沙眼衣原体15个血清型omp1基因的VS1和VS2区序列分析

目的比较分析沙眼衣原体15个血清型omp1基因VS1和VS2序列的同源性和变异性。方法巢式PCR扩增VS1-VS2基因,自动测序仪测定核苷酸序列,DNAstar生物软件进行比对分析。结果15个血清型沙眼衣原体扩增出大小453bp的VS1-VS2基因。VS1区域序列比对显示血清群B和中间群的VS1核苷酸序列相对保守,而血清群C各型VS1区域表现出较大的核苷酸变异,型间显示1～9个核苷酸替换,且发生在中心区域。血清型VS2序列较VS1存在更多的变异,血清群B中各血清型间均存在2～19个核苷酸的改变,血清群C表现为4～8个核苷酸差异,中间群的F和G型之间存在6个核苷酸差异。结论阐明VS1和VS2区核苷酸的多态性,为下一步进行该蛋白表达和构建寡核苷酸型特异性探针奠定了基础。     

MLVA subtyping of genovar E Chlamydia trachomatis individualizes the Swedish variant and anorectal isolates from men who have sex with men

This study describes a new multilocus variable number tandem-repeat (VNTR) analysis (MLVA) typing system for the discrimination of Chlamydia trachomatis genovar D to K isolates or specimens. We focused our MLVA scheme on genovar E which predominates in most populations worldwide. This system does not require culture and therefore can be performed directly on DNA extracted from positive clinical specimens. Our method was based on GeneScan analysis of five VNTR loci labelled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This MLVA, called MLVA-5, was applied to a collection of 220 genovar E and 94 non-E genovar C. trachomatis isolates and specimens obtained from 251 patients and resulted in 38 MLVA-5 types. The genetic stability of the MLVA-5 scheme was assessed for results obtained both in vitro by serial passage culturing and in vivo using concomitant and sequential isolates and specimens. All anorectal genovar E isolates from men who have sex with men exhibited the same MLVA-5 type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. The MLVA-5 assay was compared to three other molecular typing methods, ompA gene sequencing, multilocus sequence typing (MLST) and a previous MLVA method called MLVA-3, on 43 genovar E isolates. The discriminatory index was 0.913 for MLVA-5, 0.860 for MLST and 0.622 for MLVA-3. Among all of these genotyping methods, MLVA-5 displayed the highest discriminatory power and does not require a time-consuming sequencing step. The results indicate that MLVA-5 enables high-resolution molecular epidemiological characterisation of C. trachomatis genovars D to K infections directly from specimens.     

Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development

Twenty one Chlamydia trachomatis reference strains and 40 clinical isolates belonging to the lymphogranuloma venerum (LGV) and trachoma biovars were genotyped by differential restriction mapping of the major-outer-membrane-protein gene (MOMP) obtained by the polymerase-chain reaction (PCR). AluI digestion of the PCR product distinguishes eight MOMP-genotypes corresponding to 8 serovars. Six additional enzymes (NlaIII, CfoI, EcoRI, HinfI, DdeI and FokI) further permit the discrimination of 10 MOMP-genotypes corresponding to the 10 remaining serovars of the species. AluI alone allows direct typing of 78% of the clinical isolates. AluI digestion patterns of mouse C. trachomatis biovar, a C. pneumoniae and two C. psittaci strains, studied for comparison, were clearly distinguishable from one another and from the C. trachomatis LGV and trachoma strains. These results indicate that MOMP genotyping by PCR is a valuable molecular tool for studying C. trachomatis epidemiology.     

Determinants of trachoma endemicity using Chlamydia trachomatis ompA DNA sequencing

A six-year prospective study of Chlamydia trachomatis infection and ocular disease in Tanzanian village children was conducted to identify the determinants of trachoma endemicity using sequencing of ompA. Overall, 749 conjunctival samples were obtained, with 176 children sampled in both 1989 and 1995. 31.1% (233/749) were positive by PCR-enzyme immunoassay, and 76% (176/233) of the positives were sequenced in variable domains (VD) 1 to 4 (22 children in both 1989 and 1995). Twenty-six ompA genotypes of serovar A, and 19 of B/Ba were identified, and only 20% of genotypes identified in 1995 matched those found in 1989. In particular, B/Ba genotypes exhibited a 15-base region in VD 2 with increased nucleotide substitution, and these types were associated with age and water availability. Homotypic infection and infection with multiple genotypes and high chlamydial load did predict subsequent severe trachoma (odds ratio (OR) = 10.14, 95% confidence interval (CI): 1.71, 60.23; OR = 6.40, 95% CI: 0.75, 54.41; OR = 6.74, 95% CI: 0.82, 55.38, respectively). And, multitypic infection was clustered with residence of village and associated with familial cattle ownership. In conclusion, high ompA polymorphism and the inability of some hosts to clear infection with the same ompA genotype suggest two distinct but converging mechanisms of endemic severe trachoma.     

