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Sample records for optimized pentaplex pcr

  1. A new pentaplex PCR system for forensic casework analysis.

    Science.gov (United States)

    Lederer, T; Seidl, S; Graham, B; Betz, P

    2000-01-01

    In 1998 the Federal Criminal Police Office of Germany (BKA) established a central genetic database of offenders and suspects to facilitate comparisons with biological samples from future criminal offences. The five obligatory short tandem repeat (STR) loci in this database (TH01, SE33, vWA, FGA and D21S11) were co-amplified in a new PCR pentaplex analysing system together with the sex-specific locus amelogenin. Due to overlapping fragment sizes, amplification products were fluorescent dye-labelled with different colours, separated by electrophoresis and detected directly using the ABI PRISM 310 Genetic Analyzer. Reproducible and reliable results were obtained from as low as 125 pg template DNA, indicating high specificity and sensitivity of the assay. Environmental studies and enzymatic digest with DNase I revealed an excellent stability of the pentaplex system with typeable results even in cases of partially degraded DNA. Complete and reproducible DNA typing was possible in blood-stain mixtures with the minor component as low as 10%. Mean stutter peak intensities were analysed for all loci and ranged from 2.7 +/- 0.8% (TH01) to 10.6 +/- 1.6% (vWA) of the main signal intensity. Allele frequencies were determined in a North Bavarian population sample (n = 121). The combination of five systems resulted in a mean exclusion chance of 99.86% and a power of discrimination of 99.999996%. No deviation from Hardy-Weinberg equilibrium could be found.

  2. A pentaplex PCR assay for the detection and differentiation of Shigella species.

    Science.gov (United States)

    Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Singh, Kirnpal-Kaur Banga

    2013-01-01

    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 10(4) CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.

  3. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

    Science.gov (United States)

    Ojha, Suvash Chandra; Yean Yean, Chan; Ismail, Asma; Banga Singh, Kirnpal-Kaur

    2013-01-01

    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. PMID:23509722

  4. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

    Directory of Open Access Journals (Sweden)

    Suvash Chandra Ojha

    2013-01-01

    Full Text Available The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.

  5. Pentaplex PCR as screening assay for jellyfish species identification in food products.

    Science.gov (United States)

    Armani, Andrea; Giusti, Alice; Castigliego, Lorenzo; Rossi, Aurelio; Tinacci, Lara; Gianfaldoni, Daniela; Guidi, Alessandra

    2014-12-17

    Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market.

  6. A new pentaplex-nested PCR to detect five pathogenic bacteria in free living amoebae.

    Science.gov (United States)

    Calvo, L; Gregorio, I; García, A; Fernández, M T; Goñi, P; Clavel, A; Peleato, M L; Fillat, M F

    2013-02-01

    Changes in water use and anthropogenic activity have major impacts on the quality of natural aquatic ecosystems, water distribution and wastewater plants. One of the main problems is the presence of some pathogenic microorganisms that are resistant to disinfection procedures when they are hosted by free living amoeba and that in many cases are hardly detectable by culture-based procedures. In this work we report a sensitive, low-cost procedure consisting of a pentaplex-nested PCR that allows simultaneous detection of Legionella pneumophila, Mycobacterium spp., Pseudomonas spp., Vibrio cholerae and the microcystin-producing cyanobacteria Microcystis aeruginosa. The method has been used to detect the presence of these pathogenic bacteria in water and inside free living amoeba. Its validation in 72 samples obtained from different water sources from Aragon (Spain) evidences that Mycobacterium and Pseudomonas spp are prevailing as amoeba-resistant bacteria.

  7. A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA.

    Science.gov (United States)

    Meissner, C; Bruse, P; Mueller, E; Oehmichen, M

    2007-03-02

    Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing.

  8. A pentaplex PCR assay for detection and characterization of Vibrio vulnificus and Vibrio parahaemolyticus isolates.

    Science.gov (United States)

    Bhattacharyya, N; Hou, A

    2013-09-01

    Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood-related illnesses and also can cause wound infections. These bacteria often co-exist in marine and estuarine environments. However, there have been no reported protocols that can detect and characterize (i.e. pathogenic or nonpathogenic) them in a single PCR. In this study, we developed a pPCR assay with a combination of two species-specific and three pathogenic-specific PCR primers to simultaneously detect virulent (viuB in V. vulnificus and tdh/trh in V. parahaemolyticus) and nonvirulent (vvhA in V. vulnificus and tlh in V. parahaemolyticus) markers of the two species in bacterial isolates. The assay was validated by three methods. First, the pPCR was used to characterize 300 bacterial isolates consisting of seven reference strains and 293 environmental strains isolated from the Gulf of Mexico water. Results were compared with characterizations based on single-gene PCR amplifications and previously published multiplex PCR protocols. Second, 51 isolates characterized with the pPCR were analysed by 16S rRNA sequencing to confirm any false-negative/positive reaction. Finally, the effectiveness of the assay for heterogeneous bacterial samples was validated. The pPCR correctly characterized isolates from the Gulf with an efficiency of 96·6-98·7%.

  9. Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

    Science.gov (United States)

    Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

    2013-10-30

    Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

  10. Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese.

    Science.gov (United States)

    Cremonesi, Paola; Vanoni, Laura; Morandi, Stefano; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

    2011-03-30

    A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species.

  11. A Pentaplex Real-Time Polymerase Chain Reaction Assay for Detection of Four Species of Soil-Transmitted Helminths

    OpenAIRE

    Basuni, Madihah; Muhi, Jamail; Othman, Nurulhasanah; Verweij, Jaco J.; Ahmad, Maimunah; Miswan, Noorizan; Rahumatullah, Anizah; Aziz, Farhanah Abdul; Zainudin, Nurul Shazalina; Noordin, Rahmah

    2011-01-01

    Soil-transmitted helminth infections remain a major public health burden in low- and middle-income countries. The traditional diagnosis by microscopic examination of fecal samples is insensitive and time-consuming. In this study, a pentaplex real-time polymerase chain reaction (PCR) was evaluated for the simultaneous detection of Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis. The results were compared with those obtained by conventional parasitological d...

  12. 用荧光复合扩增体系检测5个X染色体短串联重复序列基因座的多态性%Polymorphism of Five X-STRs Loci with a New Pentaplex PCR

    Institute of Scientific and Technical Information of China (English)

    刘秋玲; 吕德坚; 赵虎; 李新国; 陆惠玲; 孙宏钰; 梁艳芳; 伍新尧

    2009-01-01

    [Objective] To learn about the genetic diversity,we studied the five X-chromosomal STR (X-STR) loci in Guangdong Han Nationality Groups.[Methods] The five Loci (DXS6803,DXS981,DXS6809,DXS6789,and DXS7132) were amplified in a pentaplex PCR reaction.PCR products were analyzed using capillary electrophoresis and ABI prism 3100 Genetic Analyzer,with GeneMapper ID 3.1 Analysis Software.[Results] A total of 363 individuals (181 unrelated male and 182 unrelated female) from Guangdong Han population were tested,54 alleles were observed for these loci.Polymorphism information content is 0.6935 ~ 0.8177.Power of discrimination in females was 0.8976 ~ 0.9562.Mean exclusion chance for X-STR in standard trios with daughters was 0.7805 ~ 0.8467.[Conclusion] The five loci in the multiplex system provide high polymorphism information for forensic identification and paternity testing,particularly for difficult paternity deficiency cases.%[目的] 为了研究X染色体短串联重复序列(X-STR)基因座的遗传多态性,我们检测了DXS6803、DXS981、DXS6809、DXS6789和DXS7132 5个基因座在广东汉族人群中的多态性. [方法] 利用荧光标记引物PCR技术复合扩增上述5个基因座,并用ABI PRISM 3100毛细管电泳及GeneMapper ID3.1软件进行基因分型.[结果] 在363个广东汉族无关个体(男性:181个,女性:182个)中5个基因座共检出54个等位基因.多态性信息含量为0.6935 ~ 0.8177;女性个体识别率为0.8976 ~ 0.9562.三联体非父排除率为0.7805 ~ 0.8467.[结论] 这5个基因座多态性较高,在个体识别和亲权鉴定中具有重要的应用价值.

  13. Optimized MOL-PCR for Characterization of Microbial Pathogens.

    Science.gov (United States)

    Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

    2016-01-06

    Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium.

  14. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters.

    Science.gov (United States)

    Wadle, Simon; Lehnert, Michael; Schuler, Friedrich; Köppel, René; Serr, Annerose; Zengerle, Roland; von Stetten, Felix

    2016-01-01

    Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.

  15. A G-pentaplex-based assay for Cs(+) ions in aqueous solution using a luminescent Ir(III) complex.

    Science.gov (United States)

    Lin, Sheng; Yang, Chao; Mao, Zhifeng; He, Bingyong; Wang, Yi-Tao; Leung, Chung-Hang; Ma, Dik-Lung

    2016-03-15

    A series of 5 randomly designed in-house cyclometalated Ir(III) complexes were examined for their application in G-pentaplex probes and the "proof-of-principle" concept in G-pentaplex-based Cs(+) ions detection. The G-pentaplex-forming sequence (DNA1, 5'-T(iG)4T-3', where iG=isoguanine) is present in single strand DNA form ab initio, however, the addition of Cs(+) ions lead to formation of the intermolecular G-pentaplex structure which is identified by the novel Ir(III) complex 1 afterward and produce an enhanced luminescence signal for Cs(+) ions monitoring. To the best of our knowledge, this is the first G-pentaplex probe and also the first G-pentaplex-based label-free detection platform for Cs(+) ions reported in the literature. The monitoring of spiked Cs(+) ions in natural water samples demonstrates the potential application and technical sound of this "proof-of-principle" concept sensing platform.

  16. Chaotic Inertia Weight Particle Swarm Optimization for PCR Primer Design

    Directory of Open Access Journals (Sweden)

    Cheng-Huei Yang

    2013-06-01

    Full Text Available In order to provide feasible primer sets for performing a polymerase chain reaction (PCR experiment, many primer design methods have been proposed. However, the majority of these methods require a long time to obtain an optimal solution since large quantities of template DNA need to be analyzed, and the designed primer sets usually do not provide a specific PCR product size. In recent years, particle swarm optimization (PSO has been applied to solve many problems and yielded good results. In this paper, a logistic map is proposed to determine the value of inertia weight of PSO (CIWPSO to design feasible primers. Accuracies for the primer design of the Homo sapiens RNA binding motif protein 11 (RBM11, mRNA (NM_144770, and the Homo sapiens G protein-coupled receptor 78 (GPR78, mRNA (NM_080819 were calculated. Five hundred runs of PSO and the CIWPSO primer design method were performed on different PCR product lengths and the different methods of calculating the melting temperature. A comparison of the accuracy results for PSO and CIWPSO primer design showed that CIWPSO is superior to the PSO for primer design. The proposed method could effectively find optimal or near-optimal primer sets.

  17. Multiplex PCR: Optimization and Application in Diagnostic Virology

    Science.gov (United States)

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  18. Design and optimization of reverse-transcription quantitative PCR experiments.

    Science.gov (United States)

    Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

    2009-10-01

    Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

  19. Multiplex PCR System Optimization with Potato SSR Markers

    Institute of Scientific and Technical Information of China (English)

    Wang Shao-peng; Liu Shang-wu; Li Yong; Liu Wei-ting; Lv Dian-qiu

    2012-01-01

    Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components (enzyme, MgCl2, DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 (34) was applied in the optimization of four sets of primers (STM0014, Pat, SSI, and UGP) in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL : 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 : 1 : 2 : 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..

  20. Low cost and high performance GPON, GEPON and RFoG optical network pentaplexer module design using diffractive grating approach

    Science.gov (United States)

    Chen, I.-Ju; Chi, Chang-Chia; Tarn, Chen-Wen

    2016-01-01

    A new architecture of a pentaplexer transceiver module which can be used in GPON/GEPON and RFoG triple play optical networks with supporting of the multiple optical wavelengths of 1310 nm, 1490 nm, 1550 nm, 1610 nm, and 1650 nm, is proposed. By using diffractive grating elements combing with market readily available GRIN (Gradient-Index) lens, grating, mirrors, beamsplitter, LDs (Laser Diodes), and PDs (Photodetectors), the proposed design have the advantages of low cost, high efficiency/performance, easy design and manufacturing, over the contemporary triplex transceivers which are made of multilayer filters or waveguides that increase the complexity of manufacturing and reduce the performance efficiency. With the proposed design, a pentaplexer system can accommodate GPON/GEPON, RFoG, and monitoring integration services, total five optical wavelength channels into a hybrid-integrated TO-CAN package platform with sufficient efficiency.

  1. Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

    Science.gov (United States)

    Lievens, A; Jacchia, S; Kagkli, D; Savini, C; Querci, M

    2016-01-01

    Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain.

  2. Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

    Directory of Open Access Journals (Sweden)

    A Lievens

    Full Text Available Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain.

  3. Optimized PCR assay for detection of white spot syndrome virus (WSSV).

    Science.gov (United States)

    Nunan, Linda M; Lightner, Donald V

    2011-01-01

    A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.

  4. Fast detection of genetic information by an optimized PCR in an interchangeable chip.

    KAUST Repository

    Wu, Jinbo

    2012-02-01

    In this paper, we report the construction of a polymerase chain reaction (PCR) device for fast amplification and detection of DNA. This device consists of an interchangeable PCR chamber, a temperature control component as well as an optical detection system. The DNA amplification happens on an interchangeable chip with the volumes as low as 1.25 μl, while the heating and cooling rate was as fast as 12.7°C/second ensuring that the total time needed of only 25 min to complete the 35 cycle PCR amplification. An optimized PCR with two-temperature approach for denaturing and annealing (Td and Ta) of DNA was also formulated with the PCR chip, with which the amplification of male-specific sex determining region Y (SRY) gene marker by utilizing raw saliva was successfully achieved and the genetic identification was in-situ detected right after PCR by the optical detection system.

  5. Optimization of Splicing by Overlap Extension PCR Using Nested PCR%基于巢式PCR的重叠延伸PCR优化

    Institute of Scientific and Technical Information of China (English)

    彭雷; 赵艳; 马银花

    2016-01-01

    [目的]结合巢式PCR对重叠延伸PCR进行优化。[方法]以褐飞虱Actin 1基因启动子区与EGFP表达盒区基因融合为例,通过巢式PCR对重叠延伸PCR进行优化。[结果]成功获得融合片段,经过巢式PCR优化后大大简化试验流程,缩短试验时间,并增强PCR特异性与检出率。[结论]优化了重叠延伸PCR,可更快速高效融合基因片段。%Objective] To optimize the Splicing by overlap extension PCR using nested PCR.[Method] Nested PCR was used to optimize SOE-PCR (splicing by overlap extension PCR) for combining brown planthopper (BPH) Actin 1 promoter and EGFP expression cassette. [Result] Nested PCR could greatly optimize SOE-PCR.After optimization of nested PCR, the test process was simplified, and the detection time was shortened.The PCR specificity and detection rate were enhanced.[ Conclusion] The optimized SOE-PCR can rapidly and effectively fuse the gene segment.

  6. Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I.

    Science.gov (United States)

    Thanakiatkrai, Phuvadol; Welch, Lindsey

    2012-01-01

    Here, we applied the Taguchi method, an engineering optimization process, to successfully determine the optimal conditions for three SYBR Green I-based quantitative PCR assays. This method balanced the effects of all factors and their associated levels by using an orthogonal array rather than a factorial array. Instead of running 27 experiments with the conventional factorial method, the Taguchi method achieved the same optimal conditions using only nine experiments, saving valuable resources.

  7. Real-time PCR probe optimization using design of experiments approach.

    Science.gov (United States)

    Wadle, S; Lehnert, M; Rubenwolf, S; Zengerle, R; von Stetten, F

    2016-03-01

    Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3-14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7-11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.

  8. Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid

    Directory of Open Access Journals (Sweden)

    Hackman Robert C

    2008-05-01

    the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.

  9. Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

    Directory of Open Access Journals (Sweden)

    Haug Trude M

    2010-11-01

    Full Text Available Abstract Background The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. Results Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. Conclusion Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

  10. Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR.

    Science.gov (United States)

    Nadkarni, Mangala A; Martin, F Elizabeth; Hunter, Neil; Jacques, Nicholas A

    2009-07-01

    Methods for the optimal extraction of genomic DNA for real-time PCR enumeration of oral bacteria using the muramidase, mutanolysin, were developed using a simple in vitro oral flora model comprised of the facultative anaerobic gram-positive bacteria, Lactobacillus acidophilus and Streptococcus mutans, the gram-positive anaerobe, Parvimonas micra, and the gram-negative anaerobes, Porphyromonas gingivalis, Prevotella melaninogenica and Fusobacterium nucleatum. Traditional, as well as more elaborate, methods of quantifying bacterial numbers, including colony counting and estimation of DNA content using 4',6-diamino-2-phenylindole were compared in order to validate the real-time PCR approach. Evidence was obtained that P. gingivalis nuclease activity adversely affected the extraction of double-stranded DNA from this bacterium either alone or when it formed part of a consortium with the other bacteria. This nuclease activity could be overcome by treatment of the bacteria with either 20 mM diethyl pyrocarbonate or 70% ethanol at 4 degrees C overnight. A final purification of the DNA to remove any potential PCR inhibitors was added to the protocol in order to accurately quantify the amount of DNA by real-time PCR and hence the number of bacteria in a sample.

  11. Optimization and Application of PCR Detection for Mycobacterium Leprae%麻风杆菌 PCR 检测方法的优化及应用

    Institute of Scientific and Technical Information of China (English)

    叶理; 吕成志; 靳亚丽; 王会翔; 张振国; 宋顺鹏

    2016-01-01

    目的:研建一种适用于临床中检测麻风杆菌( ML)的分子生物学方法,并优化检测条件以提高检测方法的灵敏度。方法:用麻风杆菌纯DNA作模板,对PCR反应体系中模板浓度、引物浓度等反应条件进行优化,以建立高敏感性的PCR反应体系,并与实时荧光定量PCR(Real-time PCR)方法比较,对麻风病人标本进行检测,确定优化后两法的反应体系及两法的临床检测意义。结果:经过大量的实验比对表明,反应体系引物含量为0.2μL ×100μmol时,模板浓度10-1条ML/mL可以作为稳定的可检测到条带的模板浓度的下限。对52例各型麻风患者标本的检测结果显示常规 PCR 检测阳性数(49/52)略高于Real-time PCR(47/52),但Real-time PCR操作程序简单和需时短,且成本低。结论:通过对PCR反应条件中DNA模板浓度和引物浓度的优选,提高了PCR检测麻风杆菌DNA的灵敏度;对于不同PCR方法的选择方面, Real-time PCR 比常规 PCR 简捷快速,而常规 PCR 灵敏度略高于 Real-time PCR。%Objective:To establish a molecular biological method with high sensitivity for de-tecting Mycobacterium leprae(ML) in clinics.Methods:Establish a highly sensitive PCR reaction system by optimizing the reaction conditions of PCR reaction system using the pure DNA of Myco -bacterium leprae and compare the efficacy and significance of two systems-conventional PCR ( cPCR) and real-time PCR as clinical detection methods .Results:The results indicated that 10 -1 ML/mL is the lowest DNA concentration which can be steadily detected when the primer content is 0.2 μL ×100 μmol/L.The number of positive results of cPCR (49/52) was slightly higher than that of Real-time PCR ( 47/52 ) according to the results detected from the samples of leprosy pa-tients.Conclusion:Through the optimization of reaction conditions in the PCR reaction , such as DNA template concentration and

  12. [High efficiency genome walking method for flanking sequences of cotton mitochondrial double-copy atpA gene based on optimized inverse PCR and TAIL-PCR].

