Anti-microbial resistance of Enterococci isolated from urinary tract infections in Iran

Background: During the last decade, enterococci have become important nosocomial pathogens, representing the second leading cause of urinary tract infections. This increasing prevalence has been paralleled by the occurrence of multi-drug resistant (MDR) and high-level gentamicin resistant (HLGR) strains. Methods: From September 2005 to 2006, a total of 638 enterococcal isolates were collected from urine samples among 9 medical centers in Tehran (Iran). Confirmation of species and detection of gentamicin resistance genes were done by PCR method. Anti-microbial susceptibility test was determined with disk diffusion and minimal inhibitory concentration of gentamicin among HLGR isolates assayed by microdilution methods. Results: The isolates were found to consist of Enterococcus faecalis (77.8%) and Enterococcus faecium (22.2%). The results obtained from PCR showed a high rate of agreement with phenotypic assays for both species. MDR to most prevalent anti-microbials was present in 29% and 72% of the E. faecalis and E. faecium isolates, respectively. HLGR phenotype was detected in 64% of E. faecalis and 92% of E. faecium isolates. The aac(6')-Ie-aph(2')-Ia gene were identified in 83% of E. faecalis and 100% of E. faecium HLGR isolates. E. faecalis and E. faecium isolates differed in their susceptibilities to different antibiotics. Conclusion: Emergence of multi-resistant enterococci and high level resistance to gentamicin shown by enterococcal strains is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.     

多重PCR快速检测甘蔗转基因成分研究

以单子叶植物常见外源转基因元件Ubiquitin启动子、NOS启动子、NOS终止子、Bar基因和Bt基因序列设计多重PCR引物,通过对PCR扩增体系中退火温度、Premix TaqTM浓度、引物浓度的优化,建立一种快速检测转基因甘蔗成分的多重PCR方法。结果表明:建立的体系一次能有效检测5个参数,其检测的最低DNA质量百分比可达1.0%,并在多个转基因甘蔗品种检测中得到验证。     

Development of Molecular Tools for Distinguishing Between the Highly Similar Rx1 and Rx2 PVX Extreme Resistance Genes in Tetraploid Potato

The Rx1 and Rx2 are extreme resistance genes, which have been introgressed from different species into potato cultivars and breeding lines. These two genes have a 98% and 96% sequence similarity at the nucleotide as well as at the amino acid level, respectively. Except one extra amino acid in the Rx2 gene, the high variations of the amino acid chain are due to single and double nucleotide variations, which are scattered throughout the coding regions. The high level of sequence similarity makes it complicated to identify these genes and to distinguish them from other highly similar genes, like the Gpa2 or from paralogous sequences by a single polymerase chain reaction (PCR). Here, we report the development of markers for the simple and rapid identification of the Rx1 as well as the Rx2 gene. Further, a multiplex PCR reaction is recommended for the simultaneous detection of both genes in a single reaction. Since these genes reside on different chromosomes, following their inheritance by the multiplex PCR method could help the easy incorporation of both genes into breeding lines. The detection method shown here could be routinely used in marker-assisted selection for Potato virus X extreme resistance and could enhance the effectiveness of potato breeding programs. Besides potato breeding, this method could also be effectively applied to mapping experiments as well as in research studies of resistance.     

利用多重PCR技术快速鉴定番木瓜性别

利用鉴定番木瓜雄性性别的特异基因片段(1 001 bp)的引物和鉴定番木瓜雄性、两性性别的特异基因片段(225 bp)的引物,分别以5个品种番木瓜的3种性别植株的总DNA为模板进行多重PCR扩增,结果表明:以雄株总DNA为模板得到1个1 001 bp和1个225 bp的扩增产物,以雌株总DNA为模板没有得到扩增产物,以两性株总DNA为模板得到1个225bp的扩增产物,根据多重PCR扩增结果,可以将番木瓜3种性别植株一次性鉴定出来.     

粘类小麦CMS恢复基因Rfv1快速定向转育体系的研究

为了实现粘类小麦雄性不育恢复基因Rfv1的定向转育,创制优良的粘类非1BL/1RS小麦雄性不育新恢复系,以携有粘类小麦雄性不育恢复基因Rfv1的恢复系亲本5253、5383为供体,以西农Mp1、西农Mp2(化杀杂交小麦新品种西杂1号、西杂5号的父本)为受体,采用定向回交转育方法,结合根尖细胞学镜检、复合引物多重PCR等技术,进行了恢复基因Rfv1的定向转育与检测,育成既携有Rfv1恢复基因,又具有西农Mp1、西农Mp2核基因背景的粘类非1BL/1RS小麦雄性不育新恢复系.本研究表明:(1)根尖体细胞随体鉴定和复合引物PCR分析,不仅能准确地鉴定出1BL/1RS纯合易位系,而且可以鉴定1BL/1RS·1BL/1BS R~(fv1)易位杂合体.其中复合引物PCR更适合于回交后代中大量目标种子的筛选;(2)恢复基因转育后代定株与不育系测交,依据测交恢复度可准确确定恢复基因在回交后代的延续,恢复基因定株定向转育与性状选择结合可大大提高转育后代的实际应用效果;(3)本研究提出的粘类小麦雄性不育恢复基因Rfv1定向转育体系,可有效地实现小麦由化控强优势组合向三系强优势组合的定向转化,即生理型不育途径向遗传型不育途径的定向转化,进而建拓一套杂交小麦强优势组合多途径利用新体系.     

