获得了双重PCR扩增体系（gyrB和cytK）与4重PCR扩增体系（nheA、hblC、entFM和ces）的最佳检测方案。结论筛选的蜡样芽胞杆菌鉴定基因和毒力基因能够全面、特异、简便、高效地检测蜡样芽胞杆菌,可为食品安全检测及快速诊断提供依据,在实际检验工作中具有良好的应用前景。     

基于高通量测序技术的102株铜绿假单胞菌耐药基因研究

目的通过高通量测序技术结合生物信息学分析多重耐药铜绿假单胞菌的耐药基因型。方法从246株铜绿假单胞菌中筛选出多重耐药株,采用碱裂解法提取目的菌的基因组和质粒DNA后进行高通量测序,应用生物信息学分析携带的耐药基因。结果共检出102株多重耐药株,21株广泛耐药株只对多黏菌素B敏感。102株多重耐药铜绿假单胞菌样本经高通量测序产生reads 32 088 766条,长度98nt,总测序碱基数3.1Gb,发现了8种抗菌药物耐药类型,含有21种耐药基因型,共有16个基因存在69个核苷酸的多态位点,其中有8种基因型17个位点发生非同义突变,占总单核苷酸多态性(SNP)位点的24.6%。结论运用高通量混合基因组测序技术分析多重耐药病原菌耐药基因具有可行性。     

云南省鼠疫耶尔森菌多位点可变数目串联重复序列分析

目的 通过多位点可变数目串联重复序列分析(multiple-locus variable-number tandem-repeat analysis,MLVA)方法,对云南省鼠疫菌株进行基因分型。 方法 采用聚合酶链反应(PCR)和毛细管电泳,通过BioNumerics软件进行处理,分析云南省158株鼠疫耶尔森菌基因型。 结果 选取鼠疫菌的14个VNTR位点,5个VNTR位点对云南省鼠疫菌具有多态性分析意义,利用这5个位点对云南菌株进行分析,云南158株鼠疫菌可分为2个群,3个簇,5个基因型,野鼠鼠疫菌属于A簇,丽江玉龙鼠疫菌属于B簇,家鼠鼠疫菌属于C簇,与传统的生态分型结果吻合。 结论 本次试验筛选的5个位点可以用于云南省鼠疫菌的分型,云南家鼠鼠疫菌、野鼠鼠疫菌及玉龙鼠疫菌分属不同的基因簇,玉龙鼠疫菌与野鼠鼠疫菌同属一个群,聚类结果与实际的地理相关性很好。     

一起GⅠ型诺如病毒混合感染多克隆蜡样芽胞杆菌致急性胃肠炎暴发事件实验室分析

  目的  通过一起急性胃肠炎暴发事件的实验室检测,分析暴发相关病原特征。  方法  对患者粪便、呕吐物标本以及可疑污染食品样本进行相关病原及毒力基因的实时荧光PCR筛查以及阳性菌的培养检测,对分离菌株进一步进行毒力基因分布分析和脉冲肠凝胶电泳分型分析。  结果  25例患者的粪便标本诺如病毒荧光PCR阳性,检出率为71.43%（25/35）,均为GⅠ型;5例患者的6份标本中蜡样芽胞杆菌荧光PCR和病原培养均为阳性,1名厨师粪便标本蜡样芽胞杆菌荧光PCR和培养结果均为阳性。 7件可疑食品样本中4件检出蜡样芽胞杆菌,其中5份食品样本ces基因阳性。 19株蜡样芽胞杆菌分离株脉冲场凝胶电泳（PFGE）分为14种带型,5例患者分离株中有2株PFGE带型相同且与厨师来源菌株分型一致,相同食品来源分离株的PFGE带型不完全一致。 19株分离株均不携带ces基因。  结论  诺如病毒和蜡样芽胞杆菌可能是导致本次急性胃肠炎暴发事件重要病原,以多病原快速筛查为指导的实验室检测对于病原的识别具有重要意义。     