    Science.gov (United States)

    Zhang, Xiao; Zhang, Rui; Sun, Guoqing; Shi, Ji; Meng, Zhigang; Zhou, Tao; Hou, Siyu; Liang, Chengzhen; Yu, Yuanhua; Guo, Sandui

    2012-01-01

    Cloning of flanking sequences of double-copy gene is a challenge in molecular biology. We developed a method to solve this problem by combining an optimized inverse PCR (iPCR) with TAIL-PCR. First, Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene. Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene. Based on the obtained sequences, TAIL-PCR was performed to amplify further flanking regions of the gene. With this method, we obtained all of the EcoR I restriction fragments (2.2-5.1 kb) and Hind III restriction fragments (8.5-11.7 kb) of mitochondrial atpA gene in cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton. The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.

  13. Comparative assessment of 5' A/T-rich overhang sequences with optimal and sub-optimal primers to increase PCR yields and sensitivity.

    Science.gov (United States)

    Arif, M; Ochoa-Corona, F M

    2013-09-01

    Efficient PCR amplifications require precisely designed and optimized oligonucleotide primers, components, and cycling conditions. Despite recent software development and reaction improvement, primer design can still be enhanced. The aims of this research are to understand (1) the effect on PCR efficiency and DNA yields of primer thermodynamics parameters, and (2) the incorporation of 5' A/T-rich overhanging sequences (flaps) during primer design. Two primer sets, one optimal (ΔG = 0) and one sub-optimal (ΔG = 0.9), were designed using web interface software Primer3, BLASTn, and mFold to target a movement protein gene of Tobacco mosaic virus. The optimal primer set amplifies a product of 195 bp and supports higher PCR sensitivity and yields compared to the sub-optimal primer set, which amplifies a product of 192 bp. Greater fluorescence was obtained using optimal primers compared to that with sub-optimal primers. Primers designed with sub-optimal thermodynamics can be substantially improved by adding 5' flaps. Results indicate that even if the performance of some primers can be improved substantially by 5' flap addition, not all primers will be similarly improved. Optimal 5' flap sequences are dependent on the primer sequences, and alter the primer's T m value. The manipulation of this feature may enhance primer's efficiency to increase the PCR sensitivity and DNA yield.

  14. Optimizing SSR-PCR system of Panax ginseng by orthogonal design

    Institute of Scientific and Technical Information of China (English)

    YANG Tian-tian; MU Li-qiang; WANG Jun

    2007-01-01

    An orthogonal design was used to optimize SSR-PCR amplification system using Panax ginseng genomic DNA as template. Four levels of five factors (DNA template, Taq DNA polymerase, Mg2+, primer, and dNTP) and annealing temperature have been tested separately in this system. The results demonstrated the reaction efficiency was affected by these factors. Based on the results, a stable, productive and reproducible PCR system and cycling program for amplifying a ginseng SSR locus were obtained: 20 μL system containing 1.0 U Taq DNA polymerase, 2.0 mmol·L-1 Mg2+, 0.2 mmol· L-1 dNTPs, 0.3 μmol· L-1 SSR primer, 60 ng·μL-1 DNA template, performed with a program of 94℃ for 5 min, 94℃ for 30 s, annealing at 56.3℃ for 30 s, 72℃ for 1 min, 37 cycles, finishing at 72℃ for 7 min, and storing at 4℃.

  15. Construction and optimization of an efficient amplification method of a random ssDNA library by asymmetric emulsion PCR.

    Science.gov (United States)

    Shao, Keke; Shi, Xinhui; Zhu, Xiangjun; Cui, Leilei; Shao, Qixiang; Ma, Da

    2017-03-01

    Construction of a random ssDNA sublibrary is an important step of the aptamer screening process. The available construction methods include asymmetric PCR, biotin-streptavidin separation, and lambda exonuclease digestions, in which PCR amplification is a key step. The main drawback of PCR amplification is overamplification increasing nonspecific hybridization among different products and by-products, which may cause the loss of potential high-quality aptamers, inefficient screening, and even screening failure. Cycle number optimization in PCR amplification is the main way to avoid overamplification but does not fundamentally eliminate the nonspecific hybridization, and the decreased cycle number may lead to insufficient product amounts. Here, we developed a new method, "asymmetric emulsion PCR," which could overcome the shortcomings of conventional PCR. In asymmetric emulsion PCR, different templates were separated by emulsion particles, allowing single-molecule PCR, in which each template was separately amplified, and the nonspecific hybridization was avoided. Overamplification or formation of by-products was not observed. The method is so simple that direct amplification of 40 or more cycles can provide a high-quality ssDNA library. Therefore, the asymmetric emulsion PCR would improve the screening efficiency of systematic evolution of ligands by exponential enrichment. © 2015 The Authors. Biotechnology and Applied Biochemistry published by Wiley Periodicals, Inc. on behalf of the International Union of Biochemistry and Molecular Biology, Inc.

  16. A Rapid and Sensitive Detection of Aflatoxin-producing Fungus Using an Optimized Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Bintvihok, Anong; Treebonmuang, Supitchaya; Srisakwattana, Kitiya; Nuanchun, Wisut; Patthanachai, Koranis; Usawang, Sungworn

    2016-01-01

    Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was 65°C. The optimized template and primer concentration were 1.5 μL (50 ng/μL) and 3 μL (10 μM/μL) respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at 88.0°C, 87.5°C, 83.5°C, and 89.5°C respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

  17. Orthogonal design in the optimization of a start codon targeted (SCoT) PCR system in Roegneria kamoji Ohwi.

    Science.gov (United States)

    Zeng, B; Yan, H D; Huang, L K; Wang, Y C; Wu, J H; Huang, X; Zhang, A L; Wang, C R; Mu, Q

    2016-10-24

    Roegneria kamoji Ohwi is an excellent forage grass due to its high feeding value and high resistance to some biotic and abiotic stresses. However, the start codon targeted (SCoT) polymorphism has not been conducted on R. kamoji. In this study, an orthogonal L16 (4(5)) design was employed to investigate the effects of five factors (Mg(2+), dNTPs, Taq DNA polymerase, primer, and template DNA) on the polymerase chain reaction (PCR) to determine the optimal SCoT-PCR system for R. kamoji. The results showed that the most suitable conditions for SCoT-PCR in R. kamoji included 1.5 mM Mg(2+), 0.15 mM dNTPs, 1.0 U Taq DNA polymerase, 0.4 pM primer, and 40 ng template DNA. SCoT primers 39 and 41 were used to verify the stability of the optimal reaction system, and amplification bands obtained from diverse samples were found to be clear, rich, and stable in polymorphisms, indicating that this reaction system can be used for SCoT-PCR analysis of R. kamoji. We have developed a simple and rapid way to study the mutual effects of factors and to obtain positive results through the use of an orthogonal design L16 (4(5)) to optimize the SCoT-PCR system. This method may provide basic information for molecular marker-assisted breeding and analyses of genetic diversity in R. kamoji.

  18. A Belgian Serosurveillance/Seroprevalence Study of Diphtheria, Tetanus and Pertussis Using a Luminex xMAP Technology-Based Pentaplex

    Directory of Open Access Journals (Sweden)

    Raissa Nadège Caboré

    2016-05-01

    Full Text Available Serosurveillance and seroprevalence studies are an essential tool to monitor vaccine-preventable diseases. We have developed a magnetic bead-based pentaplex immunoassay (MIA for the simultaneous detection of IgG antibodies against diphtheria toxin (DT, tetanus toxin (TT, pertussis toxin (PT, filamentous hemagglutinin (FHA and pertactin (Prn. The in-house pentaplex MIA showed a good correlation with commercial ELISAs with correlation coefficients between 0.89 for PT and 0.98 for TT. Intra- and inter-assay variability was <10%. A total of 670 anonymized serum samples collected in 2012 in Belgian adults (ages 20–29.9 years were analyzed. Geometric mean concentrations (GMC were 0.2 (0.13–0.29 IU/mL for DT, 0.63 (0.45–0.82 IU/mL for TT, 3.9 (2.6–5.8 IU/mL for PT, 16.3 (11.7–22.7 IU/mL for FHA and 15.4 (10.1–23.6 IU/mL for Prn. Antibody concentrations were below the protective level of 0.1 IU/mL in 26.4% of the sera for DT and in 8.6% of the sera for TT. Anti-PT IgG concentrations indicative of recent pertussis infection (>125 IU/mL were detected in 1.2% of the subjects. High anti-PT antibodies were not correlated with high antibodies against any of the four other vaccine antigens. This pentaplex MIA will be used for a new large-scale Belgian serosurveillance/seroprevalence study of diphtheria, tetanus and pertussis.

  19. An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

    Directory of Open Access Journals (Sweden)

    Amanda de Faveri Pitz

    2013-12-01

    Full Text Available Introduction Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR assay for the detection of Paracoccidioides brasiliensis. Methods We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. Results The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. Conclusions The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

  20. Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis.

    Science.gov (United States)

    Le Gall, Florence; Le Berre, Rozenn; Rosec, Sylvain; Hardy, Jeanne; Gouriou, Stéphanie; Boisramé-Gastrin, Sylvie; Vallet, Sophie; Rault, Gilles; Payan, Christopher; Héry-Arnaud, Geneviève

    2013-06-21

    The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.

  1. Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA misamplification

    Directory of Open Access Journals (Sweden)

    Vitor Fernandes Oliveira de Miranda

    2010-02-01

    Full Text Available The nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2 from leaves of Drosera (Droseraceae were amplified using "universal" primers. The analysis of the products demonstrated most samples were a molecular mixture as a result of unsuccessful and non-specific amplifications. Among the obtained sequences, two were from Basidiomycota fungi. Homologous sequences of Basidiomycota were obtained from GenBank database and added to a data set with sequences from Drosera leaves. Parsimony analysis demonstrated that one sequence was amplified from an Ustilaginomycetes fungus, and another from a Heterobasidiomycetes. Possibly these fungi were associated to leaves of Drosera, and not because of samples contamination. In order to provide optimization and a better specificity of PCR (polymerase chain reaction, a very successful method was demonstrated using dimethyl sulfoxide (DMSO and bovine serum albumin (BSA in reactions.Os espaçadores internos transcritos do DNA nuclear ribossomal (ITS1 e ITS2 de folhas de Drosera (Droseraceae foram amplificados com o emprego de iniciadores "universais". A análise demonstrou que a maior parte das amostras continha uma mistura resultante de amplificações não-específicas. Dentre as sequências de DNA obtidas, duas delas foram de fungos basidiomicetos. Sequências homólogas foram obtidas do GenBank e analisadas junto às sequências obtidas de folhas de Drosera. Através das análises filogenéticas de máxima parcimônia foi possível identificar uma seqüência como sendo de um Ustilaginomycetes e outra de Heterobasidiomycetes (Basidiomycota. Possivelmente esses organismos estavam associados às folhas de Drosera e assim não sejam resultantes de contaminação. Com o objetivo de otimizar e buscar uma melhor especificidade das reações de PCR, um protocolo bem sucedido foi demonstrado com o uso de dimetilsulfóxido (DMSO e soroalbumina bovina (BSA.

  2. Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes.

    Science.gov (United States)

    Ahlawat, Sonika; Sharma, Rekha; Maitra, A; Roy, Manoranjan; Tantia, M S

    2014-12-01

    New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes.

  3. Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes

    Directory of Open Access Journals (Sweden)

    Sonika Ahlawat

    2014-12-01

    Full Text Available New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242C (BMPR1B, G1189A (GDF9 and G735A (BMP15. Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes.

  4. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    OpenAIRE

    Eva Malatinkova; Maja Kiselinova; Pawel Bonczkowski; Wim Trypsteen; Peter Messiaen; Jolien Vermeire; Bruno Verhasselt; Karen Vervisch; Linos Vandekerckhove; Ward De Spiegelaere

    2014-01-01

    Introduction: In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and M...

  5. Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm.

    Science.gov (United States)

    Kirakodu, S S; Govindaswami, M; Novak, M J; Ebersole, J L; Novak, K F

    2008-01-01

    Quantitative PCR (qPCR) has recently been used to quantify microorganisms in complex communities, including dental plaque biofilms. However, there is variability in the qPCR protocols being used. This study was designed to evaluate the validity of two of these variables with the intent of developing a more standardized qPCR protocol. The two variables evaluated were (1) the use of DNA content versus actual cell counts to estimate bacterial numbers in mixed plaque samples and (2) the effectiveness of three different universal primers versus species specific primers in amplifying specific target pathogens in these samples. Results lead to the development of a standardized protocol that was shown to be highly reproducible as demonstrated by low coefficients of variation. The results also confirmed that this standardized qPCR protocol can be used as a sensitive method for quantifying specific bacterial species in human plaque samples.

  6. Detection of influenza A virus RNA in birds by optimized Real-Time PCR system

    Institute of Scientific and Technical Information of China (English)

    Ilinykh Ph A; Shestopalova EM; Khripko Yu I; Durimanov AG; Sharshov KA; Shestopalov AM

    2010-01-01

    Objective: To evaluate the use of Real-Time PCR system based on specific amplification of matrix protein gene fragment for influenza A virus RNA detection in cloacal swabs from wild birds. Methods:Sensitivity, specificity and reproducibility of analysis results were identified. Study of cloacal swabs from wild birds for influenza A virus presence was performed. Results:Reproducibility of low concentrations of virus detection in samples by Real-Time PCR was significantly higher than that of detection based on cytopathic effect of viruses grown on MDCK cell culture. Conclusions: Real-Time PCR system for influenza A virus RNA detection is developed and applied for virus surveillance study.

  7. Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR.

    Science.gov (United States)

    Walker, Roblena E; Petersen, Jeannine M; Stephens, Kenyatta W; Dauphin, Leslie A

    2010-10-01

    Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5'nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.

  8. An optimized affordable DNA-extraction method from Salmonella enterica Enteritidis for PCR experiments

    Directory of Open Access Journals (Sweden)

    Karimnasab, N.,

    2013-12-01

    Full Text Available In diagnostic and research bacteriology settings with budget and staff restrictions, fast and cost-effective genome extraction methods are desirable. If not inactivated properly, cellular and/or environmental DNA nucleases will degrade genomic material during the extraction stage, and therefore might give rise to incorrect results in PCR experiments. When crude cell extracts, proteinase K–treated templates and purified DNAs prepared by phenol-chloroform-isoamylalcohol method as well as a commercial extraction kit were subjected to the Salmonella enterica Enteritidis specific STM2 PCR, with exception of crude cell extract, PCR products from all other three methods saved their integrity for 28 days post-generation. This work aimed to find out whether improvement to boiling method can guaranty stability of PCR products. As results showed, treatment of crude cell extracts from S. Enteritidis with proteinase K offers an inexpensive, fast and effective DNA extraction method suitable for high-throughput laboratories.

  9. Optimization of PMA-PCR Protocol for Viability Detection of Pathogens

    Science.gov (United States)

    Mikkelson, Brian J.; Lee, Christine M.; Ponce, Adrian

    2011-01-01

    This presented study demonstrates the need that PMA-PCR can be used to capture the loss of viability of a sample that is much more specific and time-efficient than alternative methods. This protocol is particularly useful in scenarios in which sterilization treatments may inactivate organisms but not degrade their DNA. The use of a PCR-based method of pathogen detection without first inactivating the DNA of nonviable cells will potentially lead to false positives. The loss of culturability, by heat-killing, did not prevent amplified PCR products, which supports the use of PMA to prevent amplification and differentiate between viable and dead cells. PMA was shown to inhibit the amplification of DNA by PCR in vegetative cells that had been heat-killed.

  10. Design and sampling plan optimization for RT-qPCR experiments in plants: a case study in blueberry

    Directory of Open Access Journals (Sweden)

    Jose V Die

    2016-03-01

    Full Text Available The qPCR assay has become a routine technology in plant biotechnology and agricultural research. It is unlikely to be technically improved, but there are still challenges which center around minimizing the variability in results and transparency when reporting technical data in support of the conclusions of a study. There are a number of aspects of the pre- and post-assay workflow that contribute to variability of results. Here, through the study of the introduction of error in qPCR measurements at different stages of the workflow, we describe the most important causes of technical variability in a case study using blueberry. In this study, we found that the stage for which increasing the number of replicates would be the most beneficial depends on the tissue used. For example, we would recommend the use of more RT replicates when working with leaf tissue, while the use of more sampling (RNA extraction replicates would be recommended when working with stems or fruits to obtain the most optimal results. The use of more qPCR replicates provides the least benefit as it is the most reproducible step. By knowing the distribution of error over an entire experiment and the costs at each step, we have developed a script to identify the optimal sampling plan within the limits of a given budget. These findings should help plant scientists improve the design of qPCR experiments and refine their laboratory practices in order to conduct qPCR assays in a more reliable-manner to produce more consistent and reproducible data.

  11. Design and Sampling Plan Optimization for RT-qPCR Experiments in Plants: A Case Study in Blueberry.

    Science.gov (United States)

    Die, Jose V; Roman, Belen; Flores, Fernando; Rowland, Lisa J

    2016-01-01

    The qPCR assay has become a routine technology in plant biotechnology and agricultural research. It is unlikely to be technically improved, but there are still challenges which center around minimizing the variability in results and transparency when reporting technical data in support of the conclusions of a study. There are a number of aspects of the pre- and post-assay workflow that contribute to variability of results. Here, through the study of the introduction of error in qPCR measurements at different stages of the workflow, we describe the most important causes of technical variability in a case study using blueberry. In this study, we found that the stage for which increasing the number of replicates would be the most beneficial depends on the tissue used. For example, we would recommend the use of more RT replicates when working with leaf tissue, while the use of more sampling (RNA extraction) replicates would be recommended when working with stems or fruits to obtain the most optimal results. The use of more qPCR replicates provides the least benefit as it is the most reproducible step. By knowing the distribution of error over an entire experiment and the costs at each step, we have developed a script to identify the optimal sampling plan within the limits of a given budget. These findings should help plant scientists improve the design of qPCR experiments and refine their laboratory practices in order to conduct qPCR assays in a more reliable-manner to produce more consistent and reproducible data.

  12. Effective Natural PCR-RFLP Primer Design for SNP Genotyping Using Teaching-Learning-Based Optimization With Elite Strategy.

    Science.gov (United States)

    Cheng, Yu-Huei; Kuo, Che-Nan; Lai, Ching-Ming

    2016-10-01

    SNP (single nucleotide polymorphism) genotyping is the determination of genetic variations of SNPs between members of a species. In many laboratories, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) is a usually used biotechnology for SNP genotyping, especially in small-scale basic research studies of complex genetic diseases. PCR-RFLP requires an available restriction enzyme at least for identify a target SNP and an effective primer pair conforms numerous constraints. However, the lots of restriction enzymes, tedious sequence and complicated constraints make the mining of available restriction enzymes and the design of effective primer pairs become a major challenge. In the study, we propose a novel and available CI (Computation Intelligence)-based method called TLBO (teaching-learning-based optimization) and introduce the elite strategy to design effective primer pairs. Three common melting temperature computations are available in the method. REHUNT (Restriction Enzymes HUNTing) is first combined with the method to mine available restriction enzymes. Robust in silico simulations for the GA (genetic algorithm), the PSO (particle swarm optimization), and the method for natural PCR-RFLP primer design in the SLC6A4 gene with two hundred and eighty-eight SNPs had been performed and compared. These methods had been implemented in JAVA and they are freely available at https://sites.google.com/site/yhcheng1981/tlbonpd-elite for users of academic and non-commercial interests.