多重PCR技术检测4种大豆镰孢菌根腐病病原

镰孢菌是引起大豆根腐病的常见病原菌,传统检测病原镰孢菌的方法存在操作繁琐、时间长、成本高和 效率低等缺点,建立一种快速检测方法显得尤为重要。本研究基于翻译延伸因子序列（EF-1α）建立了检测镰孢菌 （Fusarium species）的多重PCR反应体系,检测了多重PCR体系灵敏度,模拟侵染样本验证多重PCR技术的可行性。 实验结果表明,该方法能够通过扩增片段的大小区分锐顶镰孢菌（Fusarium acuminatum）、尖孢镰刀菌（Fusarium oxysporum）、茄病镰孢菌（Fusarium solani）和禾谷镰孢菌（Fusarium graminearum）。灵敏度检测显示,最低检测基因 组DNA浓度达1×10-4 ng/μL;模拟侵染样本实验表明,使用镰孢菌和大豆基因组混合样本作为模板,在DNA浓度为 100 ng/μL时仍能准确检测出目的条带。因此。以翻译延伸因子序列为靶标建立的多重PCR技术,能够灵敏、特异 性地检测出该四种镰孢菌,可在复合侵染引起大豆镰孢菌根腐病的病原菌鉴定中准确检测。     

A Molecular Survey of Campylobacter jejuni and Campylobacter Coli Virulence and Diversity

Background: The aim of this study was to determine the prevalence of virulence-associated genes and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) analysis of Campylobacter spp. isolated from children with diarrhea in Iran. Methods: A total of 200 stool specimens were obtained from children under 5 years during July 2012 to July 2013. Detection of C. jejuni and C. coli was performed by standard biochemical and molecular methods. The presence of virulence-associated genes and genetic diversity of isolates was examined using PCR and ERIC-PCR analyses. Results: A total of 12 (6%) Campylobacter spp. were isolated from patients including 10 (4.5%) C. jejuni and 2 (1.5%) C.coli. The flaA, cadF and ciaB genes were present in 100% of isolates, while no plasmid of virB11 gene was present in their genome. The prevalence of invasion-associated marker was 100% among C. coli and was not detected in C. jejuni isolates. The distribution of both pldA and the genes associated with cytolethal distending toxin (CDT) was 58.3% in C. jejuni isolates. Seven distinct ERIC-PCR profiles were distinguished in three clusters using ERIC-PCR analysis. Genotyping analysis showed a relative correlation with geographic location of patients and virulence gene content of isolates. Conclusion: To our knowledge, this is the first molecular survey of Campylobacter spp. in Iran concerning genotyping and virulence gene content of both C. jejuni and C. coli. ERIC-PCR revealed appropriate discriminatory power for clustering C. jejuni isolates with identical virulence gene content. However, more studies are needed to clearly understand the pathogenesis properties of specific genotypes. Key Words: Campylobacter jejuni, Campylobacter coli, Ddiarrhea, Virulence factors     

Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA

BACKGROUND: The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. METHODS: The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. RESULTS AND CONCLUSION: The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.     

多重引物SSR技术鉴定玉米杂交种子纯度的研究

采用热碱法提取玉米种子DNA,并且运用多重引物(3对SSR引物)PCR对其进行SSR扩增,进行种子纯度鉴定。结果表明:①利用热碱法可以实现玉米种子的DNA快速提取,同时降低了检测成本;②利用三重引物PCR技术降低了检测结果的误差,提高了检测结果的准确性。因此,将这两种技术结合起来,将极大地推动SSR技术在玉米种子纯度鉴定中的推广和应用。     

应用单引物PCR获得以Bar基因为锚定基因的转基因水稻侧翼序列

 确定外源基因在水稻基因组上的插入位置,是转基因水稻研究的重要内容之一。为了建立一种简单、快速、准确的获取外源基因在水稻基因组上插入位置侧翼序列的方法,结合前人TAIL PCR引物设计的原则和经验,以转入水稻基因组中的Bar基因为锚定基因,设计了既能作为锚定引物又能作为随机引物使用的一系列单引物,应用这些单引物通过单引物PCR成功获得了外源基因在水稻基因组插入位点的侧翼序列。这种方法与目前较为常用的TAIL PCR相比,简化了70%左右的试验步骤,节省了60%以上的试验时间,是一种简便、快捷的获得未知侧翼序列的方法。     