Analysis of Chlamydia trachomatis serovars in endocervical specimens derived from pregnant Japanese women

The polymerase chain reaction (PCR) method has been employed to amplify a chlamydial genome encoding four variable segments of the major outer membrane protein and genotyping of different Chlamydia trachomatis serovars was successfully achieved by means of restriction fragment length polymorphism (RFLP) analysis and sequencing of amplified DNA. These methods were applied to identify the serotypes of C. trachomatis in endocervical specimens obtained from asymptomatic pregnant Japanese women at 28-30 weeks of gestation. Among the 218 specimens, 207 were serotyped 43 (19.3%) as serovar D, 53 (24.3%) as E, 24 (11.0%) as F, 39 (17.9%) as G, 15 (6. 9%) as H, 15 (6.9%) as I, five (2.3%) as J, nine (4.1%) as K and four (1.8%) as mixed. Among the 11 unclassified strains by RFLP, six (2.8%) were identified as serovar B variants and five (2.3%) were identified as D/IC-Cal-8. It was suggested that variants of endemic trachoma serovars also have affinity for the urogenital tract of Japanese pregnant women.     

SNP/RD typing of Mycobacterium tuberculosis Beijing strains reveals local and worldwide disseminated clonal complexes

The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.     

泌尿生殖道沙眼衣原体omp1基因多态性研究

从中国不同城市收集疑为沙眼衣原体(Ct)感染的泌尿生殖道标本323份,巢式PCR扩增Ctomp1基因片段(包括4个变异区),测定其中96份阳性标本omp1基因序列,根据同源性分型并分析其多态位点;根据氨基酸序列,用Mega 3软件构建进化树,分析临床株与相应参考株之间的亲缘关系。从96份沙眼衣原体阳性标本中,检出28种基因变体,其中E型最常见;同时发现Ct E、F型omp1基因高度保守,而其它基因型都显示一定的变异性。进化树分析发现,各临床株与相应参考株之间遗传距离较近。实验结果表明沙眼衣原体omp1基因呈现较大的多态性,可为其疫苗的研制及感染的防治提供重要的实验依据。     

Comparative analysis of 22 coronavirus HKU1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus HKU1

We sequenced and compared the complete genomes of 22 strains of coronavirus HKU1 (CoV HKU1) obtained from nasopharyngeal aspirates of patients with respiratory tract infections over a 2-year period. Phylogenetic analysis of 24 putative proteins and polypeptides showed that the 22 CoV HKU1 strains fell into three clusters (genotype A, 13 strains; genotype B, 3 strains and genotype C, 6 strains). However, different phylogenetic relationships among the three clusters were observed in different regions of their genomes. From nsp4 to nsp6, the genotype A strains were clustered with the genotype B strains. For nsp7 and nsp8 and from nsp10 to nsp16, the genotype A strains were clustered with the genotype C strains. From hemagglutinin esterase (HE) to nucleocapsid (N), the genotype B strains were clustered closely with the genotype C strains. Bootscan analysis showed possible recombination between genotypes B and C from nucleotide positions 11,500 to 13,000, corresponding to the nsp6-nsp7 junction, giving rise to genotype A, and between genotypes A and B from nucleotide positions 21,500 to 22,500, corresponding to the nsp16-HE junction, giving rise to genotype C. Multiple alignments further narrowed the sites of crossover to a 143-bp region between nucleotide positions 11,750 and 11,892 and a 29-bp region between nucleotide positions 21,502 and 21,530. Genome analysis also revealed various numbers of tandem copies of a perfect 30-base acidic tandem repeat (ATR) which encodes NDDEDVVTGD and various numbers and sequences of imperfect repeats in the N terminus of nsp3 inside the acidic domain upstream of papain-like protease 1 among the 22 genomes. All 10 CoV HKU1 strains with incomplete imperfect repeats (1.4 and 4.4) belonged to genotype A. The present study represents the first evidence for natural recombination in coronavirus associated with human infection. Analysis of a single gene is not sufficient for the genotyping of CoV HKU1 strains but requires amplification and sequencing of at least two gene loci, one from nsp10 to nsp16 (e.g., pol or helicase) and another from HE to N (e.g., spike or N). Further studies will delineate whether the ATR is useful for the molecular typing of CoV HKU1.     