  13. Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

    Science.gov (United States)

    Sandeu, Maurice Marcel; Moussiliou, Azizath; Moiroux, Nicolas; Padonou, Gilles G.; Massougbodji, Achille; Corbel, Vincent; Tuikue Ndam, Nicaise

    2012-01-01

    Background An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. Methods Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. Results The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, PPlasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. Conclusion This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate

  14. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    Directory of Open Access Journals (Sweden)

    Eva Malatinkova

    2014-11-01

    Full Text Available Introduction: In HIV-infected patients on combination antiretroviral therapy (cART, the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods: We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR. Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59. Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results: Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974 and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs, (R2=0.671. Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10. Conclusions: 2-LTR

  15. Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat.

    Science.gov (United States)

    Josefsen, M H; Krause, M; Hansen, F; Hoorfar, J

    2007-05-01

    We developed a 12-h Salmonella detection method, based on 8 h of preenrichment, followed by automated DNA extraction and a sensitive real-time PCR. The method was optimized to obtain the highest possible yield of cells and DNA. The growth of different Salmonella strains in various preenrichment media and the effects of adding growth-promoting and selective reagents were explored, taking into account their PCR compatibility. The effects of (i) analyzing larger volumes (1 to 5 ml) from preenriched samples and introducing wash steps prior to DNA extraction, (ii) regulating the amount of paramagnetic particles (increasing it from 60 to 90 microl) in the DNA extraction, (iii) eluting the DNA in reduced volumes (25 or 50 microl rather than 100 microl), and (iv) increasing the PCR template volume (from 5 to 20 microl) were investigated. After 8 h of preenrichment, buffered peptone water yielded the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 microl were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 microl) had no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mul improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples, with a relative accuracy of 99%, a relative sensitivity of 98%, and a relative specificity of 100%.

  16. An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails.

    Science.gov (United States)

    Caron, Y; Righi, S; Lempereur, L; Saegerman, C; Losson, B

    2011-05-31

    This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex(®) DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react.

  17. Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae.

    Science.gov (United States)

    Mogni, Virginia Y; Kahan, Mariano A; de Queiroz, Luciano Paganucci; Vesprini, José L; Ortiz, Juan Pablo A; Prado, Darién E

    2016-01-01

    The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites-i.e. tannins and oleoresins-that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS-ETS fragments. The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

  18. Detection of GM soybean by multiplex-touchdown PCR-microchip capillary electrophoresis with response surface methodology optimization.

    Science.gov (United States)

    Li, Yongxin; Su, Ning; Zheng, Bo; Ruan, Jia; Li, Yang; Luo, Chunying; Li, Yuanqian

    2015-02-01

    The combination of the molecular technique, the multivariate strategy and microchip capillary electrophoresis (MCE) was applied to rapid and sensitive analysis of genetically modified (GM) soybean in food samples. A multiplex-touchdown polymerase chain reaction (PCR) system was developed for simultaneously amplifying three target sequences in Roundup Ready soybean (RRS). Response surface methodology was introduced to determine the optimal separation condition in MCE with good resolution and short analytical time. The detection of the PCR products of RRS was completed within 4 min under the optimal conditions. The specificity of the method was evaluated by testing non-GM soybean materials and three GM maize varieties (MON810, Bt176 and Bt11). A sensitivity of 0.1% GM organisms content was obtained, which was remarkably lower than the labeling threshold for transgenic food defined as 0.9% in the European regulation. The relative standard deviation of migration time was in the range of 0.17-0.95%. The proposed method was rapid, sensitive and specific and can be used to identify and detect GM soybean in food samples.

  19. A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach.

    Directory of Open Access Journals (Sweden)

    Qian Mei

    Full Text Available BACKGROUND: The spatiotemporal expression patterns of microRNAs (miRNAs are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively. RESULTS: We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run. CONCLUSIONS: The cost-effective RT-qPCR of miRNAs with poly(A-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.

  20. Establishment and Optimization of ISSR-PCR Amplification System in Squash%南瓜 ISSR-PCR 反应体系的建立与优化

    Institute of Scientific and Technical Information of China (English)

    朱海生; 卢丽芳; 温庆放; 林义章

    2014-01-01

    为获得南瓜 ISSR 最优扩增体系,为后续南瓜的遗传多样性分析和亲缘关系鉴定奠定基础,本研究以‘密本’南瓜为筛选体系材料,采用单因素试验,对 ISSR 反应体系的 Mg2+、dNTP、Taq 酶、引物和 DNA 浓度等5个因素进行优化。研究结果表明,南瓜 ISSR 25μL 最佳反应体系为:2.5μL 10×Buffer,2.0 mmol/L Mg2+,0.3 mmol/L dNTP,1 U Taq 酶,0.48 mmol/L 引物,75 ng 的 DNA。在此基础上,对筛选出的12条引物进行退火温度的优化,最终确定每条引物的最佳退火温度。利用该反应体系,选用引物891对85个南瓜品种进行所确立扩增体系的验证,结果显示扩增产物条带清晰明亮、多态性丰富,且特异性强、重复性好,表明本研究所确定的反应体系适用于南瓜的 ISSR 分子标记。%In order to obtain the ISSR-PCR reaction system of Squash, We took an optimization experiment with single factor design which was concentrated on five factors of Mg2+, dNTP, Taq DNA polymerase, primer and template DNA by using the squash ‘miben’as the material of filter system to set up a good foundation of analy-sing the genetic diversity and relationship identification of Squash. The test result showed that the greatest 25 μL ISSR reaction system which contained 2.5 μL 10×Buffer, 2.0 mmol/L Mg2+, 0.3 mmol/L dNTP, 1 U Taq DNA polymerase, 0.48 mmol/L primer, 75 ng template DNA. On this basis, optimized annealing temperature of 12 primers which have been screened, and ultimately determined the optimal annealing temperature of each primer. 85 squashes were amplified with primer 891 under the optimized reaction conditions. The test result showed the amplification products which were clear and bright, abundant polymorphism, strong specificity and good repeatability. Accordingly, it was suitable for ISSR analysis of squash.

  1. The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

    Directory of Open Access Journals (Sweden)

    Pesta David

    2003-06-01

    Full Text Available Abstract Background DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein. We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions The technique detailed

  2. Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

    Science.gov (United States)

    Orue, Andrea; Rieber, Manuel

    2016-01-01

    Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting. PMID:27632281

  3. Optimization of ISSR-PCR system for chenopodium album L%藜ISSR-PCR反应体系的优化

    Institute of Scientific and Technical Information of China (English)

    张恒庆; 朱立南; 杜洋; 贾俊玲; 谭琨

    2011-01-01

    In this study, the factors which affect the ISSR-PCR reaction were detected by TaKaRa PCR reaction system of Chenopodium album L. The purified template DNA was extracted from fresh young leaves, the effect of content of Mg2+,templates DNA and Taq DNA polymerase were tested. According to the results of amplifications in gradient it was determined that the optimization ISSR-PCR reaction system in rolumes of 25 mL contained 2.5 μL to XPCR buffer, 0.2 mrnol/L mixture of dNTPs,0.2 mmol/L primer, 1. % mmol/L Mg2+ ,40 ng of DNA and 1.5 unit Taq DNA Polymerase.%采用TaKaRa的PCR反应系统,以从藜(Chenopodium album L)幼叶中提取纯化后的总DNA为模板,进行UBC 859号引物的ISSR-PCR扩增实验,分别测试了镁离子、模板DNA和Taq DNA聚合酶含量对反应结果的影响,通过各因子的浓度梯度组合实验,确定了在25 mL体积的PCR反应中:酶配套缓冲液2.5μL,dNTPs0.2μmmol/L,引物0.2mmol/L,Mg2+1.5mmol/L,模板含量为40 ng,Taq DNA聚合酶为1.5U是藜最佳的ISSR-PCR反应体系.

  4. Development of an optimized method for the detection of airborne viruses with real-time PCR analysis

    Directory of Open Access Journals (Sweden)

    Legaki Euaggelia

    2011-07-01

    Full Text Available Abstract Background Airborne viruses remain one of the major public health issues worldwide. Detection and quantification of airborne viruses is essential in order to provide information regarding public health risk assessment. Findings In this study, an optimized new, simple, low cost method for sampling of airborne viruses using Low Melting Agarose (LMA plates and a conventional microbial air sampling device has been developed. The use of LMA plates permits the direct nucleic acids extraction of the captured viruses without the need of any preliminary elution step. Molecular detection and quantification of airborne viruses is performed using real-time quantitative (RT-PCR (Q(RT-PCR technique. The method has been tested using Adenoviruses (AdVs and Noroviruses (NoVs GII, as representative DNA and RNA viruses, respectively. Moreover, the method has been tested successfully in outdoor experiments, by detecting and quantifying human adenoviruses (HAdVs in the airborne environment of a wastewater treatment plant. Conclusions The great advantage of LMA is that nucleic acids extraction is performed directly on the LMA plates, while the eluted nucleic acids are totally free of inhibitory substances. Coupled with QPCR the whole procedure can be completed in less than three (3 hours.

  5. Strategies for Amplification of Trinucleotide Repeats: Optimization of Fragile X and Androgen Receptor PCR.

    Science.gov (United States)

    Papp; Snyder; Sedra; Guida; Prior

    1996-06-01

    Background: Trinucleotide repeat regions are heritable unstable elements that change in copy number from generation to generation. Amplification of these triplet repeats is an important diagnostic tool for molecular medicine. However, these repeats are often difficult to amplify and may require the use of different cosolvents or amplification strategies. Methods and Results: We used the fragile X and androgen receptor triplet repeat regions to demonstrate a series of conditions that may be used to optimize the amplification of repeat sequences. Conclusions: For androgen receptor, we show that predigestion of the template DNA was sufficient to generate consistent amplification. In the case of fragile X we found that predigestion, when combined with use of betaine as a destabilizing additive, was superior to other methods and yielded consistent amplification of normal and premutation alleles in both isotopic and nonisotopic reactions.

  6. Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

    DEFF Research Database (Denmark)

    Søndergaard, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer;

    commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied...... and robustness of a PCR assay, as some polymerases are more resistant to inhibitors, and thus be a simple strategy for assay optimization. Identifying an optimal polymerase can even render costly and time-consuming sample preparation unnecessary. The aim of this study was to evaluate the performance of 16...... different DNA extraction methods for Campylobacter. Results show that VeriQuest qPCR master mix have the best general performance, while the AmpliTaq Gold and HotMasterTaq DNA polymerases performed well with meat samples and poorly with fecal samples. Tth DNA polymerase performed well only with the purest...

  7. BLV-CoCoMo-qPCR-2: improvements to the BLV-CoCoMo-qPCR assay for bovine leukemia virus by reducing primer degeneracy and constructing an optimal standard curve.

    Science.gov (United States)

    Takeshima, Shin-nosuke; Kitamura-Muramatsu, Yuri; Yuan, Yuan; Polat, Meripet; Saito, Susumu; Aida, Yoko

    2015-05-01

    Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. Because BLV infection can remain clinically silent, the proviral load is an important index for estimating disease progression. CoCoMo-qPCR, an assay developed to estimate BLV proviral load, allows the highly sensitive detection of BLV originating in different countries. Here, we developed a modified version of the CoCoMo-qPCR assay, the "BLV-CoCoMo-qPCR-2" assay, which uses optimized degenerate primers. We also constructed a new plasmid standard. Finally, we used both assays to examine DNA samples from BLV-infected cattle and compared the results.

  8. End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: performance under optimal and suboptimal conditions.

    Science.gov (United States)

    Ramachandran, Sumathi; Xia, Guo-Liang; Ganova-Raeva, Lilia M; Nainan, Omana V; Khudyakov, Yury

    2008-08-01

    An approach for determination of hepatitis C virus (HCV) quasispecies by end-point limiting-dilution real-time PCR (EPLD-PCR) is described. It involves isolation of individual coexisting sequence variants of the hypervariable region 1 (HVR1) of the HCV genome from serum specimens using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak study provided insights into the epidemiological relationships between incident and chronic cases. When applied to samples from a longitudinal study of infected patients, HVR1 sequences from each sampling time-point were observed to group as distinct phylogenetic clusters. Melting peak analysis conducted on EPLD-PCR products generated from these patients could be used for evaluation of HVR1 sequence heterogeneity without recourse to clonal sequencing. Further, to better understand the mechanism of single-molecule PCR, experiments were conducted under optimal and suboptimal annealing temperatures. Under all temperature conditions tested, HVR1 variants from the major phylogenetic clusters of quasispecies could be amplified, revealing that successful HVR1 quasispecies analysis is not contingent to dilution of starting cDNA preparations to a single-molecule state. It was found that EPLD-PCR conducted at suboptimal annealing temperatures generated distributions of unique-sequence variants slightly different from the distribution obtained by PCR conducted at the optimal temperature. Hence, EPLD-PCR conditions can be manipulated to access different subpopulations of HCV HVR1 quasispecies, thus, improving the range of the quasispecies detection. Although EPLD-PCR conducted at different conditions detect slightly different quasispecies populations, as was shown in this study, the resulted samples of quasispecies are completely suitable for molecular epidemiological investigation in different clinical and epidemiological settings.

  9. Optimization and clinical validation of a Real-Time PCR protocol for direct detection of Trichomonas vaginalis in pooled urine samples

    Directory of Open Access Journals (Sweden)

    WHA Zandijk

    2009-12-01

    Full Text Available Background and Objectives: A new Real- Time PCR protocol for the detection of Trichomonas vaginalis in pooled urine"nsamples has been optimized and validated."nMaterials and Methods: The amplification protocol, targeting a 2kb repeated gene in the T. vaginalis genome, was optimized"nby varying PCR parameters. As a reference method, a Real-Time PCR protocol targeting the beta-tubulin gene (Y. Versluis"net al, 2006, Int J STD AIDS 17:642 was used. Clinical validation was performed with pooled urine samples obtained from"npatients of the sexually transmitted diseases clinic of a university hospital (n=963; from February – June 2007."nResults: Positive samples with the new optimized technique is 1.1% (n=10, while the beta-tubulin real-time PCR method"ngenerated four positives (0.3%."nConclusion: The new RT- PCR protocol is a sensitive (1.000 and specific (0.993 procedure to detect and to identify T."nvaginalis in urine samples.

  10. Establishment and optimization of ISSR-PCR and SRAP-PCR reaction systems of Thymus L.%百里香属植物ISSR-PCR和SRAP-PCR 体系的确立及优化

    Institute of Scientific and Technical Information of China (English)

    权俊萍; 袁菊红; 穆红梅; 张明霞; 李乃伟; 夏冰

    2008-01-01

    通过对 PCR 扩增体系中的模板 DNA 用量,Mg2+、dNTPs和引物浓度的单因子实验,分别建立了适合百里香属(Thymus L.)植物总 DNA 的 ISSR-PCR 及 SRAP-PCR 优化反应体系.ISSR-PCR 优化反应体系为:反应总体积20μL,含模板DNA 30 ng、Mg2+ 3.75 mmol·L-1、dNTPs 0.20 mmol·L-1、引物0.3 mmol·L-1和Taq DNA 聚合酶1 U.SRAP-PCR 优化反应体系为:反应总体积20μL,含模板DNA 30 ng、Mg2+ 2.50 mmol·L-1、dNTPs 0.10mmol·L-1、引物0.4 mmol·L-1和 Taq DNA 聚合酶l U.运用优化的反应体系对百里香属植物地椒(T. quinque-costatus Celak.)、地椒的亚洲变种[T. quinquecostatus var.asiaticus(Kitagawa)C.Y.Wu et Y.C.Huang]和百里香(T. mongolicus Ronn.)15个居群的总DNA进行了ISSR和SRAP初步分析,获得了较好的扩增结果.

  11. 研究土壤微生物多样性的PCR条件优化%Optimization of PCR Conditions for Soil Microbial Diversity Study

    Institute of Scientific and Technical Information of China (English)

    邸宁; 马焕普; 刘志民

    2012-01-01

    [Objective] The research aimed to optimize the test condition of PCR-DGGE method to analyze genetic diversity of soil microorganism. [Method] The amplified results of PCR were compared by improving high salt method for extracting soil DNA, improving primer design, changing the annealing temperature and amplification system of PCR reaction process. [Result] The result of extracting soil micro-bial DNA was better by high salt method which was improved. A single band was obtained and operation was easy when choosing 20 μl system for PCR amplification. There was no nonspecific amplification when choosing annealing temperature at 55 ℃, and cycle number of 35 was easy for following DGGE analysis. [Conclusion] The optimized PCR reaction system has high specificity and reliability.%[目的]对PCR-DGGE法的试验条件进行优化,以更好地分析土壤微生物遗传多样性。[方法]改良高盐法提取土壤DNA,改进引物设计,改变PCR反应过程中退火温度、扩增体系,比较PCR扩增结果。[结果]经过改良的高盐法提取的土壤微生物DNA效果更好。PCR扩增选择20μl体系条带单一,易于操作。退火温度选择在55℃时无非特异性扩增,35个循环次数易于后续DGGE分析。[结论]已经优化的PCR基因扩增体系特异性高且稳定可靠。

  12. Optimization of triplex real time PCR for detecting Staphylococcus aureus mecA, pvl and nuc genes.

    Science.gov (United States)

    Vremeră, Teodora; Iancu, Luminiţa Smaranda; Logigan, Cătălina; Năstase, Eduard; Miftode, Egidia; Luncă, Cătălina; Dorneanu, Olivia

    2011-01-01

    Multiplex polymerase chain reaction (PCR) allows simultaneous detection of two or more genes, using the same reaction conditions, and so it is possible the rapid detection of methicillin resistant Staphylococcus aureus strains (MRSA) in clinical specimens. This study aimed to implement, for the first time in our laboratory, a triplex real time PCR (RT-PCR) technique for detection of genes encoding resistance to oxacillin and synthesis of Panton Valentine leukocidin (pvl), a pathogenicity factor characteristic for community acquired strains (CA-MRSA). The application of this method will permit the epidemiological surveillance of circulating strains and early application of prevention measures.

  13. CAT基因突变热点区域的PCR扩增方法%Optimization of PCR Amplification Parameters for Mutational Hotspots in the Human Catalase Gene

    Institute of Scientific and Technical Information of China (English)

    李毅; 赵华; 赵红宇; 章锦才

    2009-01-01

    Objective To study the speciality and sensitivity of PCR for mutational hotspots in the human catalase gene. Methods Blood samples were taken from volunteers for genomic DNA preparation. Polymerase chain reaction (PCR) amplification of sixteen gene segments encompassing mutational hotspots in the human catalase was carried out. Touchdown and Hot-start PCR was imple-mented to improve the efficiency of gene amplification. Results High and constant amplification effi-ciency is obtained using touchdown and hot-start PCR. Conclusion The stable and reproducible PCR amplification parameters for mutational hotspots in the human catalase gene were optimized.%目的 探讨过氧化氢酶基因突变热点区域PCR扩增方法 ,提高PCR反应的特异性和灵敏度,有助于快速检测CAT基因相关疾病.方法 从人静脉血液标本提取人血液基因组DNA,设计引物扩增特定的CAT基因片段,联合应用热启动PCR和降落PCR技术.结果 建立了重复性好,分辨率高的PCR反应体系.结论 建立了适用于CAT基因突变热点区域的PCR反应体系,有助于快速检测CAT基因相关痰病.