2007-2020年上海市布鲁氏菌分离株分型方法研究

  目的  对2007 — 2020年上海市人间布鲁氏菌分离株进行分子特征分析,了解菌株基因分型和聚类,为布鲁氏菌病（布病）的预防和控制提供依据。  方法  收集2007 — 2020年上海市人感染布鲁氏菌临床分离株20株;采用生化方法和AMOS-PCR进行鉴定;运用多位点序列分型（MLST）、多位点可变数目串联重复序列分析（MLVA）和基于全基因组测序的单核苷酸多态性（SNP）分析3种方法进行分子分型;并与参考株进行聚类分析。  结果  20株布鲁氏菌均为ST8型羊种布鲁氏菌;被MLVA-8分为4个基因型,MLVA-16分为17个基因型,panel 2B具有多态性;SNP分析表明,20株临床分离株亲缘关系较近,与羊种布鲁氏菌聚类。  结论  2007 — 2020年上海市人感染布病分离株主要为ST8型羊种布鲁氏菌,分子分型方法可为上海市布鲁氏菌的溯源研究提供依据。     

Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis

Specific identification of Bacillus anthracis and differentiation from closely related Bacillus cereus and Bacillus thuringiensis strains is still a major diagnostic problem. Commercially available diagnostic kits targeting plasmid-markers cannot differentiate between B. anthracis, non-anthracis Bacillus species harbouring anthrax-specific virulence plasmids, and plasmidless B. anthracis strains. A TaqMan PCR assay was designed targeting sequences of gene locus BA_5345 of the B. anthracis strain Ames. Specificity was determined by using a panel of 328 Bacillus strains; sensitivity was determined by probit analysis. All B. anthracis isolates (n=92) were specifically detected by using the genomic TaqMan PCR assay whereas 236 strains belonging to 19 Bacillus species other than B. anthracis were PCR negative. The detection limit was determined to be 12.7 copies per reaction (95% confidence interval 10.2-17.5 copies). Here we present an extensively evaluated and - to our current knowledge - specific TaqMan PCR assay for the detection of B. anthracis based on a chromosomal marker.     

DnaJ sequences of Bacillus cereus strains isolated from outbreaks of hospital infection are highly similar to Bacillus anthracis

Bacillus cereus is becoming an important nomosomial pathogen because of frequent isolation from blood cultures and from severe systemic infections. To differentiate highly pathogenic outbreak strain of B. cereus from other sources of the Bacillus cereus, we attempted to analyze their dnaJ sequences. Assays indicated that dnaJ sequence similarity of all of 52 blood culture isolates of B. cereus ranged from 92.8% to 100%. The distance between B. anthracis and B. cereus except six outbreak isolates ranged from 3.8% to 6.4%. The dnaJ sequences of six outbreak strains of B. cereus (GTC 02891, GTC 02896, GTC 02916, GTC 02917, GTC 03221, and GTC 03222) were closely related to those of B. anthracis (99.2%-99.5% sequence similarity). Ba813 sequences were only found in the six outbreak strains of B. cereus. The other pathogenic factors of B. anthracis were not found in these six outbreak strains, with the exception of GTC 02891 (cap-positive). The six outbreak strains formed clear β-hemolytic colonies on a sheep blood agar plate. Our findings suggest that outbreak strains of B. cereus isolated from blood cultures are likely to have the risk of causing serious infection, and dnaJ and Ba813 are important markers to identify such strains. Phylogenetic analysis of dnaJ and MLST revealed that the six outbreak strains of B. cereus are closely related to B. anthracis.     