小麦和玉米籽粒赤霉菌产毒类型的PCR检测

为了建立一种准确、快速鉴定赤霉菌毒素的方法,根据赤霉菌毒素生物合成调控路径T ri5-T ri6基因保守序列,设计了一对通用引物,用于检测和鉴别产生脱氧雪腐镰刀菌烯醇(DON)和雪腐镰刀菌烯醇(N IV)毒素的赤霉菌特异DNA片断。结果表明,产生毒素DON的赤霉菌具有一个300 bp的特异DNA片断,而产生毒素N IV的则为360 bp。PCR检测与HPLC或GC/M S化学分析结果完全一致。利用这一方法分析检测了小麦、玉米籽粒中赤霉菌产毒类型,发现除健康籽粒外,在所有轻微、中度、严重感染赤霉病的小麦或玉米籽粒中,均同时检测到DON及N IV的两种DNA片断,表明这一对通用引物对两种毒素特异DNA片断的扩增效率相同。这些结果说明,该技术是一种经济、快速、可靠的PCR检测技术,可广泛用于赤霉菌及其毒素检测鉴定中。     

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

Background:

Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.

Results:

The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.

Conclusion:

Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)     

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×104 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.     

应用抑制消减杂交技术 构建花生果皮差异表达文库

为构建花生果皮差异表达基因消减文库，分离花生果皮差异表达cDNA片段，本研究采用抑制消减杂交技术，以花生种仁cDNA为Driver，果皮cDNA为Tester，进行两次消减杂交和两次抑制性PCR，将第二次PCR产物与pGEM-T easy 载体相连，电击转化大肠杆菌，成功构建果皮差异表达cDNA文库。所长出菌落中89.2%为白色克隆。采用PCR法对重组子进行鉴定，发现其中单一扩增条带的克隆约占83.5%， 片段大小集中分布于250～750bp之间。     

小麦春化基因新等位变异Vrn-B3d的鉴定

春化基因的遗传多样性决定了小麦广泛的适应性,挖掘和鉴定春化基因的新等位变异,可为广适小麦育种奠定基础。本研究以普通小麦地方品种和尚头为材料,采用生物信息学和分子克隆手段获得 VRN-B3基因,全长3 818 bp。与隐性等位变异 vrn-B3相比,和尚头中 VRN-B3基因在启动子区有一个160 bp的插入片段,为 VRN-B3基因的一种新等位变异,暂命名为 Vrn-B3d。经PlantCare网站预测,该插入片段包含CAAT-box、G-box和TATA-box三种顺式作用元件。基于 Vrn-B3d差异序列,开发一对检测新变异的分子标记,并构建能够同时区分 VRN-B3位点5种等位变异的高效多重PCR体系。通过检测262份中国微核心种质资源,发现93.5％的品种携带 vrn-B3,5.7％的品种携带 Vrn-B3b,并在新疆春麦和红冬麦品种中发现新等位变异 Vrn-B3d,说明该变异主要分布在新疆地区。温室表型和基因型相关性分析结果表明,与 vrn-B3相比, Vrn-B3d能使小麦推迟两天抽穗。本研究丰富了小麦春化基因遗传变异的多样性,开发了分子标记并构建多重PCR体系,为小麦育种提供了优异种质资源和分子检测有效方法。     

A Single-Tube,Functional Marker-Based Multiplex PCR Assay for Simultaneous Detection of Major Bacterial Blight Resistance Genes Xa21,xa13 and xa5 in Rice

In marker-assisted breeding for bacterial blight(BB) resistance in rice, three major resistance genes, viz., Xa21, xa13 and xa5, are routinely deployed either singly or in combinations. As efficient and functional markers are yet to be developed for xa13 and xa5, we have developed simple PCR-based functional markers for both the genes. For xa13, we designed a functional PCR-based marker, xa13-prom targeting the In Del polymorphism in the promoter of candidate gene Os8N3 located on chromosome 8 of rice. With respect to xa5, a multiplex-PCR based functional marker system, named xa5 FM, consisting of two sets of primer pairs targeting the 2-bp functional nucleotide polymorphism in the exon II of the gene TFIIA5(candidate for xa5), has been developed. Both xa13-prom and xa5 FM can differentiate the resistant and susceptible alleles for xa13 and xa5, respectively, in a co-dominant fashion. Using these two functional markers along with the already reported functional PCR-based marker for Xa21(p TA248), we designed a single-tube multiplex PCR based assay for simultaneous detection of all the three major resistance genes and demonstrated the utility of the multiplex marker system in a segregating population.     