  14. Cost-effective optimization of real-time PCR based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases

    DEFF Research Database (Denmark)

    Fachmann, Mette Sofie Rousing; Josefsen, Mathilde Hasseldam; Hoorfar, Jeffrey

    2015-01-01

    PCR master mix performed best for both meat and fecal samples (LODs of 102 and 104 CFU ml-1 in the purest and crudest DNA extractions, respectively) compared with Tth (LOD=102 -103 and 105 -106 CFU ml-1 ). AmpliTaqGold and HotMasterTaq both performed well (LOD=102 -104 CFU ml-1 ) with meat samples and poorly......AIMS: The aim of this study was to cost-effectively improve detection of foodborne pathogens in PCR inhibitory samples through the use of alternative DNA polymerases. METHODS AND RESULTS: Commercially available polymerases (n=16) and PCR master mixes (n=4) were screened on DNA purified from...... bacterial cells in two validated real-time PCR assays for Campylobacter and Salmonella. The five best performing (based on: limit of detection (LOD), maximum fluorescence, shape of amplification curves, and amplification efficiency) were subsequently applied to meat and fecal samples. The VeriQuest q...

  15. Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6).

    Science.gov (United States)

    Canto, Cynthia Liliane Motta do; Sumita, Laura Massami; Machado, Adriana Freire; Tateno, Adriana; Cunha, Eveline Vieira da; Machado, Clarisse Martins

    2008-01-01

    HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/microL (equivalent to 2465.8 molecules/microL) to 10-9 (equivalent to 2.46 molecules/microL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/microL and for the nested PCR was 2.46 molecules/microL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

  16. PCR反应体系的设计及优化%Design and Optimization of PCR Reaction System

    Institute of Scientific and Technical Information of China (English)

    纪朋艳

    2016-01-01

    PCR是分子生物学中常用的实验技术手段,在揭示生命现象规律方面发挥了至关重要的作用。目前在进行PCR实验操作时,因为PCR反应体系设计不合理而导致实验失败的事例较多。因此,从优化PCR反应体系的角度出发,对提高PCR反应效率进行探讨。%Polymerase Chain Reaction (PCR) technology is commonly used in molecular biology experiments, which played a vital role in terms of reveals life phenomenon law. Now in the PCR laboratory procedures, more and more PCR experiment were failure examples because the not reasonable design PCR reaction system. Therefore, this article discussed the perspective of the PCR reaction system to improve the efifciency of the PCR reaction.

  17. Otimização da técnica da PCR para a detecção de Actinobacillus pleuropneumoniae Optimization of PCR technique for detection of Actinobacillus pleuropneumoniae

    Directory of Open Access Journals (Sweden)

    Karina Koerich de Souza

    2008-11-01

    sensitivity when compared with traditional methods of bacteriological isolation, but they can suffer influence of substances that reduce the specificity of the test and resulting in inespecific amplifications. In order to reduce inespecific amplifications, observed when applied the PCR technique for the gene cpx in tonsil's tissue samples, the optimization was performed, in which different annealing temperatures were analyzed and introduced, in the technique, an antibody that binds to the enzyme Taq DNA Polimerase, increasing its specificity. In parallel, an experiment was performed in order to verify the inhibiting effect of the tonsil's tissue on the PCR results. For that, portions of tonsil from animals negative to the A. pleuropneumoniae were artificially contaminated with the reference sample of the sorotype 5B. The addition antibody for the enzyme Taq DNA Polimerase and the increase of the primers anneling temperature to 57°C reduced the inespecific amplifications. The results obtained in the experiment demonstrated a possible inhibiting effect of the tonsil's tissue in the PCR amplifications. Besides, amplifications depend on at least 675 UFC present in the aliquot of samples that will be used in PCR (equivalent to 1.35 x 10(5 UFC mL-1, therefore, samples tissue's fragments in initial infections and/or with few cells can result in false-negative.

  18. Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Krause, Michael; Hansen, F.

    2007-01-01

    no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mu l improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples...... the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 mu l were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 mu l) had...

  19. Sequence Optimized Real-Time RT-PCR Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus

    Science.gov (United States)

    2017-03-21

    density macroarrays [15], high density resequencing arrays [16], 55 padlock probes with colorimetric readout [17], LAMP [18], and polymerase chain reaction ...Announcement). Briefly, the S segment of each virus was amplified using the 83 SuperScript III One-Step RT- PCR system with Platinum Taq DNA Polymerase High...Platinum One-Step Quantitative RT- PCR System) # Rxns = Reagents Stock [Final] 1rxn 28 2X Reaction Mix 2 1 10 280 Nuclease-free water 1.7 47.6 100 µM F

  20. CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A.

    Directory of Open Access Journals (Sweden)

    Laetitia Fabre

    Full Text Available BACKGROUND: Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms. METHODOLOGY: Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats, as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers. PRINCIPAL FINDINGS: We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species. CONCLUSIONS: The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.

  1. A Novel Teaching-Learning-Based Optimization for Improved Mutagenic Primer Design in Mismatch PCR-RFLP SNP Genotyping.

    Science.gov (United States)

    Cheng, Yu-Huei

    2016-01-01

    Many single nucleotide polymorphisms (SNPs) for complex genetic diseases are genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in small-scale basic research studies. It is an essential work to design feasible PCR-RFLP primer pair and find out available restriction enzymes to recognize the target SNP for PCR experiments. However, many SNPs are incapable of performing PCR-RFLP makes SNP genotyping become unpractical. A genetic algorithm (GA) had been proposed for designing mutagenic primer and get available restriction enzymes, but it gives an unrefined solution in mutagenic primers. In order to improve the mutagenic primer design, we propose TLBOMPD (TLBO-based Mutagenic Primer Design) a novel computational intelligence-based method that uses the notion of "teaching and learning" to search for more feasible mutagenic primers and provide the latest available restriction enzymes. The original Wallace's formula for the calculation of melting temperature is maintained, and more accurate calculation formulas of GC-based melting temperature and thermodynamic melting temperature are introduced into the proposed method. Mutagenic matrix is also reserved to increase the efficiency of judging a hypothetical mutagenic primer if involve available restriction enzymes for recognizing the target SNP. Furthermore, the core of SNP-RFLPing version 2 is used to enhance the mining work for restriction enzymes based on the latest REBASE. Twenty-five SNPs with mismatch PCR-RFLP screened from 288 SNPs in human SLC6A4 gene are used to appraise the TLBOMPD. Also, the computational results are compared with those of the GAMPD. In the future, the usage of the mutagenic primers in the wet lab needs to been validated carefully to increase the reliability of the method. The TLBOMPD is implemented in JAVA and it is freely available at http://tlbompd.googlecode.com/.

  2. Optimization of ISSR-PCR Reaction System on Ranunculus nephelogenes var. nephelogenes and Primer Selection%云生毛茛 ISSR-PCR 体系优化与引物筛选

    Institute of Scientific and Technical Information of China (English)

    石琳; 胡延萍; 王建科; 王钧; 许小宁; 李毅; 王莉

    2016-01-01

    This work is aimed to establish a stable ISSR-PCR system for Ranunculus nephelogenes var. nephelogenes. A L16(45) orthogonal design was used to screen 5 main parameters(Mg2+,dNTP,Taq DNA polymerase,primer,and template DNA);then the process of ISSR-PCR reaction was optimized,and consequently the optimal reaction system and amplifying procedure for R. nephelogenes var. nephelogenes was established ;further,the optimized reaction system and amplifying procedure was verified. Results showed that the optimal concentrations in 20 μL reaction mixture were template DNA 30 ng,Mg2+ 1.95 mmol/L,Taq DNA polymerase 0.04 U/μL,dNTP 0.150 mmol/L, and primer 0.5 μmol/L. The optimal reaction procedure was :pre-denaturalization for 5 mins at 94℃,denaturalization for 20 s at 94℃, renaturation for 1 min at 49.6-60.6℃,followed by 38 cycles of extension for 100 s at 72℃,and final extension for 6 min at 72℃. Under the above optimized reaction system and procedure,16 amplified primers were screened from 100 ISSR primers,and the optimal annealing temperature for each primer was determined. Verification in R. nephelogenes var. nephelogenes of different populations with the optimized ISSR system confirmed that bands amplified were clear and steady,thus the established ISSR-PCR could favor further studies on the genetic diversity of R. nephelogenes var. nephelogenes.%旨在建立稳定可靠的云生毛茛 ISSR-PCR 反应体系。采用正交试验设计方法,对影响云生毛茛 ISSR-PCR 扩增结果的 Mg2+、dNTP、Taq DNA 聚合酶、引物、模板 DNA 五个因素进行优化筛选,对反应程序进行优化,建立适用于云生毛茛的最佳反应体系和扩增程序,并对反应体系和扩增程序进行验证;在此基础上筛选多态性好的 ISSR 引物,采用梯度法筛选各个引物的最适退火温度。结果表明,云生毛茛20μL ISSR-PCR 的最佳反应体系为:模板 DNA 30 ng,Mg2+1.95 mmol/L,Taq DNA 聚合酶0.04 U/μL,dNTP 0

  3. Condition optimization and primer screening for ISSR-PCR of Demodex%蠕形螨ISSR-PCR的反应体系优化及引物筛选

    Institute of Scientific and Technical Information of China (English)

    刘艳荣; 刘付红; 肖克源; 郭淑玲; 冯玉新; 袁方曙

    2012-01-01

    目的 以蠕形螨的DNA为模板,对蠕形螨简单重复序列锚定PCR (ISSR-PCR)反应体系优化并对ISSR引物进行筛选,为ISSR分析奠定基础.方法 用自然沉降法收集山羊蠕形螨,用基因组DNA提取试剂盒提取山羊蠕形螨DNA,并以此为模板,以(CA)8RG(R为1分子的嘧啶)为引物,利用正交实验法,从Mg2+浓度、引物用量、dNTPs、DNA模板用量和TaqDNA聚合酶以及退火温度对蠕形螨ISSR-PCR反应体系进行优化,并以优化的反应体系筛选ISSR-PCR的引物.结果 ISSR-PCR 25 μL最佳反应体系包括:0.4 mmol/L Mg2+、30 pmol引物、30~60 ng模板DNA、2u Taq酶,最佳退火温度为49℃、循环35次.筛选出10条条带清晰、多态性好、重复性高的引物.结论 筛选出蠕形螨ISSR-PCR最佳反应体系和理想的引物,为蠕形螨的分子生物学研究奠定了基础.%Objective To optimize conditions and screening for primers for inter-simple sequence repeat-anchored (IS-SR-PCR) of Demodex, using genome DNA as the template, preparing for the genetic diversity research of Demodex with ISSR mark. Methods After mites were rinsed and precipitated in double distilled water (DDW), the genome DNA of Demodex was extracted with a kit. Then experiments were carried out using DNA of Demodex as the template, and (CA) 8 RG as the primer. The orthogonal array was conducted, and the involved factors were as follows: concentrations of Mg2+ , dNTPs, primers, template DNA and Taq DNA polymerase and return temperature. An optimal reaction system of ISSR-PCR of Demodex was established, through which an optimal primer was obtained. Results An optimal 25 (xL of ISSR-PCR amplification system contained 2.0 mmol/L of Mg2+ , 0. 4 mmol/L of dNTPs, 30-60 ng of template DNA, 30 pmol of primers, and 2U of Taq DNA polymerase. The optional return temperature for the primer was 49 t, and the cycle index was 35. 10 primers were selected, with clear bands, good polymorphism and high repetition. Conclusion

  4. Establishment and Optimization of RSAP-PCR Reaction Conditions in Pleurotus ostreatus%侧耳RSAP-PCR体系的建立和优化

    Institute of Scientific and Technical Information of China (English)

    潘祖桐; 宋慧; 刘麒; 李胜南; 李媛媛; 刘晓龙

    2013-01-01

    In this study, single factor and orthogonal experiments were used to establish and optimize the RSAP - PCR reaction system of Pleurotus ostreatus. Then, experiment tests were done to check the stability of the RSAP - PCR reaction system using a different pair of primers. Finally, a 25 μL optimized reaction system was obtained: 30 ng DNA, 2.0 μL 10 × PCR buffer, Mg2+ 2.5 mmol/L, dNTPs 0.25 mmol/L, Primers 0.32 μmol/L, Taq DNA polymerase 2 U, ddH2O 13.2 μL.%采用单因素试验和正交设计相结合进行了侧耳(Pleurotus ostreatus)RSAP-PCR反应体系的建立和优化,并对优化后体系的稳定性进行了验证.讨论了Mg2+浓度、dNTPs浓度、引物浓度、Taq聚合酶用量和模板DNA含量对侧耳RSAP-PCR反应的影响,最终得出总体积为25μL的反应体系:30 ng模板DNA,2.0 μL 10倍PCR buffer,Mg2+ 2.5 mmol/L,dNTPs o.25 mmol/L,引物0.32 μmol/L,Taq DNA聚合酶2U,ddH2O 13.2 μL.

  5. Development and Optimization of Multiplex PCR-Based Marker of Ms Locus in Onion%洋葱 Ms 位点多重 PCR 标记的开发与优化

    Institute of Scientific and Technical Information of China (English)

    王振宝; 吴雄; 霍雨猛; 刘冰江; 缪军; 杨妍妍; 高莉敏; 孔素萍; 程斐; 陈宁

    2014-01-01

    以本课题组已获得的洋葱Ms位点侧翼序列为基础,设计、筛选引物获得了一个兼容的多重PCR分子标记,并对其反应体系和反应程序进行了优化。优化后的扩增体系:10×PCR buffer (Mg2+free)2.5μL、25 mmol/L MgCl24μL、2.5 mmol/L dNTP 6μL、DNA模板1μL (约50 ng)、10μmol/L引物各1μL、5 U/μL rTaq聚合酶0.6μL、用灭菌双蒸水补齐至25μL;反应程序:94℃预变性5 min;94℃变性30 s,65.4℃退火45 s,72℃延伸1 min,35个循环;最后72℃延伸10 min。优化后的体系和程序可以检测到清晰的目的条带,通过一次PCR反应即可鉴定Ms位点的3种基因型( MsMs、Msms、msms),操作简单,稳定性好。%Through designing and screening primers on the basis of flanking sequence of Ms locus, a compatible multiplex PCR-based marker ( MK4 ) was obtained , and its reaction system and procedure were optimized.The optimal PCR reaction system consisted of 2.5μL of 10 ×PCR buffer (Mg2+free), 4μL of 25 mmol/L MgCl2, 6 μL of 2.5 mmol/L dNTP, 1 μL of DNA template (about 50 ng), 1 μL of each of 10 mmol/L primer, 0.6μL of 5 U/μL rTaq DNA polymerase , and finally adding ddH 2 O to 25μL.The PCR re-action procedure was initial denaturing at 94℃ for 5 min, followed by 35 cycles of denaturing at 94℃ for 30 s, annealing at 65.4℃for 45 s, extending at 72℃for 1 min, and then extending at 72℃for 10 min in the end.Three genotypes ( MsMs, Msms, msms) of Ms locus could be clearly distinguished by MK 4 based on the optimal reaction conditions and procedure .The reaction was simple and had good stability .

  6. Touchdown General Primer (GP5+/GP6+ PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

    Directory of Open Access Journals (Sweden)

    Simmons-Arnold Linda

    2005-11-01

    Full Text Available Abstract Background The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. Methods Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng–100 ng diluted in a background of C-33A DNA (100 ng-2 μg. Secondly, the detection of small quantities (15ag-1.5pg of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56 diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. Results HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 μg or 1 μg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (~28 copies HPV-16 in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 μg or 1 μg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA than by a touchdown approach (15fg detection limit. HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72% abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97% vaginal lesions. Multiple infections were also detectable using a touchdown

  7. Qualitative and event-specific real-time PCR detection methods for Bt brinjal event EE-1.

    Science.gov (United States)

    Randhawa, Gurinder Jit; Sharma, Ruchi; Singh, Monika

    2012-01-01

    Bt brinjal event EE-1 with cry1Ac gene, expressing insecticidal protein against fruit and shoot borer, is the first genetically modified food crop in the pipeline for commercialization in India. Qualitative polymerase chain reaction (PCR) along with event-specific conventional as well as real-time PCR methods to characterize the event EE-1 is reported. A multiplex (pentaplex) PCR system simultaneously amplifying cry1Ac transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthase (nos) terminator, aminoglycoside adenyltransferase (aadA) marker gene, and a taxon-specific beta-fructosidase gene in event EE-1 has been developed. Furthermore, construct-specific PCR, targeting the approximate 1.8 kb region of inserted gene construct comprising the region of CaMV 35S promoter and cry1Ac gene has also been developed. The LOD of developed EE-1 specific conventional PCR assay is 0.01%. The method performance of the reported real-time PCR assay was consistent with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with the LOD and LOQ values of 0.05%. The developed detection methods would not only facilitate effective regulatory compliance for identification of genetic traits, risk assessment, management, and postrelease monitoring, but also address consumer concerns and resolution of legal disputes.

  8. Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize.

    Science.gov (United States)

    Félix-Urquídez, Dalmira; Pérez-Urquiza, Melina; Valdez Torres, José-Benigno; León-Félix, Josefina; García-Estrada, Raymundo; Acatzi-Silva, Abraham

    2016-01-05

    Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%-100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio.

  9. 水蛭ISSR-PCR反应体系的建立及优化%Establishment and optimization of ISSR-PCR reaction system for Hirudo nipponica

    Institute of Scientific and Technical Information of China (English)

    张香东; 刘庚; 常素慧; 白秀娟; 杨洪雁; 朱洪强

    2013-01-01

    Objective To establish and optimize the ISSR-PCR reaction system and amplification procedure for Hirudo nipponica and to provide the basis for its genetic diversity research. Methods Using Primer ISSR825, the orthogonal test design was adopted to optimize the ISSR-PCR amplification system on H. nipponica in five factors (Mg2+, dNTP, primer, Taq DNA polymerase, and template DNA) at four levels, and the suitable anneal temperature of the primer was selected. Results The suitable ISSR-PCR system (25 μL reaction volume) in H. nipponica included 10×PCR buffer (2.5 μL), Mg2+ (2.0 mmol/L), dNTP (0.25 mmol/L), primer (0.8 μmol/L), Taq DNA polymerase (2.5 U), and template DNA (2.0 ng/μL); The suitable anneal temperature was 49.7 ℃. Conclusion The established and optimized ISSR reaction system is stable and reliable, which could provide the basis for the analysis of genetic diversity, germplasm resources, and genetic relationship of H. nipponica.%目的 建立并优化水蛭ISSR-PCR反应体系及扩增程序,为水蛭进行遗传多样性研究提供依据.方法 使用引物ISSR825,采用正交优化方法对影响PCR反应的Mg2+、dNTP、引物、TaqDNA聚合酶、模板DNA进行了体系优化,同时对引物的最佳退火温度进行了选择.结果 在25 μL总体积中,其中包括10×PCR缓冲液2.5 μL、Mg2+ 2.0 mmol/L、dNTP0.25 mmol/L、引物0.8 μmol/L、Taq DNA聚合酶2.5 U、模板DNA 2.0 ng/μL,最佳退火温度为49.7℃.结论 所建立的最佳ISSR-PCR反应体系稳定可靠,可用于水蛭遗传多样性评价、不同种源鉴定及亲缘关系分析.