Treatment of experimental anthrax with recombinant capsule depolymerase

Bacillus anthracis produces an antiphagocytic gamma-linked poly-D-glutamic acid capsule that is required for virulence. Capsule depolymerase (CapD) is a membrane-associated poly-gamma-glutamate-specific depolymerase encoded on the B. anthracis capsule plasmid, pX02, that is reported to contribute to virulence by anchoring the capsule to the peptidoglycan and partially degrading high-molecular-weight capsule from the bacterial surface. We previously demonstrated that treatment with CapD effectively removes the capsule from anthrax bacilli, rendering them susceptible to phagocytic killing in vitro. Here we report that CapD promoted in vivo phagocytic killing of B. anthracis bacilli by mouse peritoneal neutrophils and that parenteral administration of CapD protected mice in two models of anthrax infection. CapD conferred significant protection compared with controls when coinjected with encapsulated bacilli from fully virulent B. anthracis Ames or the nontoxigenic encapsulated strain Delta Ames and when injected 10 min after infection with encapsulated bacilli from B. anthracis Ames. Protection was also observed when CapD was administered 30 h after infection with B. anthracis Delta Ames spores, while significant protection could not be demonstrated following challenge with B. anthracis Ames spores. These data support the proposed role of capsule in B. anthracis virulence and suggest that strategies to target anthrax bacilli for neutrophil killing may lead to novel postexposure therapies.     

2006年河南省南阳市流行性乙型脑炎病毒的分离与分子特征分析

目的在河南省南阳市采集蚊虫标本,进行西尼罗病毒基因检测,分离流行性乙型脑炎(乙脑)病毒并分析分离株的分子生物学特征。方法 2006年从南阳市采集蚊虫标本,将标本研磨处理后进行巢式PCR检测西尼罗病毒核酸,用细胞培养法对标本进行病毒分离,通过血清学与分子生物学方法鉴定新分离到的乙脑病毒,扩增分离株PrM和E基因区,使用生物学软件进行序列和系统发生分析。结果共采集蚊虫标本6231只,巢式PCR检测西尼罗病毒结果均为阴性。从标本中分离到7株病毒,经鉴定均为乙脑病毒。基因分型显示7株病毒均为基因Ⅰ型,新分离株在E基因之间的核苷酸同源性为98.5%～100%,氨基酸同源性为98.4%～100%。新分离株与P3株和SA14-14-2的E基因核苷酸同源性分别为87.7%～88.2%和87.3%～87.9%。新分离株与P3株相比存在10个共同氨基酸差异位点,但在决定毒力的关键位点不同于SA14-14-2。结论河南省南阳市2006年乙脑病毒流行株为基因Ⅰ型,毒株的毒力基因没有明显改变,所有标本均未检测到西尼罗病毒阳性。     

Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta-lactamase genes.

Escherichia coli HKY56, which demonstrated resistance to various beta-lactams except carbapenems, was isolated from the throat swab of an inpatient in 1994. Conjugal transfer of cephem resistance from HKY56 to E. coli CSH2 was not successful. Three cefotaxime-resistant E. coli clones harboring plasmid pMRE001, pMRE002, or pMRE003, each of which carried a 3.4-, 5.8-, or 6.2-kb EcoRI fragment insert, respectively, were obtained from HKY56. Although restriction analysis suggested their different origins, these clones showed similar profiles of resistance to various beta-lactams. The sequence of 10 amino acid residues at the N terminus of beta-lactamase purified from E. coli HB101(pMRE001) was identical to that of Toho-1. This Toho-1-like beta-lactamase-1 (TLB-1) was able to hydrolyze cefoperazone and cefotaxime efficiently, but it failed to hydrolyze cephamycins. A Toho-1-specific DNA probe was hybridized with three distinct EcoRI fragments derived from the chromosomal DNA of strain HKY56, and these fragments corresponded to DNA inserts carried by pMRE001, pMRE002, and pMRE003, respectively. PCR and Southern hybridization analysis suggested that all six cephem-resistant E. coli strains, strains HKY273, HKY285, HKY288, HKY305, HKY316, and HKY335, which were isolated in 1996 at the same hospital where strain HKY56 had been isolated, also possessed multiple Toho-1-like beta-lactamase (TLB) genes, and the hybridization patterns obtained with the Toho-1-specific probe were quite similar among these six isolates. The DNA fingerprinting patterns observed by pulsed-field gel electrophoresis revealed that among the E. coli isolates tested, all isolates except HKY56 possessed a similar genetic background. These findings suggested that E. coli strains that carry chromosomally multiplied TLB genes may have been proliferating and transmitted among patients in the same hospital.     