双色荧光杂交芯片在检测 P 1A 1M P I 基C 因多态性中的应用

目的：应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术检测CYPIA1 MspI基因多态性。方法：收集江苏汉族人群原发性肺癌患者75例和相应对照77例,应用双色荧光杂交芯片技术检测了152例样本的CYPIAI基因MspI基因多态性,并应用PCR-RFLP技术验证双色荧光杂交芯片的特异性。结果：152例样本的CYPIAI基因双色荧光杂交芯片技术分型结果与PCR-RFLP结果完全相符,两种方法的基因型分型结果具有很好的一致性。结论：双色荧光杂交芯片技术是一个高通量SNP检测的良好工具,特异性高,在大规模人群SNP筛检中具有良好的发展前案。     

Control of Trachoma from Achham District,Nepal: A Cross-Sectional Study from the Nepal National Trachoma Program

Background

The WHO seeks to control trachoma as a public health problem in endemic areas. Achham District in western Nepal was found to have TF (trachoma follicular) above 20% in a 2006 government survey, triggering 3 annual mass drug administrations finishing in 2010. Here we assess the level of control that has been achieved using surveillance for clinical disease, ocular chlamydia trachomatis infection, and serology for antibodies against chlamydia trachomatis protein antigens.

Methods

We conducted a cross-sectional survey of children aged 1–9 years in communities in Achham District in early 2014 including clinical examination validated with photographs, conjunctival samples for Chlamydia trachomatis (Amplicor PCR), and serological testing for antibodies against chlamydia trachomatis protein antigens pgp3 and CT694 using the Luminex platform.

Findings

In 24 randomly selected communities, the prevalence of trachoma (TF and/or TI) in 1–9 year olds was 3/1124 (0.3%, 95% CI 0.1 to 0.8%), and the prevalence of ocular chlamydia trachomatis infection was 0/1124 (0%, 95% CI 0 to 0.3%). In 18 communities selected because they had the highest prevalence of trachoma in a previous survey, the prevalence of TF and/or TI was 7/716 (1.0%, 95% CI 0.4 to 2.0%) and the prevalence of ocular chlamydia trachomatis infection was 0/716 (0%, 95% CI 0 to 0.5%). In 3 communities selected for serological testing, the prevalence of trachoma was 0/68 (0%, 95% CI 0 to 5.3%), the prevalence of ocular chlamydia trachomatis infection was 0/68 (0%, 95% CI 0 to 0.5%), the prevalence of antibodies against chlamydia trachomatis protein antigen pgp3 was 1/68 (1.5%, 95% CI 0.04% to 7.9%), and the prevalence of antibodies against chlamydia trachomatis protein antigen CT694 was 0/68 (0%, 95% CI 0 to 5.3%).

Conclusion/Significance

This previously highly endemic district in Nepal has little evidence of recent clinical disease, chlamydia trachomatis infection, or serological evidence of trachoma, suggesting that epidemiological control has been achieved.     

Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice

Ames dwarfism is caused by a homozygous single nucleotide mutation in the pituitary specific prop-1 gene, resulting in combined pituitary hormone deficiency, reduced growth and extended lifespan. Thus, these mice serve as an important model system for endocrinological, aging and longevity studies. Because the phenotype of wild type and heterozygous mice is undistinguishable, it is imperative for successful breeding to accurately genotype these animals. Here we report a novel, yet simple, approach for prop-1 genotyping using PCR-based allele-specific amplification (PCR-ASA). We also compare this method to other potential genotyping techniques, i.e. PCR-based restriction fragment length polymorphism analysis (PCR-RFLP) and fluorescence automated DNA sequencing. We demonstrate that the single-step PCR-ASA has several advantages over the classical PCR-RFLP because the procedure is simple, less expensive and rapid. To further increase the specificity and sensitivity of the PCR-ASA, we introduced a single-base mismatch at the 3' penultimate position of the mutant primer. Our results also reveal that the fluorescence automated DNA sequencing has limitations for detecting a single nucleotide polymorphism in the prop-1 gene, particularly in heterozygotes.     