  10. Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity

    Science.gov (United States)

    Li, B; Matter, EK; Hoppert, HT; Seeley, RJ; Sandoval, DA

    2014-01-01

    BACKGROUND Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular endpoints. Real time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to be overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs. re-fed and chow vs. high fat diet (HFD) conditions. DESIGN Male Long-Evans rats were treated under four conditions, chow/fasted, chow/re-fed, HFD/fasted, and HFD/re-fed. Expression stabilities of the13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum, and ileum using ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. RESULTS Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. Unique subsets of reference genes out of the 13 candidate genes were identified, each of which is specific for one type of rat tissue (hypothalamus, duodenum, jejunum, or ileum) under a different combination of diet and feeding condition. CONCLUSIONS Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB, or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues play an integral role in regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene

  11. Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity.

    Science.gov (United States)

    Li, B; Matter, E K; Hoppert, H T; Grayson, B E; Seeley, R J; Sandoval, D A

    2014-02-01

    Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions. Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered. Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression

  12. 松阿扁叶蜂SSR-PCR反应体系的优化%Optimization of SSR-PCR System for Acantholyda posticalis

    Institute of Scientific and Technical Information of China (English)

    金娜; 南小宁; 贺虹

    2012-01-01

    以SDS-蛋白酶K法提取的松阿扁叶蜂(Acantholyda posticalis Matsumura)基因组DNA为模板,利用L16(45)正交设计对影响松阿扁叶蜂SSR-PCR反应的主要参数DNA模板、Taq DNA聚合酶、Mg2+、引物和dNTPs进行优化.结果表明,松阿扁叶蜂SSR-PCR最优的反应体系为25 μL体系中含1.00U Taq DNA 聚合酶、3.00 mmol/L Mg2+、3.75 mmol/L dNTPs、25.00 ng/μL DNA模板和10.00 μmol/L引物.%In order to establish the SSR-PCR amplification system using genomic DNA of Acantholyda posticalis which was extracted by SDS-proteinase K method as template, orthogonal design was used to optimize main PCR factors such as template DNA, Taq DNA polymerase, Mg2+, primer and dNTPs. The results showed that the optimized PCR system included 1.00 U Taq DNA polymerase, 3.00 mmol/L Mg2+, 3.75 mmol/L dNTPs, 25.00 ng/μL DNA template and 20.00 μmol/L each primer in the total volume 25 μL.

  13. Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus.

    Science.gov (United States)

    Abarshi, M M; Mohammed, I U; Wasswa, P; Hillocks, R J; Holt, J; Legg, J P; Seal, S E; Maruthi, M N

    2010-02-01

    Sampling procedures and diagnostic protocols were optimized for accurate diagnosis of Cassava brown streak virus (CBSV) (genus Ipomovirus, family Potyviridae). A cetyl trimethyl ammonium bromide (CTAB) method was optimized for sample preparation from infected cassava plants and compared with the RNeasy plant mini kit (Qiagen) for sensitivity, reproducibility and costs. CBSV was detectable readily in total RNAs extracted using either method. The major difference between the two methods was in the cost of consumables, with the CTAB 10x cheaper (0.53 pounds sterling=US$0.80 per sample) than the RNeasy method (5.91 pounds sterling=US$8.86 per sample). A two-step RT-PCR (1.34 pounds sterling=US$2.01 per sample), although less sensitive, was at least 3-times cheaper than a one-step RT-PCR (4.48 pounds sterling=US$6.72). The two RT-PCR tests revealed consistently the presence of CBSV both in symptomatic and asymptomatic leaves and indicated that asymptomatic leaves can be used reliably for virus diagnosis. Depending on the accuracy required, sampling 100-400 plants per field is an appropriate recommendation for CBSD diagnosis, giving a 99.9% probability of detecting a disease incidence of 6.7-1.7%, respectively. CBSV was detected at 10(-4)-fold dilutions in composite sampling, indicating that the most efficient way to index many samples for CBSV will be to screen pooled samples. The diagnostic protocols described below are reliable and the most cost-effective methods available currently for detecting CBSV.

  14. 甘草RAPD-PCR反应体系正交优化研究%Optimization of RAPD-PCR Reaction System for Glycyrrhiza uralensis Based on Orthogonal Design

    Institute of Scientific and Technical Information of China (English)

    张增福; 董建力; 李明

    2011-01-01

    [Objective] The aim was to obtain the optimum RAPD-PCR reaction system for Glycyrrhiza uralensis. [ Method] Orthogonal design was adopted to screen the suitable concentration of major factors (dNTPs, primer, Taq polymerase, DNA template) in PCR reaction system. [ Result] The optimal protocol was accomplished by orthogonal design in 25 μl reaction volume containing 10 × PCR buffer solution(include MgCl2) 2.5 μl, 10 mmol/L dNTPs 2.5 μl, 50 ng DNA template 2.0 μl, 10 μmol/L primer 2. 0 μl, 5 U/μl Taq polymerase 0.4 μl; the optimum annealing temperature was 34 ℃. [ Conclusion] Orthogonal design was an effective method for the optimization of RAPD-PCR reaction system for C. Uralensis.%[目的]建立一套适合甘草分子学研究的RAPD-PCR反应体系.[方法]以甘草种质为试材,采用正交试验法设计,对影响RAPD-PCR扩增的主要因素dNTPs、引物、Taq酶和DNA模板进行优化筛选.[结果]总体积25μl的甘草RAPD-PCR最佳反应体系为:10 ×PCR缓冲液(含MgCl2 )2.5 μl,10 mmol/L dNTPs 2.5 μl,50 ng DNA 2.0μl,10 μmol/L引物2.0μl,5 U/μl Taq酶0.4μl.对引物的退火温度进行了梯度筛选,34℃时扩增效果较好.[结论]进行甘草RAPD-PCR反应体系的正交优化非常有效.

  15. Development and Optimization of a Fluorescent Differential Display PCR System for Analyzing the Stress Response in Lactobacillus sakei Strains

    Directory of Open Access Journals (Sweden)

    Giovanni Salzano

    2009-11-01

    Full Text Available Lactobacillus sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors. We studied the differential expression of genome in response to different stresses by the fluorescent differential display (FDD technique. This study resulted in the development and optimization of an innovative technique, with a high level of reproducibility and quality, which allows the identification of gene expression changes associated with different microbial behaviours under different growth conditions.

  16. Optimization and head-to-head comparison of MISSR-PCR, ERIC-PCR, RAPD and 16S rRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China.

    Science.gov (United States)

    Mo, Q H; Wang, H B; Tan, H; An, S L; Feng, Z L; Wang, Q; Lin, J C; Yang, Z

    2015-01-01

    To establish a new genotyping method for Vibrio cholerae and compare it with other methods. In the current study, a modified inter simple sequence repeat-polymerase chain reaction (MISSR-PCR) system was developed via several rounds of optimisation. Comparison study was then conducted between MISSR-PCR and three other methods, including enterobacterial repetitive intergenic consensus sequences-based PCR (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) and 16S rRNA evolutionary clock, for the detection and genetic tracing of Vibrio cholerae isolated from seafood in China. The results indicated that the MISSR-PCR system could generate the highest polymorphic fingerprinting map in a single round PCR and showed the best discriminatory ability for Vibrio cholerae genotyping by clearly separating toxigenic/nontoxigenic strains, local/foreign strains, and O1/O139/non-O1/non-O139 serogroup strains, comparing to ERIC-PCR, RAPD and 16S rRNA evolutionary clock. Moreover, the MISSR-PCR is superior to previously described traditional simple sequence repeat based PCR method on genotyping by more clearly separating different clusters. To the best of our knowledge, this is the first head-to-head comparison of four detection and genotyping methods for Vibrio cholerae The MISSR-PCR system established here could serve as a simple, quick, reliable and cost-effective tool for the genotyping and epidemiological study.

  17. Optimization and head-to-head comparison of MISSR-PCR, ERIC-PCR, RAPD and 16S rRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China

    Directory of Open Access Journals (Sweden)

    Q H Mo

    2015-01-01

    Full Text Available Purpose: To establish a new genotyping method for Vibrio cholerae and compare it with other methods. Materials and Methods: In the current study, a modified inter simple sequence repeat-polymerase chain reaction (MISSR-PCR system was developed via several rounds of optimisation. Comparison study was then conducted between MISSR-PCR and three other methods, including enterobacterial repetitive intergenic consensus sequences-based PCR (ERIC-PCR, randomly amplified polymorphic DNA (RAPD and 16S rRNA evolutionary clock, for the detection and genetic tracing of Vibrio cholerae isolated from seafood in China. Result: The results indicated that the MISSR-PCR system could generate the highest polymorphic fingerprinting map in a single round PCR and showed the best discriminatory ability for Vibrio cholerae genotyping by clearly separating toxigenic/nontoxigenic strains, local/foreign strains, and O1/O139/non-O1/non-O139 serogroup strains, comparing to ERIC-PCR, RAPD and 16S rRNA evolutionary clock. Moreover, the MISSR-PCR is superior to previously described traditional simple sequence repeat based PCR method on genotyping by more clearly separating different clusters. Conclusion: To the best of our knowledge, this is the first head-to-head comparison of four detection and genotyping methods for Vibrio cholerae The MISSR-PCR system established here could serve as a simple, quick, reliable and cost-effective tool for the genotyping and epidemiological study.

  18. 万寿菊SS-PCR反应体系的优化%Optimization in SSR-PCR Reaction System of Marigold

    Institute of Scientific and Technical Information of China (English)

    杨帆; 曾丽; 赵子刚; 张嫔; 龚霄雯

    2011-01-01

    实验材料为色素万寿菊雄性不育两用系'9901AB',通过对SSR-PCR反应体系中的主要因素dNTPs、引物及模板DNA进行最优条件筛选,建立了适合于万寿菊雄性不育两用系的SSRPCR反应体系:2.5μL 10×buffer、40 ng模板DNA、2μL 10μmol·μL1引物、1.0 U Taq酶、1.2μL2.5 mmol·μL-1dNTP,ddH2()补足体系至25μL;P(R循环程序为:94℃预变性2 min;38个循环(94℃变性30 s、56℃退火30 s、72℃延伸1 min),72℃延伸10 min.采用优化后的SSR-PCR体系,将已公布的向日葵15对引物运用于万寿菊雄性不育两用系材料上,结果表明万寿菊与向日葵之间可以共享部分SSR引物信息.%The experimental material is marigold yellow pigment male sterile line '9901AB'. The SSR-PCR was established within 25 μL reaction volumes containing 2. 5 μL 10 × PCR amplification buffer, 40 ng template DNA,2 μL 10 μmol · μL-1 primers, 1.0 U Taq DNA polymerase, 1.2 μL 2. 5 mmol ·μL-1 dNTP and made to volume with sterile double-distilled water by screening the optimal main factors that dNTP,primers and template DNA in SSR-PCR reaction system. The SSR-PCR was built using the conditions: 94 ℃ for 2 min; 38 cycles of 94 ℃ for 30 s,56 ℃ for 30 s,72 ℃ for 1 min; 72 ℃ for 10 min. Results show that some of the SSR primers developed from sunflower (Helianthus annuus ) could be directly used in marigold genome study.

  19. Optimization of DNA isolation and PCR protocol for analysis and evaluation of genetic diversity of the medicinal plant, Anemopsis californica using RAPD.

    Science.gov (United States)

    Lizette Del-Toro-Sánchez, C; Villaseñor-Alvarado, S; Zurita-Martínez, Florentina; Castellanos-Hernández, O A; Rodríguez-Sahagún, Araceli; Isabel Torres-Morán, M; Rojas-Bravo, D; Gutiérrez-Lomelí, M

    2013-06-01

    Anemopsis californica is a perennial herbaceous plant that has been utilized as a medicinal plant for the treatment of various diseases. The present work was carried out with the objective of optimizing a method of extraction of the genomic DNA of A. californica and a PCR protocol and later to evaluate the existing genetic diversity among the genotypes deriving from different origins. For DNA extraction, we tested four procedures: with the CTA B-2 protocol, we obtained the highest yield (61.5±2.2 μg DNA/g of leaf tissues) and the best quality (A260/280 1.83±0.022). To estimate genetic variability, we utilized the randomly amplified polymorphism DNA (RAPD) technique, employing 20 oligonucleotides, of which only 18 generated reproducible banding patterns, producing 123 polymorphic bands generated, thus obtaining a polymorphism rate of 93.93% among the genotypes analyzed. The Jaccard similarity coefficient generated a variation ranging from 0.325-0.921, indicating a high level of genetic variation among the studied genotypes. An Unweighted pair-group method with arithmetic mean (UPGMA) group analysis indicated six distinct groups. The present optimized method for DNA isolation and RAPD protocol may serve as an efficient tool for further molecular studies.

  20. Evaluation and subsequent optimizations of the quantitative AmpliSens Florocenosis/Bacterial vaginosis-FRT multiplex real-time PCR assay for diagnosis of bacterial vaginosis.

    Science.gov (United States)

    Rumyantseva, Tatiana; Shipitsyna, Elena; Guschin, Alexander; Unemo, Magnus

    2016-12-01

    Traditional microscopy-based methods for diagnosis of bacterial vaginosis (BV) are underutilized in many settings, and molecular techniques may provide opportunities for rapid, objective, and accurate BV diagnosis. This study evaluated the quantitative AmpliSens Florocenosis/Bacterial vaginosis-FRT multiplex real-time PCR (Florocenosis-BV) assay. Vaginal samples from a previous study including unselected female subjects (n = 163) and using Amsel criteria and 454 pyrosequencing for BV diagnosis were examined with the Florocenosis-BV test and additionally tested for the presence and quantity of Gardnerella vaginalis clades 3 and 4. The Florocenosis-BV assay demonstrated 100% and 98% sensitivity compared with the Amsel criteria and 454 pyrosequencing, respectively, with 91% specificity. The modified Florocenosis-BV assay (detecting also G. vaginalis clades 3 and 4) resulted in 100% sensitivity vs the Amsel criteria and 454 pyrosequencing with specificity of 86% and 88%, respectively. Further optimizations of thresholds for the quantitative parameters used in the kit resulted in 99-100% accuracy vs Amsel criteria and 454 pyrosequencing for selected parameters. The Florocenosis-BV assay is an objective, accurate, sensitive, and specific method for BV diagnosis; however, the performance of the test can be further improved with some minor optimizations. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  1. Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products.

    Science.gov (United States)

    Hu, B; Sellers, J; Kupec, J; Ngo, W; Fenton, S; Yang, T-Y; Grebanier, A

    2014-01-01

    Host cell DNA contamination occurs during the production of biopharmaceuticals and must be controlled and monitored for the purity and safety of the drug products. A sodium iodide-based DNA extraction and a subsequent real time PCR assay were developed and validated for the quantitative measurement of residual host cell DNA impurity in monoclonal antibody therapeutic products. A sodium iodide-based commercial kit was optimized for the removal of interfering matrices. Several incubation steps from the kit protocol were combined and a neutralization buffer was introduced to protein digestion step to eliminate any precipitation from the detergent. The elimination of the two washing steps significantly reduced assay variability from loss of DNA pellets. The optimized DNA extraction procedure can recover DNA close to 100% for DNA concentrations from 10 to 100,000pg/mL. Of the published sequences of repetitive interspersed nuclear elements, we identified a nucleotide mismatch from the published CHO probe. Correction of this nucleotide increased DNA amplification by a thousand fold. The optimized assay was further validated for the quantitation of residual CHO DNA according to ICH guidelines with preset assay acceptance criteria. The method met all assay acceptance criteria and was found linear, accurate and precise for the quantitation of residual CHO in the linear range of 10-100,000pg DNA/mL. LOQ was measured at 10pg DNA/mL and LOD at 1pg DNA/mL. No matrix interference to our validated assay was detected from bioreactor harvest, Protein A eluate or eluate from ion exchange columns.

  2. Optimization of CAPS -PCR Reaction System in Processing Tomato%加工番茄CAPS遗传标记反应体系的优化

    Institute of Scientific and Technical Information of China (English)

    王柏柯; 帕提古丽; 杨生保; 杨涛; 张贵仁; 余庆辉

    2011-01-01

    [目的]以加工番茄M82、IL7 1-5为材料,研究加工番茄CAPS分析中PCR反应体系的主要成分对CAPS扩增结果的影响.[方法]以公开发表并检验稳定的CAPS标记c2_at5g20180为引物,PCR反应采用25 μL反应体积,含模板DNA 100 ng,2.5 UTaq DNA聚合酶,1.5 mmol/L MgCl2,0.2 mmool/L dNTPs,0.15μmmol/L引物.在保持其它因素一致的条件下,通过5个梯度,变化单一因子,筛选最优参数.反应程序为:94℃预变性3 min;94℃变性40 s,55℃退火40 s,72℃延伸90 s,共37个循环,最后72℃延伸10 min,4℃保存产物.[结果]通过体系优化,成功扩增出清晰稳定的PCR产物.[结论]在总体积为25 μL的PCR反应中,含50 ng模板DNA,0.75 UTaq DNA聚合酶,Mg2+终浓度为1.5 mmol/L,dNTP浓度为0.16 mmol/L,引物浓度为0.2 μmol/L时效果最好.%[ Objective]The purpose of this project was to study the the effects of major elements in PCR reaction system on CAPS by using tomato species M82 and IL7 -5 as the test materials. [ Method ] This study took C2_at5g20180 as the primer, whose sequence was openly published and examined to be steady. The PCR Reaction System was 25 (xL, including 100 ng DNA template, 2.5 U DNA Polymerases, 1. 5 mmol/L Mg2+ , 0.2 mmol/L dNTPs, 0.15 ujnmol/L primer. The effects of major amplification reaction components in PCR reaction system on CAPS analysis were studied. When keeping other factors consistent, through five gradients, the study changed the single factor, and screened the optimal parameters. Response procedures were; 941 pre degeneration 3minutes, then 941 degeneration 40 seconds, 55°C annealing 40 seconds, 72°C extend 90 seconds, 37 times cycle, the last 72°C extend 10 minutes, and at 4°C to save the product. [Result] Through the system optimization, the clear stability production of PCR was amplificated. [Conclusion] The results showed that in PRC reaction in total volume of 25 jxL, including50 ng template of DNA and 0. 75 U Taq DNA polymerase, Mg2

  3. Optimization and Validation of Real Time PCR Assays for Absolute Quantification of toxigenic Vibrio cholerae and Escherichia coli

    DEFF Research Database (Denmark)

    Ferdous, J.; Hossain, Z. Z.; Tulsiani, S.