Comparison of Antimicrobial Susceptibility of Campylobacter Strains Isolated from Food Samples and Patients with Diarrhea

Background:

Campylobacter infections may lead to serious conditions, including septicemia or other invasive forms of the disease, which require rapid and accurate laboratory diagnosis and subsequently appropriate antimicrobial therapy. The aim of this study was to compare the species distribution and antimicrobial susceptibility pattern of Campylobacter spp. strains isolated from patients and food samples.

Methods:

Biochemical identification was performed on 15 clinical and 30 food isolates of Campylobacter recovered onto Brucella agar containing 5% sheep blood. PCR was carried out to confirm the identity of Campylobacter spp. using primers for cadF, hipO, and asp genes of Campylobacter. To determine antibiotic sensitivity of isolates, Kirby-Bauer assay was carried out using 16 different antibiotic discs.

Results:

PCR assay and biochemical tests confirmed all 45 isolates as Campylobacter: 20 (44.44%) as C. jujeni, 10 (22.22%) as C. coli, and 15 (33.34%) as other Campylobacter strains. The maximum resistance was observed to cefotaxime and imipenem (each 86.49%) and the maximum sensitivity to erythromycin (48.65%).

Conclusion:

C. jujeni is dominant among isolates from clinical and food samples. In addition, tetracycline remains the first-line therapeutic agent against Campylobacter infections in Iran. Key Words: Campylobacter, Genetic diversity, Drug resistance     

杧果炭疽病菌 3 个果胶裂解酶基因序列特征及受漆酶基因Lac1 影响的分析

果胶裂解酶是炭疽菌在侵染寄主过程中降解寄主细胞壁的一类重要水解酶。本研究在杧果炭疽病菌中克隆获得了 3 个果胶裂解酶基因 Cgpel1、Cgpel2 和 Cgpel3，DNA 全长分别为 1 037、1 498、1 089 bp，cDNA 全长分别为 975、 1 380、978 bp，分别编码 324、459、325 个氨基酸，均含 1 个果胶裂解酶保守结构域，均有典型的信号肽，不存在跨膜结构。其二级结构中 α-螺旋分别占 16.05%、20.26%、16.92%，延伸链分别占 28.09%、21.79%、29.54%，β-转角分别占 5.86%、8.93%、7.38%，无规则卷曲分别占 50.00%、49.02%、46.15%。Cgpel1、Cgpel2 和 Cgpel3 的氨基酸序列分别与 C. tofieldiae（KZL77240.1）、草莓炭疽菌（C. gloeosporioides）（XP-007274932.1）、C. incanum（KZL84476.1）果胶裂解酶序列相似度在 93%以上。qRT-PCR 分析发现，3 个果胶裂解酶基因在整个侵染过程中均持续高效表达，但在漆酶基因 Lac1 敲除突变体中，表达均下降约 90%。可见 Cgpel1、Cgpel2 和 Cgpel3 是果胶裂解酶基因家族成员，序列差异较大，在侵染过程中起着重要的作用，且其表达受漆酶基因 Lac1 影响。     

杧果炭疽病菌 3 个果胶裂解酶基因序列特征及受漆酶基因 Lac1 影响的分析

果胶裂解酶是炭疽菌在侵染寄主过程中降解寄主细胞壁的一类重要水解酶。本研究在杧果炭疽病菌中克隆获 得了 3 个果胶裂解酶基因 Cgpel1、Cgpel2 和 Cgpel3,DNA 全长分别为 1 037、1 498、1 089 bp,cDNA 全长分别为 975、 1 380、978 bp,分别编码 324、459、325 个氨基酸,均含 1 个果胶裂解酶保守结构域,均有典型的信号肽,不存在跨 膜结构。其二级结构中 α-螺旋分别占 16.05%、20.26%、16.92%,延伸链分别占 28.09%、21.79%、29.54%,β-转角分 别占 5.86%、8.93%、7.38%,无规则卷曲分别占 50.00%、49.02%、46.15%。Cgpel1、Cgpel2 和 Cgpel3 的氨基酸序列 分别与 C. tofieldiae（KZL77240.1）、草莓炭疽菌（C. gloeosporioides）（XP-007274932.1）、C. incanum（KZL84476.1） 果胶裂解酶序列相似度在 93%以上。qRT-PCR 分析发现,3 个果胶裂解酶基因在整个侵染过程中均持续高效表达,但 在漆酶基因 Lac1 敲除突变体中,表达均下降约 90%。可见 Cgpel1、Cgpel2 和 Cgpel3 是果胶裂解酶基因家族成员,序 列差异较大,在侵染过程中起着重要的作用,且其表达受漆酶基因 Lac1 影响。