Genotyping of Chlamydia trachomatis directly from urogenital and conjunctiva samples using an ompA gene pyrosequencing-based assay

The aim of the present study was to develop and validate a multitarget pyrosequencing-based protocol for basic Chlamydia trachomatis genotyping directly from clinical samples and to characterize the distribution of genotypes among Slovenian sexually active population. The newly developed combination of assays that targets the variable domains VD-I and VD-IV of the C. trachomatis ompA gene, was optimized and validated with 11 reference C. trachomatis strains and by comparison to complete ompA conventional sequencing. In addition, 183 clinical specimens which were previously diagnosed as C. trachomatis positive were evaluated by pyrosequencing. The pyrosequencing products showed a 100% match to corresponding sections of the respective conventional ompA sequences. Based on our results the most frequent genotype in urogenital samples was E (51.1%) followed by F (21.4%), G and K (6.9%), D (6.1%), H (3.8%), J (2.3%) and Ia and Ja (0.8%). In conjunctiva samples the genotype distribution was E (63.3%), D and F (13.3%), K (6.7%) and G (3.3%). Pyrosequencing thus proved itself to be a rapid method for C. trachomatis typing, which is important for better understanding the pathogenesis and epidemiology of this pathogen.     

Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed,Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5⁺/6⁺ (BSGP)5⁺/6⁺. To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.     

碳青霉烯类耐药肺炎克雷伯菌基因型检测及同源性分析

目的了解福建医科大学附属第一医院耐碳青霉烯类肺炎克雷伯菌（KPC）的耐药基因分型及同源性分布情况。方法收集临床分离的29株KPC,改良Hodge试验检测碳青霉烯酶表型。PCR检测碳青霉烯酶blaKPC、blaIMP-1、blaVIM-1、blaOXA-48及blaNDM-基因;阳性结果进行DNA测序,并进行BLAST比对确定其基因型。采用肠杆菌科基因间重复序列（ERIC—PCR）对KPC进行同源性分析。结果29株KPC中Hodge试验阳性27株,阳性率为93．1％（27／29）。PCR扩增和DNA测序显示28株为blaKPC-2,株为blaIMP-1,未检出blaVIM-2、blaOXA-48和blaNDM-1基因。同源性分析发现KPC主要存在两种型别：28株A1和1株A2。结论blaKPC-2型碳青霉烯酶是该院KPC的主要型别,A1型已在该院流行,应加强院感监测和预防。ERIC—PCR是一种简便、快捷的基因分型技术,适合同源性的初步分型筛查。     

Population-based genetic and evolutionary analysis of Chlamydia trachomatis urogenital strain variation in the United States

Chlamydia trachomatis is a major cause of ocular and sexually transmitted diseases worldwide. While much of our knowledge about its genetic diversity comes from serotyping or ompA genotyping, no quantitative assessment of genetic diversity within serotypes has been performed. To accomplish this, 507 urogenital samples from a multicenter U.S. study were analyzed by phylogenetic and statistical modeling. No B, Da, or I serotypes were represented. Based on our analyses, all but one previous urogenital B serotype was identified as Ba. This, coupled with the lack of B serotypes in our population, suggests that B has specific tropism for ocular mucosa. We identified a Ba/D recombinant (putative crossover nucleotide 477; P < 0.0001) similar to a B/D mosaic we described previously from an African trachoma patient. Computational analyses of the Ba/D recombinant indicated that upstream changes were less important for tissue tropism than downstream incorporation of the D sequence. Since most serotypes had nonsynonymous/synonymous ratios of <1.0, the major outer membrane protein, encoded by ompA, has many functional constraints and is under purifying selection. Surprisingly, all serotype groups except for J had a unimodal population structure indicating rapid clonal expansion. Of the groups with a unimodal structure, E and Ia and, to a lesser extent, G and K were prevalent, had infrequent incorporation of mutations, and, compared to other groups, had a relatively greater degree of diversifying selection, consistent with a selective sweep of mutations within these groups. Collectively, these data suggest a diverse evolutionary strategy for different serogroups of the organism.     

Typing Chlamydia trachomatis: from egg yolk to nanotechnology

A historical review is provided of the various methods used for half a century to differentiate and type Chlamydia trachomatis strains. Typing of C. trachomatis is an important tool for revealing transmission patterns in sexual networks, and enabling association with clinical manifestations and pathogenicity. Serotyping using the major outer membrane protein (MOMP) has been the mainstay of epidemiological work for several decades. However, the development of nucleic acid amplification techniques (NAAT) and easy access to sequencing have shifted the focus from MOMP serotypes to omp1 genotypes. However, insufficient epidemiological resolution is achieved by characterization of both MOMP and omp1 . This calls for new high-resolution genotyping methods applying for example a multilocus variable number tandem repeat assay (MLVA) or multilocus sequence typing (MLST). The futuristic nanotechnology already seems at hand to further simplify and automate the high-resolution genotyping method based on NAAT and sequencing of various targets in the C. trachomatis genome. Thereby, a high throughput can be achieved and more epidemiological information can be obtained. However, it is important to realize that culture of C. trachomatis may still be needed to detect and characterize new variants of C. trachomatis .     