    2016-01-01

    Quantitative real-time PCR (qPCR) is a dynamic and cogent assay for the detection and quantification of specified nucleic acid sequences and is more accurate compared to both traditional culture based techniques and ‘end point’ conventional PCR. Serial dilution of bacterial cell culture provides ...

  4. Optimization and Validation of Real Time PCR Assays for Absolute Quantification of toxigenic Vibrio cholerae and Escherichia coli

    DEFF Research Database (Denmark)

    Ferdous, J.; Hossain, Z. Z.; Tulsiani, S.

    2016-01-01

    Quantitative real-time PCR (qPCR) is a dynamic and cogent assay for the detection and quantification of specified nucleic acid sequences and is more accurate compared to both traditional culture based techniques and ‘end point’ conventional PCR. Serial dilution of bacterial cell culture provides ...

  5. 怀地黄ISSR扩增条件优化的研究%Optimization of ISSR-PCR amplification in Huai Rehmannia glutinosa

    Institute of Scientific and Technical Information of China (English)

    周延清; 景建洲; 李振勇; 浩健; 贾敬芬

    2004-01-01

    Huai Rehmannia glutinosa Libosch. genomic DNA,extracted from its fresh young leaves by CTAB method,was used as ISSR-PCR amplification template.The effects of main elements,i.e.temperature,Taq polymerase dosage,dNTP concentration,Mg2+ concentration,primer concentration and template DNA concentration on ISSR-PCR amplication were tested by single factor experiment,respectively,to determine its optimal levels and establish the following optimal reaction system for ISSR analysis in Huai Rehmannia glutinosa Libosch.:PCR reaction volume of 25 μL,1.5~1.0 U Taq DNA polymerase,3.0 mmol/L Mg2+,1 × Taq DNA polymerase buffer (10 mmol/L Tris-HCl,50 mmol/L KCl,0.1% Trion X-100,pH9.0 ),60 ng template DNA,0.4 μmmol/L primer,0.4 mmol/L each of dATP,dGTP,dCTP and dTTP.Proper annealing temperature was 53~55℃.This has laid the good foundation for ISSR analysis under way in Huai Rehmannia glutinosa Libosch..%用CTAB法提取怀地黄嫩叶DNA,进行简单重复间序列标记(ISSR)分析.通过单因子实验分别研究了退火温度、Taq酶单位、Mg2+浓度、dNTP浓度、引物浓度和模板DNA浓度对ISSR-PCR反应的影响,找出各自的合适条件,而且每一个合适条件确定以后都被作为后续研究的一个条件.通过各个因子的组合研究建立了适宜于怀地黄ISSR分析的扩增体系:25 μL PCR反应体积,1×Taq DNA酶缓冲液(10 mmol/L Tris-HCl,50 mmol/L KCl,0.1% Trion X-100,pH9.0 ),2.5 mmol/L MgCl2,1.5~1.0 U Taq酶,60 ng模板DNA,0.4 μmmol/L引物,各0.4 mmol/L的dATP、dGTP、dCTP和dTTP.合适的退火温度为53~55℃.为用ISSR技术分析鉴定怀地黄种质资源奠定了良好的基础.

  6. Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Reis Patricia P

    2010-06-01

    Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

  7. CODEHOP PCR and CODEHOP PCR primer design.

    Science.gov (United States)

    Staheli, Jeannette P; Boyce, Richard; Kovarik, Dina; Rose, Timothy M

    2011-01-01

    workflow of a typical CODEHOP PCR assay design and optimization and give a specific implementation example along with "best-practice" advice.

  8. The Study on Optimization of Arbitrary-primer PCR Reaction System in Eucalypt%桉树随机引物PCR反应体系优化研究

    Institute of Scientific and Technical Information of China (English)

    邓紫宇; 张照远; 项东云; 唐庆兰; 陈健波; 卢翠香; 叶露

    2012-01-01

    Using DNA of Eucalypt as template,the effect of dNTP concentration,Mg2+ concentration and annealing temperature on the response of Arbitrary-primer PCR was examined by applying single factor experimenting method.The results demonstrated that the optimal condition of reaction for Eucalypt was 2.0 μL Buffer(10 x),1.28 μL Mg2+(25 mmol/L),0.4 μL dNTP(10 mmol/L),2.0 μL Primer(10 μmol/L),0.2 μL Taq(5 U/μL),2.0 μL DNA.The optimization of the amplification process consisted of a pre-denaturation step at 94℃ for 2 minutes,followed by 35 cycles of denaturation at 94℃/30 s,annealing at 36℃/30 s and extension at 72℃/2 min,plus a final extension of 10 min at 72°C.%以桉树DNA为模板,采用单因素试验方法研究dNTP浓度、Mg2+浓度及退火温度对桉树随机引物PCR反应结果的影响。试验结果表明,最优的反应体系为:20μL的PCR反应体积中,10×Buffer2.0μL,Mg2+(25 mmol/L)1.28μL,dNTP(10 mmol/L)0.4μL,Primer(10μmol/L)2.0μL,Taq(5 U/μL)0.2μL,DNA2.0μL;最优扩增程序:94℃预变性2 min,然后进行35个循环(94℃变性30 s,36℃退火30 s,72℃延伸2 min),最后72℃延伸10 min。

  9. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas

    2010-05-01

    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  10. 山东地方绵羊MHC-DRB3基因PCR-RFLPs反应体系的优化%Optimization of PCR-RFLPs Reaction System for Shandong Province Indigenous Sheep MHC-DRB3 Gene Analysis

    Institute of Scientific and Technical Information of China (English)

    尚友国; 宋美玲; 王建民

    2005-01-01

    本文探讨了影响山东地方绵羊MHC-DRB3基因PCR-RFLPs扩增的因素,实验结果表明:抗凝剂、模板的浓度、纯度、Mg2+的浓度、引物的浓度、变性温度、Taq聚合酶、退火温度等因素对扩增结果的影响,为此我们对影响扩增结果的各个因素和反应程序进行了研究和浓度梯度的对比试验,建立了绵羊MHC-DRB3基因PCR-RFLPs的最佳反应体系和反应程序.

  11. Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

    Science.gov (United States)

    Jacchia, Sara; Nardini, Elena; Savini, Christian; Petrillo, Mauro; Angers-Loustau, Alexandre; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

    2015-02-18

    In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

  12. High efficiency genome walking method for flanking sequences of cotton mitochondrial double-copy atpA gene based on optimized inverse PCR and TAIL-PCR%优化的反向PCR结合TAIL-PCR法克隆棉花线粒体atpA双拷贝基因及其侧翼序列

    Institute of Scientific and Technical Information of China (English)

    张晓; 郭三堆; 张锐; 孙国清; 史计; 孟志刚; 周焘; 侯思宇; 梁成真; 于源华

    2012-01-01

    双拷贝基因及其侧翼序列的克隆是分子生物学中的一个难点.将优化的反向PCR (Inverse PCR,iPCR)与TAIL-PCR相结合,有效地克隆双拷贝基因及其侧翼序列.先用Southern blotting方法确定一种能获得合适长度片段的限制性内切酶,然后用优化的iPCR方法对该酶切产物进行自连和扩增,将2个拷贝的侧翼序列区分开.根据iPCR结果,进一步用TAIL-PCR扩增更远侧翼的序列.利用这套方法,获得了棉花可育胞质和不育胞质线粒体双拷贝atpA基因的所有EcoRI限制片段(2.2~5.1 kb)和HindⅢ限制片段(8.5~11.7 kb),克隆到2个拷贝各自的侧翼序列.研究结果说明,优化的iPCR与TAIL-PCR相结合是克隆双拷贝基因及其侧翼序列的一种高效方法.%Cloning of flanking sequences of double-copy gene is a challenge in molecular biology We developed a method to solve this problem by combining an optimized inverse PCR (iPCR) with TAIL-PCR. First, Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene. Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene. Based on the obtained sequences, TAIL-PCR was performed to amplify further flanking regions of the gene. With this method, we obtained all of the EcoR I restriction fragments (2.2-5.1 kb) and Hind III restriction fragments (8.5-11.7 kb) of mitochondrial atpA gene in cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton. The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.

  13. Optimization of PCR-DGGE reaction system on rhizosphere soil microorganism for Pinus densiflora Sieb.et Zucc%赤松根际土壤微生物PCR-DGGE反应体系优化

    Institute of Scientific and Technical Information of China (English)

    唐丽娜; 朴春根; 杭秋瑜; 金东淳

    2013-01-01

    为建立赤松根际土壤微生物的PCR-DGGE反应体系,以赤松根际土壤为材料,利用PCR-DGGE技术,对反应体系中的几个重要因素不同梯度进行优化,包括模板DNA、dNTPs、引物及Mg2+的用量.在25 μLPCR反应体系中,模板DNA浓度为10 ng,dNTPs浓度为0.15 mM,引物浓度为0.32 μM,Mg2+浓度为1.0 mM时,其PCR效果最佳.用该反应体系扩增出来的PCR产物,在DGGE凝胶上能够清晰地分辨出赤松根际土壤细菌和真菌多样性.这为进一步研究赤松根际土壤微生物,特别是研究赤松外生菌根奠定了基础.

  14. 长春花ISSR-PCR 反应体系的正交优化%Research on the Orthogonal Optimization of ISSR-PCR Reaction System of Madagascar Periwinkle

    Institute of Scientific and Technical Information of China (English)

    刘峰; 顾志敏; 陈析丰; 金杨; 马伯军

    2010-01-01

    [目的]筛选最适的长春花ISSR-PCR 反应体系.[方法]采用正交试验方法,对影响长春花ISSR-PCR反应体系的引物浓度、dNTP浓度、Mg~(2+)浓度、Taq DNA聚合酶浓度和模板DNA浓度5个因素在4个水平上进行正交试验,建立适合长春花ISSR-PCR的反应体系.[结果]长春花ISSR-PCR 反应体系的最佳条件:引物浓度0.50 μmol/L,dNTP浓度0.10 mmol/L,Mg~(2+)浓度 3.00 mmol/L,Taq 酶浓度0.75 U/25 μl,DNA模板浓度350 ng/25 μl.采用该反应体系筛选出12对适合于长春花ISSR-PCR反应的引物.[结论]利用优化系统进行长春花的ISSR-PCR反应,可获得稳定性高、重复性好、背景清晰的电泳结果.

  15. Modeling of 5 ' nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica

    DEFF Research Database (Denmark)

    Knutsson, R.; Löfström, Charlotta; Grage, H.;

    2002-01-01

    , the threshold cycle (C-T) and the fluorescence intensity by a normalized reporter value (DeltaR(n)). The C-r response was identified as the most suitable for detection modeling to describe the PCR performances of different samples. DNA extracted from S. enterica serovar Enteritidis was studied in double......-distilled H2O (ddH(2)O) and in two different enrichment media (brain heart infusion and BPW) with two PCR mixtures based on AmpliTaq Gold or rTth. A descriptive model was proposed and fitted to the available experimental data. Equivalent PCR performances for the two PCR mixtures were obtained when DNA....../microwell for the AmpliTaq Gold mixture. To verify the improved amplification capacity of the rTth mixture, BPW was inoculated with 1 CFU of S. enterica serovar Enteritidis per ml and the mixture was incubated at 30degreesC. Samples for PCR were withdrawn every 4 h during a 36-h enrichment. Use of the rTth mixture...

  16. Establishment and Optimization of ISSR-PCR Reaction System for Male Sterile Lines of Marigold%万寿菊雄性不育系ISSR-PCR体系建立与优化

    Institute of Scientific and Technical Information of China (English)

    伍亚平; 唐道城

    2013-01-01

    The experimental designs with a single-factor and orthogonal test was used to optimize the ISSR-PCR reaction procedures of nine male sterile lines in marigold in four factors including the concentration of Taq DNA polymerase, dNTP, Primers and Mg2+. Then, the template concentration and ISSR primers were screened and validated based on the optimized ISSR-PCR system, the annealing temperature of the primers were detected by gradient PCR approach as well. The optimal concentration of each factors for ISSR-PCR amplification system of marigold male sterile lines were determined as Mg2+1.5 mmol/L, dNTPs 0.20 mmol/L, primer 0.5μmol/L, template DNA 60 ng and Taq DNA polymerase 1 U in reaction volume of 25μL.%  以9个万寿菊雄性不育系总DNA为材料,利用单因素试验和正交试验设计对Mg2+、dNTPs、引物和Taq酶的浓度进行筛选,然后在此基础上筛选模板DNA浓度及ISSR引物并对引物退火温度进行梯度检测,优化得到最佳的ISSR-PCR反应体系,各个因素的最佳水平为:在25μL反应体系中,Mg2+浓度为1.5 mmol/L、dNTPs浓度为0.20 mmol/L、引物浓度为0.5μmol/L、模板DNA为60 ng、Taq DNA聚合酶含量为1 U.

  17. Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format

    DEFF Research Database (Denmark)

    Carracedo, A; Beckmann, A; Bengs, A

    2001-01-01

    390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories.Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male).All the laboratories...... reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples...

  18. Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli.

    Science.gov (United States)

    van Frankenhuyzen, Jessica K; Trevors, Jack T; Flemming, Cecily A; Lee, Hung; Habash, Marc B

    2013-11-01

    Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by realtime polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5-1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods.

  19. Factor optimization for the reaction of real-time fluorescent quantitative PCR%实时荧光定量PCR反应体系的优化研究

    Institute of Scientific and Technical Information of China (English)

    袁坤; 张丹; 刘新琼; 张向明

    2013-01-01

    为了降低实时荧光定量PCR(Real-Time Fluorescent Quantitative PCR,FQ-PCR)技术的实验成本,提高利用效率,对影响FQ-PCR较敏感的因素二甲基亚砜(DMSO)、甘油和Mg2+等进行了优化试验.结果表明,10μL反应体系的最优组分为:1×rTaqBuffer,0.16 mmol/L dNTPs,0.24μmol/L Primer,2.5 mmol/L Mg2+,5%甘油,5% DMSO,1×SYBR Green I,0.8 U rTaq酶,100 ng模板.该反应体系为经济型FQ-PCR的广泛应用奠定了基础.

  20. 法国蜜环菌ISSR-PCR反应体系的优化%Optimization of inter-simple sequence repeat PCR (ISSR-PCR) reaction system for Armillaria gallica

    Institute of Scientific and Technical Information of China (English)

    孙立夫; 张艳华; 裴克全; 杨国亭; 宋玉双; 秦国夫; 宋瑞清

    2009-01-01

    本文用单因子试验分析了基于ISSR(Inter-Simple Sequence Repeat)分子标记研究法国蜜环菌系统发生学的PCR(Polymerase Chain Reaction)扩增反应条件,并进行了引物筛选,同时对各个引物的退火温度以及甲酰胺对扩增效果的影响进行了讨论.为利用ISSR标记技术研究蜜环菌生物种的系统发生学、遗传多样性及种质资源提供了参考.

  1. The Optimization of Bacterial Colonies PCR-DGGE Primers inPig Nasal Cavity%猪鼻腔中细菌菌落PCR-DGGE引物条件优化

    Institute of Scientific and Technical Information of China (English)

    陈江

    2015-01-01

    变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)技术是最先用于 DNA 突变检测的一种电泳技术。为获得最佳的PCR-DGGE上样模板,本实验分别采用十六烷基三甲基溴化铵(CTAB)法和Power Soil DNA Isolation Kit试剂盒抽提细菌基因组DNA,分别以Primer(338F/518R)/Primer(P2/P3)为16Sr DNA V3扩增引物,采用温度梯度PCR对引物扩增条件进行优化。结果显示:在Tm 56℃,通过CTAB法抽提细菌基因组DNA,采用primer(338F/518R)扩增16Sr DNA V3目的产物特异性最佳。%Denaturing gradient gel electrophoresis (DGGE) technology is the first electrophoresis technique to detect DNA mutations .In order to obtainthe optimalof PCR-DGGE template, cetyltrimethyl ammonium bromide (CTAB) method and the Power Soil DNA Isolation Kitare used to extractbacterialmetagenome. Besides, Primer(338F/518R)and Primer(P2/P3)were respectivelyused for 16Sr DNA V3 amplification, temperature gradient PCRwere optimizedfor primer amplification conditions. Results showed thatextracting bacteriametagenome by CTAB and usingprimer A (338 f / 518 r) , are the best way of 16Sr DNA amplification productin Tm 56℃.

  2. HIV-1包膜单基因Nest-PCR体系优化及包膜基因准种扩增%Optimization of HIV-1 envelope gene PCR amplification system and amplification of envelope gene quasispecies

    Institute of Scientific and Technical Information of China (English)

    全红; 刘松; 张昊天; 任彩云; 庄敏; 凌虹; 李妍

    2012-01-01

    目的 确定HIV-1包膜(envelope,env)单基因扩增(single genome amplification,SGA)的最优反应体系,获得env单基因扩增产物.方法 采用SGA及Nest-PCR扩增HIV-1 env全长基因,并对Nest-PCR反应体系中各成分量进行优化;在此基础上,适当稀释HIV-1感染者cDNA模板进行Nest-PCR,以获得PCR扩增产物阳性率不超过30%的模板稀释度.结果 SGA Nest-PCR扩增HIV-1 env全长基因,引物浓度为0.3 μmol/L,dNTP浓度为0.25 μmol/L时可获得特异性高的env全长基因;凝胶回收纯化及稀释第一轮PCR产物可有效提高特异性条带的产量.采用优化反应体系成功从一名HIV-1感染者体内扩增出18株env单基因序列.结论 优化了HIV-1 env基因SGA Nest-PCR的反应体系,并从HIV-1感染者获得了病毒包膜基因准种,为后续进行准种分析奠定了基础.%Objective To optimize the PCR conditions for single genome amplification (SGA) of HIV-1 envelope (env) gene. Methods The efficiency of the SGA-PCR was assessed based on the amplification results of env gene using the non-diluted cDNA template, the optimal dilution of cDNA template that may result in no more than 30% positive amplification was predicted. Results The elements of SGA and Nested-PCR such as the amounts of primers were 0. 3 μmol/L, dNTP were 0. 25 μmol/L, purification of products of the first-round PCR reactions and diluting cDNA templates increased the amount of interest products effectively. One HIV-1 infected individual after the env gene SGA, 18 interest products confirmed by sequencing env sequence. Conclusion Optimization of the HIV-1 env gene the SGA Nest-PCR reaction system, and the viral envelope gene quasispecies from HIV-1 infection, laid the foundation for subsequent analysis of the quasispecies.

  3. Optimism

    Science.gov (United States)

    Carver, Charles S.; Scheier, Michael F.; Segerstrom, Suzanne C.

    2010-01-01

    Optimism is an individual difference variable that reflects the extent to which people hold generalized favorable expectancies for their future. Higher levels of optimism have been related prospectively to better subjective well-being in times of adversity or difficulty (i.e., controlling for previous well-being). Consistent with such findings, optimism has been linked to higher levels of engagement coping and lower levels of avoidance, or disengagement, coping. There is evidence that optimism is associated with taking proactive steps to protect one's health, whereas pessimism is associated with health-damaging behaviors. Consistent with such findings, optimism is also related to indicators of better physical health. The energetic, task-focused approach that optimists take to goals also relates to benefits in the socioeconomic world. Some evidence suggests that optimism relates to more persistence in educational efforts and to higher later income. Optimists also appear to fare better than pessimists in relationships. Although there are instances in which optimism fails to convey an advantage, and instances in which it may convey a disadvantage, those instances are relatively rare. In sum, the behavioral patterns of optimists appear to provide models of living for others to learn from. PMID:20170998

  4. PCR thermocycler

    Science.gov (United States)

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  5. Optimization

    CERN Document Server

    Pearce, Charles

    2009-01-01

    Focuses on mathematical structure, and on real-world applications. This book includes developments in several optimization-related topics such as decision theory, linear programming, turnpike theory, duality theory, convex analysis, and queuing theory.