The Bacillus cereus containing sub-branch most closely related to Bacillus anthracis, have single amino acid substitutions in small acid-soluble proteins, while remaining sub-branches are more variable

Hoffmaster et al. [Hoffmaster AR, Ravel J, Rasko DA, Chapman GD, Chute MD, Marston CK, et al. Identification of anthrax toxin genes in Bacillus cereus associated with illness resembling inhalation anthrax. Proc Natl Acad Sci U S A 2004;101:8449-54; Hoffmaster AR, Hill KK, Gee JE, Marston CK, De BK, Popovic T, et al. Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes. J Clin Microbiol 2006;44:3352-60] phylogenetically divided Bacillus cereus strains into 10 branches by amplified fragment length polymorphism (AFLP) with Branch F including all Bacillus anthracis strains and pneumonia-causing strains of B. cereus. There are four sub-branches within Branch F, referred to here as F1-A, F1-B, F2-A and F2-B. The B. anthracis strains are found within sub-branch F1-B. Concerning, the currently available B. cereus pneumonia-causing isolates, one was found to categorize within sub-branch F1-B and two within F2-B. In the following work the sequence variation between B. cereus strains was determined by MALDI-TOF MS and MS-MS for each strain of B. cereus in Branch F. ESI-MS was performed on selected strains for confirmation. Small acid-soluble proteins (SASPs) of B. cereus strains found in F1-B showed a single amino acid substitution, while strains in the other three sub-branches were more variable generally showing one or two amino acid substitutions. The single substitutions always occurred in the C-terminus. Double substitutions occurred in both N and C termini. Of the pneumonia-causing strains, one exhibited a single amino acid substitution, while the other two exhibited a two amino acid substitution.     

一起疑似炭疽疫苗菌株毒力恢复事件中炭疽芽孢杆菌的毒力鉴定与分析

目的 对某校疑似炭疽疫苗菌株毒力恢复事件（事件）中,教学实验使用的炭疽芽孢杆菌毒力及其环境污染情况进行鉴定和调查,为合理处理该事件提供参考依据。 方法 调查事件发生的情况、采集实验用培养物、冻存菌株和实验室环境样本。 对实验用培养物进行噬菌体裂解和青霉素敏感试验鉴定;选取炭疽芽孢杆菌的rpoB基因和毒力相关的pagA和capC基因,应用TaqMan荧光探针法对采集样本进行检测。 结果 实验用培养物的噬菌体裂解试验显示有明显的噬菌斑,青霉素敏感试验显示在青霉素纸片周围有明显的抑菌环,并且培养物和冻存菌株rpoB和pagA基因检测为阳性,capC基因检测为阴性;教学场所的环境样本rpoB、pagA和capC基因检测均为阴性。 结论 该事件中实验用培养物和冻存菌株均为缺少capC基因的减毒炭疽芽孢杆菌,未发现毒力恢复情况;教学场所中无炭疽芽孢杆菌的污染。      

Bcr基因断裂丛集区多态性分析

本研究旨在探讨bcr基因的断裂丛集区在中国人群中的单核苷酸多态性（SNP）及该多态性与慢性髓系白血病（CML）之间的关系。利用DNA库（DNApooling）结合变性高效液相色谱（dHPLC）对长度为3．12kb的bcr基因断裂丛集区进行序列变异的筛查分析，并通过测序对筛查结果进行了验证。结果表明，该区域有6处新的多态性位点和2处与参照序列不一致的碱基，研究获得了这些多态位点在所调查的人群中的多态性频率，其中1处SNP位于外显子13中，可引起错义突变。对CML和对照组的多态性频率的比较表明，这些新发现的SNP在两组间无显著差异。结论：bcr基因主要断裂丛集区存在一定的序列多态性，主要为SNP，但未能证实其与CML之间存在相关性。