中国荷斯坦牛POU1F1基因与PRL基因的多态性及其聚合效应对产奶性状的影响

文章采用DNA测序、PCR-RFLP和CRS-PCR技术对979头中国荷斯坦牛POU1F1基因与PRL基因进行研究,发现了3个新SNPs,分别是POU1F1基因第二外显子G1178C、PRL基因5侧翼区A906G和A1134G。采用SAS统计软件GLM程序,利用最小二乘法拟合线性模型,分析基因多态性与产奶性状的关系。结果表明:POU1F1基因1178位点GC基因型在产奶量、乳蛋白量、乳脂量方面均为优良基因型。PRL基因5侧翼区906位点AG基因型在产奶量方面为优良基因型,1134位点不同基因型产奶性状差异不显著。对PRL基因5侧翼区的906位点和POU1F1基因的1178位点进行基因互作分析,结果在乳脂率、乳蛋白率、产奶量、乳蛋白量和乳脂量方面各基因型组合之间均未观察到显著差异,说明基因聚合效应并不是单基因效应的简单相加,基因聚合效应在分子育种中具有更重要的意义。     

Nucleotide polymorphisms associated with Internal Transcribed Spacer (ITS) regions of ocular isolates of Aspergillus flavus

PURPOSE: To analyse the genetic similarity among ocular isolates of Aspergillus flavus by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing. MATERIALS AND METHODS: Seven ocular isolates of A. flavus from 5 patients (3 from paient 1, and four isolates from patients no. 2, 3, 4, and 5 respectively) consisting of 2 Aqueous Humor (AH), 2 Vitreous fluid (VF), 1 eviscerated material, 1 corneal button were included in the study. The three specimens from 1 were one each of AH, VF and corneal button. The fungal isolates were amplified using primers targeting ITS region and the amplicons were subjected to PCR-RFLP using Hae-III enzyme and DNA sequencing to analyse the genetic similarity. RESULTS: A. flavus isolates yielded a specific product of 595 bp after amplification. All the seven A. flavus isolates showed similar pattern of digestion with Hae-III . However, DNA sequencing of ITS amplicons revealed 97.7% genetic similarity and 2.3% dissimilarity with nucleotide polymorphisms -- single, double and multiple pertaining to inversion, substitution, insertion and deletion in comparison with that of standard strain of A. flavus ATCC 16883 [Accession Number ]. A. flavus isolated from AH, VF and corneal button from the same patient showed similar nucleotide polymorphisms as against other isolates which exhibited distinct polymorphisms. This pattern of nucleotide polymorphisms in A. flavus isolates is novel and first time reported in literature to the best of our knowledge. CONCLUSION: DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates.     

Effect of UV radiation on thermotolerance,ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains

After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1, VS2, VS3 and VS4 strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Efforts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0.20 g l(-1) more than the parent strains at 42 degrees C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0.30 g l(-1) at 30 degrees C and 0.10 and 0.20 g l(-1) at 40 degrees C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3) was 8.20 and 1.20 g l(-1) more than the parent strains at 42 degrees C using 150 g l(-1) glucose. More ethanol was produced by mutants (VS1 and VS3) than the parents at high glucose concentrations of 5.0 and 6.0 g l(-1) at 30 degrees C and 13.0 and 3.0 g l(-1) at 42 degrees C, respectively. These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in VS1 and VS3 yeast strains.     

PCR-RFLP技术用于中国登革2型病毒株基因型快速分型

利用RT-PCR的方法,对中国5株登革2型病毒的prM-E基因进行扩增,选择合适的限制性内切酶将扩增产物特异地切割成若干长度不同的片段,经5％聚丙烯酰胺凝胶电泳后,对获得的银染带型进行分析。结果表明FJ-10株和FJ-11株属同一基因型,D2-43株和D2-44株属同一基因型,D2-04株与上述4株基因型不同,这与核苷酸测序分析结果完全一致。在此基础上建立PCR-RFLP技术用于登革2型病毒基因型快速分型,具有省时、操作简单、不需使用同位素等特点,为登革热疫区实验室和临床诊断提供了一种快速有效的分子生物学方法。