  6. LENGTH-HETEROGENEITY POLYMERASE CHAIN REACTION (LH-PCR) AS AN INDICATOR OF STREAM SANITARY AND ECOLOGICAL CONDITION: OPTIMAL SAMPLE SIZE AND HOLDING CONDITIONS

    Science.gov (United States)

    The use of coliform plate count data to assess stream sanitary and ecological condition is limited by the need to store samples at 4oC and analyze them within a 24-hour period. We are testing LH-PCR as an alternative tool to assess the bacterial load of streams, offering a cost ...

  7. Improvement of Genomic DNA Extraction and Optimization of ISSR-PCR Amplification for Passion Fruit%西番莲基因组DNA的提取及ISSR-PCR的优化

    Institute of Scientific and Technical Information of China (English)

    吴田; 谢江; 蓝增全

    2011-01-01

    By comparing five DNA extraction methods, an efficient method suitable for ISSR-PCR of passion fruit (Passiflora edulis) was identified. The factors influencing ISSR-PCR for passion-fruit were optimized. The result showed that the modified SDS-Ⅱ procedure was most suitable for ISSR-PCR amplification for passion fruit. Detected on 2% agarose, 4.12 amplification bands on the average were observed in the ISSR-PCR products. The optimum amplification conditions for ISSR-PCR of passion fruit were 5 ng DNA template, 0. 2 mmol/L dNTPs, 0. 5 μmol/L primer, 0. 2 U Taq DNA polymerase and 1 × buffer (Mg2+ ) in 20μL reaction volumes.%对5种DNA提取方法进行比较,得到一种效率较高的、且适用于西番莲ISSR-PCR的DNA提取方法.同时,对影响西番莲ISSR-PCR的因子进行优化.结果表明:改良的SDS法2提取的DNA最适宜进行西番莲的ISSR-PCR扩增.ISSR-PCR产物在2%琼脂糖凝胶上检测,发现PCR扩增的平均条带数为4.12条.西番莲的ISSR-PCR的最优体系为20μL PCR反应液体系中含有1×buffer(Mg2+),0.2 mmol/L dNTPs,0.5 μmol/L引物,0.2 U Taq DNA聚合酶,5 ng DNA模板.

  8. Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures.

    Science.gov (United States)

    Minas, Konstantinos; McEwan, Neil R; Newbold, Charles Jamie; Scott, Karen P

    2011-12-01

    The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.

  9. 单核细胞增生性李斯特菌iap基因克隆和PCR条件优化%Cloning of iap gene of Listeria monocytogenes with PCR optimization

    Institute of Scientific and Technical Information of China (English)

    贾芙蓉; 罗雁非; 方玲; 郑岚; 刘伟林; 潘洪涛; 何成彦

    2010-01-01

    目的 构建单核细胞增生性李斯特菌Listeria monocytogenes 54002-4株p60蛋白iap基因原核克隆载体.方法 利用自行设计的引物通过梯度PCR优化扩增条件,扩增出单核细胞增生性李斯特菌54002-4株的iap基因.在iap基因的5'端和3'端分别引入BamH Ⅰ和Xho Ⅰ 2个酶切位点,琼脂糖凝胶电泳分析、回收PCR产物,并将回收纯化的PCR产物与pMD 18-T载体进行连接.将该重组质粒转化人大肠杆菌JM109感受态细胞,经1 mmol/L IPTG诱导4~6 h后,观察转化效果.结果 培养无色菌落细菌,提取质粒,经PCR鉴定和核苷酸序列测定后确定获得阳性重组质粒pMD18-T-Iap.结论 通过梯度PCR摸索出最佳反应条件,建立PCR优化条件和方法,经转化获得iap基因克隆载体.%Objective To construct a prokaryotic cloning vector of invasion- associated protein (iap) gene p60 of Listeria monocytogenes. Methods Under gradient PCR amplification conditions, Listeria monocytogenes 54002-4 strain iap gene was amplified with our in-house primers. BamH Ⅰ and Xho Ⅰ sites were induced to 5' and 3' terminals of iap gene, respectively. PCR products were then analyzed and collected by agarose gel electrophoresis. Purified PCR product was connected with the vector pMD18-T. The recombinant plasmid was transformed into Escherichia coli JM109 competent cells, with transformation effect observed after 1mmol/L IPTG induction for 4-6 h. Results Colorless bacterial colony was cultured and verified to produce positive recombinant plasmid pMD18-T-Iap by PCR and nucleotide sequencing. Conclusions PCR optimization conditions and methods are established through gradient PCR. Iap gene cloning vector is obtained after transformation.

  10. 苔藓植物matK基因PCR反应体系的建立和优化%Establishment and Optimization of matK gene-PCR Amplification System for Bryophytes

    Institute of Scientific and Technical Information of China (English)

    郑红建; 张安世

    2012-01-01

    [Objective] To establish and optimize the matK gene-PCR reaction system for Bryophytes. [ Method ] Using genomic DNA of Bryo-phytes extracted via an improved CTAB method, single factor analysis was performed to investigate the impacts of DNA template concentration, primer concentration, 2 x Taq MasterMix concentration on matK gene-PCR amplification and to optimize this system for Bryophytes. [Result] The matK gene-PCR amplification(10.0 μl) suitable for Bryophytes was determined to be composed of 0.5 μl DNA, 0.2 μl primer and 5.6 μl 2 × Taq MasterMix. [ Conclusion] The study laid basis for analyzing the molecular systematics of Bryophytes.%[目的]建立和优化适合于苔藓植物matK基因的PCR反应体系.[方法]以鳞叶藓为材料,利用改良CTAB法提取了基因组DNA,利用单因素试验分析了DNA模板、引物和2×Taq MasterMix的浓度对苔藓植物matK基因PCR反应的影响,对适合苔藓植物的matK基因PCR反应条件进行了优化.[结果]适合苔藓植物的matK基因PCR反应体系为10.0 μl,其中含DNA模板0.5μl,正反引物各0.2μl,2×Taq MasterMix 5.6 μl.[结论]为苔藓植物的分子系统学等研究奠定了基础.

  11. Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

    Science.gov (United States)

    Leung, Eric T Y; Zheng, L; Wong, Rity Y K; Chan, Edward W C; Au, T K; Chan, Raphael C Y; Lui, Grace; Lee, Nelson; Ip, Margaret

    2011-07-01

    Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

  12. 米黑根毛霉脂肪酶基因密码子优化及重叠延伸PCR合成%Codon Optimization of Rhizomucor miehei Lipase Gene and Its Synthesis by Overlapping PCR

    Institute of Scientific and Technical Information of China (English)

    苗长林; 罗文; 吕鹏梅; 李惠文; 杨玲梅; 袁振宏

    2013-01-01

    [Objective]The aim was to optimize the codon of Rhizomucor miehei lipase (RML) gene and synthesize its sequence by overlapping PCR. [ Method ] According to the bias in codon choice of the high expression gene in Pichia pastoris, the gene encoding the mature portion of Rhizomucor miehei lipase with high specific activity was optimized. And according to the coding sequence of optimized amino acids, 30 oligonucleotide fragments at length of 48 bp were synthesized, respectively, and the full-length gene was amplified by overlap extension PCR method. [Result] The synthetic gene sequences were aligned with the designed sequence by gel electrophoresis, restriction enzyme digestion and sequencing analysis. [Conclusion] This study provided foundation for the construction and subsequent expression of the lipase gene in Pichia pastoris.%[目的]对米黑根毛霉脂肪酶基因的密码子进行优化,并采用重叠延伸PCR合成设计的基因序列.[方法]根据毕赤酵母(Pichia astoris)密码子使用偏好性,对米黑根毛霉脂肪酶成熟肽基因进行全面密码子优化,根据优化后的氨基酸编码序列设计合成30条长度约为48bp的寡核苷酸片段,并采用重叠延伸PCR法扩增合成全长基因序列.[结果]经凝胶电泳和酶切鉴定、测序分析表明,合成的目的基因与设计的序列相一致.[结论]该研究为脂肪酶基因在毕赤酵母中的构建及后续表达奠定了基础.

  13. Neurocryptococcosis: diagnosis by PCR method.

    Science.gov (United States)

    Paschoal, Regina Célia; Hirata, Mário Hiroyuki; Hirata, Rosário Crespo; Melhem, Márcia de Souza Carvalho; Dias, Amanda Latercia Tranches; Paula, Claudete Rodrigues

    2004-01-01

    Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

  14. 适用于川芎的ISSR-PCR反应体系的建立及优化(英文)%Optimization of ISSR-PCR Reaction Conditions for Genomic DNA of Ligusticum chuanxiong hort.

    Institute of Scientific and Technical Information of China (English)

    王岚; 唐琳

    2012-01-01

    [Objective] To investigate the impacts of ISSR-PCR amplification factors, for the establishment and optimization of ISSR-PCR reaction system for Ligusticum chuanxiong hort. [Method] Using genomic DNA of Chuanxiong leaf extracted via an improved CTAB method as template, single factor analysis was performed to investigate the impacts of DNA template concentration, Mg2+ concentration, dNTPs concentration, primer concentration, Taq DNA polymerase concentration on ISSR-PCR amplification and to optimize this system for Ligusticum chuanxiong hort. [Result] The ISSR-PCR amplification(25 μl) suitable for Ligusticum chuanxiong hort. was determined to be composed of 2.5 μl of 10×reaction buffer, 2.1 mmol/L MgCl2, 300 μmol/L dNTPs, 0.4 μmol/L primer, 1.0 U Taq DNA polymerase and 20-40 ng genomic DNA. [Conclusion] Our study laid basis for analyzing the genetic diversity of Ligusticum chuanxiong hort. resources distributed in 17 different areas of China.%[目的]建立和优化适合川芎的ISSR-PCR反应体系。[方法]以川芎叶片为材料,利用改良CTAB法提取了叶片基因组DNA,利用单因素试验分析了DNA模板、Mg2+、dNTP、引物和TaqDNA聚合酶的浓度对ISSR-PCR反应的影响,对适合川芎的ISSR-PCR反应条件进行了优化。[结果]确定了适合川芎的ISSR-PCR反应体系为25.0μl,其中含2.5μl10×TaqDNA聚合酶缓冲液、2.1mmol/LMgCl2、300μmol/LdNTP、0.4μmol/L引物、1.0UTaqDNA聚合酶和20~40ng模板DNA。[结论]为进一步对全国17个不同分布区的川芎资源进行遗传多样性研究奠定了基础。

  15. Apolipoprotein E genotyping by multiplex amplification refractory mutation system PCR with optimized conditions%优化的多重扩增阻滞突变系统-PCR对载脂蛋白E基因的简易分型

    Institute of Scientific and Technical Information of China (English)

    刘静; 刘建伟; 叶玲

    2011-01-01

    Objective To set up a simple method for apolipoprotein E (ApoE) genotyping by modifying the conditions of multiplex amplification refractory mutation system PCR (multi-ARMS-PCR). Methods Based on the principle of multi-ARMS-PCR and considering the faults existed in some published papers, new primers were designed to improve PCR conditions. DNA genome of peripheral white blood cells was used as the template. Four allele-specific oligonucleotide upstream primers, one common downstream primer and a pair of internal positive control primers were constructed. Multi-ARMS-PCR were performed with combination of different primers in 2 reaction tubes synchronously. Amplified multiplex products were electrophoresed on agarose gels containing ethidium bromide. Six ApoE genotypes were distinguished by the band sizes. Results Using the new primers,amplification efficiency and specificity were significantly increased and the misclassification was diminished due to removing the interference of non-specific bands. Conclusion The optimized multi-ARMS-PCR is an easy, time-saving, efficient and economical method for determination of ApoE genotypes applied in a minimally equipped laboratory.%目的 通过改进和优化多重扩增阻滞突变系统-PCR(multi-ARMS-PCR)条件,建立载脂蛋白E(ApoE)基因的简易分型方法.方法 基于multi-ARMS-PCR的原理和特点,针对文献报道方法中存在的缺陷和错误,重新设计或改进引物.以外周血白细胞基因组DNA为模板,应用4个等位基因特异性寡核酸上游引物、1个通用下游引物和一对内参引物,分A,B两个管同步进行多重PCR反应.PCR扩增产物经过琼脂糖凝胶电泳分离-EB染色,根据电泳带型的差异,实现对ApoE 6种基因型的判定.结果 新引物显著提高了扩增效率和反应特异性,排除了非特异条带的干扰,减少了ApoE基因分型的错判.结论 采用优化后的multi-ARMS-PCR方法对ApoE基因型进行鉴定,具有操作简便、时间短

  16. 南方红豆杉ISSR -PCR反应体系的建立与优化%An orthogonal design to optimize ISSR-PCR reaction system for Taxus wallichiana var.chinensis

    Institute of Scientific and Technical Information of China (English)

    廖盼华; 汪庆; 李亚; 姚淦; 杨如同; 王淑安

    2012-01-01

    Several important parameters influencing ISSR-PCR amplification on DNA from Taxus wallichiana var. Chinensis were studied with an orthogonal design by four factors and four levels to establish the optimum ISSR reaction system. The optimal ISSR-PCR reaction system was established as a total volume of 20μL consisting of 60ng template DNA, 10 x PCR buffer 2. 0μX, 25 mmol/L MgCl2 l.0μX, 2.5 mmol/L dNTPs l.0μL, 10μmol/L primer 1.0 uX and Taq DNA polymerase 0.2U. The suitable PCR procedure is one cycle denaturing at 94℃ for 4min,then, 41 cycles which involves denaturing at 941 for 30s,annealing at 54.5℃ for 45s and extending at 72℃ for 80s, one cycle extending at 72℃ for 5 min.and kept at4℃o%以南方红豆杉DNA为模板,采用正交试验设计对影响南方红豆杉ISSR -PCR扩增的参数进行了优化.结果表明较佳的南方红豆杉ISSR-PCR的反应体系(20 μL)为模板DNA 60 ng,10×PCR buffer 2.0μL,25 mmol/L的MgCl2 1.0 μL,2.5 mmol/L的dNTPs 1.0 μL,10μmol/L的引物1.0 μL和反应酶0.2U.适宜的扩增条件为94℃预变性4 min,41个PCR循环(94℃变性30 s,54.5℃退火45 s),72℃延伸80 s,72℃延伸5 min,4℃保存.

  17. Optimization of SRAP-PCR reaction system and primer screening in Bougainvillea%三角梅SRAP-PCR反应体系的建立及引物筛选

    Institute of Scientific and Technical Information of China (English)

    武晓燕; 唐源江; 曹雯静

    2012-01-01

    利用L9(34)正交试验设计,对影响PCR反应的TaqDNA聚合酶量、dNTP浓度、Mg2+浓度和引物浓度4个因素以及模板DNA浓度进行了三角梅SRAP-PCR扩增反应条件优化研究,并对引物进行了全面筛选.三角梅SRAP-PCR优化反应体系结果为:2.5 μL 10×PCR buffer、60 ng模板DNA、TaqDNA聚合酶1.0U、dNTP 0.25 mmol/L、Mg2+ 2.5 mmol/L、引物0.3μmol/L,总体积25 μL.运用该结果从208对引物组合中共筛选出扩增条带清晰,多态性丰富的SRAP引物27对.优化体系的建立及其引物的筛选为今后利用SRAP标记技术进行三角梅遗传分析、图谱构建、基因定位与种质资源鉴定奠定了技术基础.%Bougainvillea is one of most important ornamental trees and shrubs in the world, but the related SRAP-PCR application on it has been hardly reported at present. Based on the L9(34) orthogonal experimental design, the optimum SRAP-PCR reaction system for Bougainvillea was established as follows: 2. 5 μLl0×PCR Buffer, 60ng template DNA, 1. 0 U TaqDNApolymerase, 0. 25 mmol/L dNTP, 2.5 mmol/L Mg2+ and 0. 3 μmol/L primer, the total reaction volume was 25 μL. 27 primer pairs were screened out from 208 SRAP primer pairs combinations based on their stable amplification, clear banding patterns and good polymorphism by utilizing the SRAP reaction system. The optimal SRAP-PCR reaction system was established and primers were screened, which would provide the basis for genetic analysis, map construction, gene localization and i-dentification of germplasm resources for Bougainvillea.

  18. 油梨基因组DNA提取、SSR-PCR反应体系优化及引物筛选%DNA Extraction,Optimization of SSR-PCR Reaction System and Primer Screening of Persea americana

    Institute of Scientific and Technical Information of China (English)

    周海兰; 李绍鹏; 李卫亮; 贺军虎; 包冬红; 李茂富

    2016-01-01

    旨在建立稳定可靠的油梨(Persea americana Mill)叶片DNA的提取方法和SSR-PCR反应体系及筛选出稳定的油梨SSR多态性引物,为开展油梨种质SSR分子标记提供遗传研究的基础。以油梨叶片为试材,比较3种油梨叶片DNA提取方法;利用L16(45)正交实验设计对油梨SSR-PCR反应体系进行优化;利用优化的反应体系筛选引物;同时,选取5对多态性引物对45份油梨种质进行PCR扩增,进一步检测该优化体系的稳定性。结果表明,常规2×CTAB法、改良2×CTAB法和植物DNA提取试剂盒法等3种DNA提取方法中,改良2×CTAB法对油梨基因组DNA的提取效果最佳;获得最优反应体系为:20μL总反应体系中,含约40 ng DNA模板、1.5 mmol/L Mg2+、0.15 mmol/L dNTPs、0.5 U Taq DNA聚合酶、0.5μmol/L引物;以此体系为基础进行引物筛选,从73对油梨SSR引物中筛选出了30对扩增条带清晰的多态性引物,说明该反应体系可用于油梨SSR标记的进一步研究;稳定性检测获得的谱带清晰,表明该优化反应体系是稳定可靠的。由此可见,改良的2×CTAB法可用于油梨叶片DNA的大量样本提取,优化后的SSR-PCR反应体系及筛选出的30对多态性引物可用于油梨SSR标记的进一步研究。%This study is to establish a stable and reliable DNA extraction method,optimize the SSR-PCR reaction system,and screen the stable polymorphism primers for avocado(Persea americana Mill)SSR for providing the genetic foundation to conduct the SSR molecular marker of germplasm in avocados. Taking avocado’leaves as the study material,3 avocado DNA extraction methods were compared,based on the L16(45)orthogonal experiment design,the SSR-PCR reaction system in avocados was optimized,and then by optimized reaction system the SSR primers were screened. To further test the stability of the optimized SSR-PCR system,the germplasms in 45 pieces of avocados were amplified by

  19. Pre-PCR processing: strategies to generate PCR-compatible samples.

    Science.gov (United States)

    Rådström, Peter; Knutsson, Rickard; Wolffs, Petra; Lövenklev, Maria; Löfström, Charlotta

    2004-02-01

    Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

  20. Reverse-transcription PCR (RT-PCR).

    Science.gov (United States)

    Bachman, Julia

    2013-01-01

    RT-PCR is commonly used to test for genetic diseases and to characterize gene expression in various tissue types, cell types, and over developmental time courses. This serves as a form of expression profiling, but typically as a candidate approach. RT-PCR is also commonly used to clone cDNAs for further use with other molecular biology techniques (e.g., see Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates and Circularized RT-PCR (cRT-PCR): analysis of RNA 5' ends, 3' ends, and poly(A) tails).

  1. The polymerase chain reaction (PCR): general methods.

    Science.gov (United States)

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  2. 适用于川芎的ISSR-PCR反应体系的建立及优化%Establishment and Optimization of ISSR-PCR Amplification System for Ligusticum chuanxiong Hort

    Institute of Scientific and Technical Information of China (English)

    王岚; 唐琳

    2011-01-01

    [Objective] This study was to establish and optimize the ISSR-PCR reaction system for Ligusticum chuanxiong Hort. [ Method] Using genomic DNA of Chuanxiong leaf extracted via an improved CTAB method, single factor analysis was performed to investigate the impacts of DNA template concentration, Mg2+ concentration, dNTPs concentration, primer concentration, Taq DNA polymerase concentration on ISSR-PCR amplification and to optimize this system for Ligusticum chuanxiong Hort. [ Result] The ISSR-PCR amplification(25 μl) suitable for Ligusticum chuanxiong Hort. Was determined to be composed of 2. 5 μl of 10 × reaction buffer, 2.1 mmol/L MgCl2, 300 μmol/L dNTPs, 0.4 μmol/L primer, 1.0 U Taq DNA polymerase and 20 -40 ng genomic DNA. [ Conclusion] Our study laid basis for analyzing the genetic diversity of Ligusticum chuanxiong Hort. Resources distributed in 17 different areas of China.%[目的]建立和优化适合川芎的ISSR-PCR反应体系.[方法]以川芎叶片为材料,利用改良CTAB法提取了叶片基因组DNA,利用单因素试验分析了DNA模板、Mg2+、dNTP、引物和Taq DNA聚合酶的浓度对ISSR-PCR反应的影响,对适合川芎的ISSR-PCR反应条件进行了优化.[结果]适合川芎的ISSR-PCR反应体系为25.0μl,其中含2.5μl10×Taq DNA聚合酶缓冲液、2.1 mmol/L MgCl2、300μmol/L dNTP、0.4 μmol/L引物、1.0 U Taq DNA聚合酶和20 ~40 ng模板DNA.[结论]为进一步对全国17个不同分布区的川芎资源进行遗传多样性研究奠定了基础.

  3. Optimizing of double PCR system for detecting transgenic ingredients in soybean products%豆制品中转基因成分的二重PCR检测体系的优化研究

    Institute of Scientific and Technical Information of China (English)

    许晓丹; 史永翠; 刘畅; 王存芳

    2014-01-01

    目的:由于转基因大豆及其制品日益增多,其安全性亦受到了越来越多消费者的关注。为满足消费者的知情选择权,本研究建立一种快速检测外源基因的方法。方法以转基因豆制品中存在的CaMV35S启动子和NOS终止子为检测目标,优化了二重 PCR 反应体系和条件,包括引物比例和酶的用量;运用聚丙烯酰胺凝胶电泳和琼脂糖凝胶电泳对PCR产物进行检测,并对聚丙烯凝胶电泳的银染条件进行探索研究。结果建立了运用聚丙烯酰胺凝胶电泳更好地区分出外源基因CaMV35S启动子和NOS终止子的目的条带的方法。结论本实验中的二重PCR和聚丙烯酰胺凝胶电泳方法适用于对转基因豆制品中这两个外源基因的检测,为转基因豆制品检测提供了指导。%Objective Account of the increasing amount of transgenic soybean and soybean products, the safety problem has received more and more attention from consumers. In order to satisfy consumers’ right of in-formed choice, a rapid method of detecting genetically modified products was established in this study. Methods CaMV35S promoter and NOS terminator in soybean products were taken as detection target. The double PCR reac-tion system and conditions were optimized, including primer ratio and enzyme amount. Agarose gel and polyacryla-mide gel electrophoresis were applied to analyze PCR products, and the silver staining conditions for polyacrylamide gel electrophoresis were explored. Results The polyacrylamide gel electrophoresis was established and could dis-tinguish CaMV35S promoter and NOS terminator better than agarose gel. Conclusion The results showed that double PCR and polyacrylamide gel electrophoresis in this experiment were suitable for the analytic detection of two foreign genes in transgenic soybean products, and it could be a guidance for detecting transgenic food.

  4. Optimization of PCR reaction system in Luzhou-flavor Daqu microbial DNA%浓香型大曲中微生物总DNA的PCR优化

    Institute of Scientific and Technical Information of China (English)

    潘明; 袁城金; 王世宽

    2012-01-01

    The DNA in Daqu extracted by the SDS-enzymic technique and diagnostic method, was diluted with gradiented concentration, and finally was amplificated. The SDS-enzymic technique can extract 300 times larger amount DNA than the diagnostic method. The optimal amplification condition was that to didute 50 times, and add the BSA in a concentration of 0.6 ng/ul. It could extract plenty of total DNA by the SDS-enzymic technique, and it can amplificate the total DNA in a effective way when diluted the total DNA and add some BSA.%利用SDS-酶法、试剂盒法提取大曲总DNA,对粗提DNA梯度稀释稀释,同时按浓度梯度加入BSA进行PCR扩增.SDS-酶法提取总DNA的量是试剂盒法的300倍;对粗提DNA稀释50倍同时添加BSA浓度为0.6ng/μL的PCR扩增效果最佳.利用SDS-酶法能够最大量的提取曲药中总DNA,对总DNA进行稀释同时加入BSA能够有效的进行PCR扩增.

  5. Sex Determination Using PCR

    Science.gov (United States)

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  6. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Science.gov (United States)

    Schrick, Livia; Nitsche, Andreas

    2016-01-01

    PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  7. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Directory of Open Access Journals (Sweden)

    Livia Schrick

    2016-01-01

    Full Text Available PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  8. 人工合成小麦 HMW-GS Dtx1.5亚基的 AS-PCR 鉴定程序优化及遗传分析%Optimization of AS-PCR Identification System for HMW-GS Dtx1.5 of Wheat Syndeme Developed with Durum Wheat and Aegilops tauschii and Its Genetic Analysis

    Institute of Scientific and Technical Information of China (English)

    万洪深; 温雯; 李俊; 杨武云

    2014-01-01

    Synthetic hexaploids have gradually been used as a bridge-tool for common wheat improvement to introduce elite gene sources from its wild relatives with strong resistance to biotic/abiotic stresses, increased yield potential and good making quality. The high molecular weight glutenin subunits (HMW-GS) Dtx1.5 from Aegilops tauschii is thought to be associated with fine making quality. In this study, the reaction condition of the Dtx1.5 AS-PCR system has been optimized step by step, and the distribution of the HMW-GS Dtx1.5 in the F2 and F9 RIL populations derived from Chuanyu 12 crossed with synthetic wheat Syn780 was also investigated using this AS-PCR system. The sharp and stable fragments amplified by this optimum AS-PCR system indicated its availability and convenience in the identification of HMW-GS Dtx1.5. And the observed distribution of Dtx1.5 obeyed Mendelian law of segregation in both low (F2) and advanced (F9) generation populations, suggesting the stable inheritance of Dtx1.5. Therefore, the Dtx1.5 from Aegilops tauschii could be used in common wheat breeding for quality and the optimum AS-PCR system could enhance the efficiency of selection by molecular markers.%人工合成小麦综合了自然界中现有四倍体小麦(Triticum turgidun)和粗山羊草(节节麦, Aegilops tauschii)的丰富遗传变异,是将野生祖先种中优异的抗病、抗逆基因成功利用到普通小麦育种中的重要桥梁。源于粗山羊草的高分子量谷蛋白(HMW-GS) Dtx1.5亚基,是一种新的优质亚基。本研究对 HMW-GS Dtx1.5亚基 AS-PCR 反应体系及扩增程序进行了逐步的优化,并利用普通小麦川育12与人工合成小麦 Syn780(含有Dtx1.5亚基)杂交 F2代(低代)和 F9重组自交系(高代)群体对 Dtx1.5亚基的遗传规律进行了分析。结果表明:优化后目的扩增条带清晰、稳定、可靠,可以用于 Dtx1.5亚基的分子鉴定;Dtx1.5亚基在普通小麦与人工合成小麦杂交的低代

  9. Addressing PCR Biases in Environmental Microbiology Studies

    Science.gov (United States)

    Sipos, Rita; Székely, Anna; Révész, Sára; Márialigeti, Károly

    Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition of the sample in the PCR product. Strategies of primer selection and PCR thermal profile design are discussed in detail.

  10. 异地板蓝根基因组DNA指纹图谱建立及RAPD-PCR反应体系优化%Establishment of Offsite Radix Isatidis Genomic DNA Fingerprint and Optimization of RAPD-PCR Reaction System

    Institute of Scientific and Technical Information of China (English)

    姜颖; 杨欣; 于英君

    2014-01-01

    Objective:RAPD method is used to study Radix Isatidis genomic DNA genetic difference and the true and false of the medicinal materials in different regions.Methods:Use RAPD technology for Radix Isatidis genomic DNA genetic diversity analyses at the molecular level from different regions, and optimize the Radix Isatidis RAPD-PCR reaction system.Results:Fingerprints showed in different regions Radix Isatidis resource had rich genetic diversity, and could distinguish between true and false, and the pros and cons of herbs.Conclusion:RAPD technique for the identification of the authenticity and the pros and cons of Chinese herbal medicines Radix Isatidis provides a practical significance for clinical safety and provide a theoretical basis for rational drug use.And Radix Isatidis best RAPD-PCR reaction system has been established.%目的:采用RAPD法研究不同地区板蓝根基因组DNA遗传差异性,药材的真假。方法:利用RAPD( random amplified polymorphic DNA)技术,在分子水平上对不同地区板蓝根基因组DNA进行了遗传差异性分析,同时优化板蓝根RAPD-PCR反应体系。结果:指纹图谱显示出不同地区板蓝根资源丰富的遗传多样性,可以辨别出药材的真假和优劣。结论:RAPD技术为鉴别中药材板蓝根真伪和优劣提供了现实意义,为临床安全和合理用药提供理论依据,并建立适合板蓝根RAPD-PCR的最佳反应体系。

  11. Overcoming inhibition in real-time diagnostic PCR.

    Science.gov (United States)

    Hedman, Johannes; Rådström, Peter

    2013-01-01

    PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR

  12. Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6 Otimização da PCR em tempo real - Sybr Green para detecção do Herpes Vírus Humano tipo 6 (HHV-6

    Directory of Open Access Journals (Sweden)

    Cynthia Liliane Motta do Canto

    2008-02-01

    Full Text Available HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL to 10-9 (equivalent to 2.46 molecules/µL. Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3' and by conventional nested PCR using primers HHV1 (outer: 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer: 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner and HHV4 (inner 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.HHV-6 é o agente etiológico do Exantema Súbito e considerado a sexta doença mais comum na infância. Em indivíduos imunocomprometidos, a reativação da infecção latente pode causar doença aguda ou morte. Padronizamos PCR em Tempo Real utilizando a química Sybr Green na detecção do HHV-6 e comparamos os resultados com a PCR convencional. Um fragmento de 214 pb foi clonado através do kit pGEM-T do sistema Promega. Com este clone, foram feitas diluições seriadas em um pool de leucócitos negativos a partir de 10-6 ng/µL (equivalente a 2465,8 moleculas/µL até 10-9 (equivalente a 2,46 moleculas/µL. As diluições foram amplificadas por PCR em Tempo Real utilizando Sybr Green, com

  13. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

    DEFF Research Database (Denmark)

    Boessenkool, Sanne; Epp, Laura S.; Haile, James Seymour

    2012-01-01

    , or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR...

  14. 实时定量RT-PCR检测肝癌miRNA表达中内参的选择%Selection of optimal internal controls for miRNA expression in human hepatocellular carcinoma using real-time quantitative RT-PCR

    Institute of Scientific and Technical Information of China (English)

    党裔武; 陈罡; 容敏华

    2012-01-01

    目的 探讨肝癌(hepatocellular carcinoma,HCC)组织采用实时定量RT-PCR(RT-qPCR)技术检测miRNA表达最佳内参的选择.方法 采用实时RT-qPCR技术检测7个内参RNU6B、RNU48、RNU66、RNU44、RNU43、5s rRNA和18s rRNA在70例HCC及相应癌旁肝组织中的表达.并使用NormFinder及geNorm软件分析最优化的内参.结果 7个内参表达结果存在差异.NormFinder及geNorm软件均显示RNU6B+RNU48组合为肝组织的最佳内参.结论 使用实时定量RT-qPCR研究目标miRNA相对定量表达时,为保证数据的精确性和客观性,在实验前对内参进行慎重的筛选是必不可少的.NormFinder及geNorm软件可用于各种实验因素下适宜内参的选择.%Purpose To investigate the selection of optimal internal controls for miRNA expression in human hepatocellular carcinoma ( HCC ) using real-time quantitative RT-PCR ( RT-qPCR ). Methods Expression of 7 controls ( RNU6B, RNU48, RNU66, RNU44, RNU43 , 5s rRNA and 18s rRNA ) was detected with real-time RT-qPCR in 70 cases of HCC and their adjacent non-cancerous liver tissues. The optimal internal controls were selected by NormFinder and geNorm softwares. Results The genes studied displayed a wide expression range with different Cq values. Both NormFinder and geNorm indicated that the optimal internal controls for liver tissue were the combination of RNU6B and RNU48. Conclusions In order to ensure the accuracy and objectivity of normalized data in RT-qPCR studies adopting relative miRNA quantification, careful selection and validation of endogenous controls are advocated as a mandatory step. NormFinder and GeNorm softwares can be used to screen out the most stable controls under different experimental conditions.

  15. 柱花草SRAP-PCR体系优化及其遗传多样性分析%Optimization of SRAP-PCR system and its application in genetic diversity analysis of Stylosanthes

    Institute of Scientific and Technical Information of China (English)

    张伟丽; 刘凤民; 刘艾

    2011-01-01

    Two different genotypes of Stylosanthes germplasm, S. Guianensis cv. Reyan No. 2 and S. Guianensis cv. Reyan No. 13 were used as material for studying the effects of concentrations of Mg2+ , dNTPs, DNA polymerase, primers and DNA template on the SRAP - PCR reactions and optimizing the establishment of SRAP molecular marker system in Stylosanthes. The optimum system was established as follows: template DNA 40 ng, Mg2+ 2. 5 mmol/L, dNTPs 0. 2 mmol/L, polymerase 1 U, primer 0. 3 μmol/L, the total reaction volume was 25 μL. The genetic diversity and relationships of nine Stylosanthes germplasms were analyzed by using this optimum system. Forty-eight primers were studied for an analysis of genetic diversity by SRAP in Stylosanthes germplasms. Fourteen effective primers selected from 48 primers combination were used for SRAP - PCR, and 150 of the 154 DNA fragments amplified, showed polymorphisms. The average binds from each primer was 11. 0 and the average percentage of polymorphic bands was 97. 36%. The Nei's genetic similarity coefficient of the tested accessions ranged from 0. 386 to 0. 882 by software NTSYSpc 2. 1 based on SRAP results, and the average Nei's coefficient was 0. 631, average the genetic distance(GD) was 0. 369. Based on the presence of bands, nine Stylosanthes germplasm were classified into there major groups by UPGMA cluster analysis, group Ⅰ , group Ⅱ and group Ⅲ. Group I included S. Hamata cv. Verano, and group Ⅱ included S. Scabra cv. Seca. Group Ⅲ included S. Guianensis cv. Reyan No. 5, S. Guianensis cv. Reyan No. 10, S. Guianensis cv. Reyan No. 13, S. Guianensis cv. White cook, S. Guianensis cv. Reyan No. 2, S. Guianensis cv. Graham and S. Guianensis tardio CIAT1283. This research should provide a scientific basis at the molecular level for further study and the application of nine Stylosanthes germplasms. The most of the varieties with relative relationship in their pedigrees and similar biological characteristics were clustered into

  16. pcrEfficiency: a Web tool for PCR amplification efficiency prediction

    Directory of Open Access Journals (Sweden)

    Mallona Izaskun

    2011-10-01

    Full Text Available Abstract Background Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair. Results We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence. Conclusions pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at http://srvgen.upct.es/efficiency.html under the GPL v2 license.

  17. Apta-PCR.

    Science.gov (United States)

    Pinto, Alessandro; Polo, Pedro Nadal; Rubio, Miriam Jauest; Svobodova, Marketa; Lerga, Teresa Mairal; O'Sullivan, Ciara K

    2016-01-01

    Real-time Apta-PCR is a methodology that can be used for a wide variety of applications ranging from food quality control to clinical diagnostics. This method takes advantage of the combination of the sensitivity of nucleic acid amplification with the selectivity of aptamers. Ultra-low detection of target analyte can potentially be achieved, or, improved detection limits can be achieved with aptamers of low-medium affinity. Herein, we describe a generic methodology coined real-time Apta-PCR, using a model target (β-conglutin) and a competitive format, which can be adapted for the detection of any target which an aptamer has been selected for.

  18. Recovery of DNA of Giardia intestinalis cysts from surface water concentrates measured with PCR and real time PCR

    Directory of Open Access Journals (Sweden)

    Adamska M.

    2011-11-01

    Full Text Available The most important restriction for the detection in water samples is the low concentration of Giardia intestinalis cysts, additional difficulty is the presence of PCR inhibitors. We have carried out trials in order to assess the sensitivity of semi-nested PCR and TaqMan real time PCR on the basis of DNA extracted from G. intestinalis cysts coming from spiked environmental and distilled water samples, filtrated with the use of Filta-Max® equipment (1623 Method. Removal of inhibitors was carried out with addition of BSA in different concentrations. During the filtration and concentration of water samples, losses of cysts have been recorded. Moreover, addition of BSA to the PCR and real time PCR mix increases the sensitivity of reaction. The optimal concentration of BSA for semi-nested PCR was 15 and 20 ng/μl, whereas for real time PCR 5 ng/μl.

  19. Explanatory chapter: PCR primer design.

    Science.gov (United States)

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Determining the optimal number of individual samples to pool for quantification of average herd levels of antimicrobial resistance genes in Danish pig herds using high-throughput qPCR

    DEFF Research Database (Denmark)

    Clasen, Julie; Mellerup, Anders; Olsen, John Elmerdahl

    2016-01-01

    The primary objective of this study was to determine the minimum number of individual fecal samples to pool together in order to obtain a representative sample for herd level quantification of antimicrobial resistance (AMR) genes in a Danish pig herd, using a novel high-throughput qPCR assay....... The secondary objective was to assess the agreement between different methods of sample pooling. Quantification of AMR was achieved using a high-throughput qPCR method to quantify the levels of seven AMR genes (ermB, ermF, sulI, sulII, tet(M), tet(O) and tet(W)). A large variation in the levels of AMR genes...

  1. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    Science.gov (United States)

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.

  2. Engineered DNA Polymerase Improves PCR Results for Plastid DNA

    Directory of Open Access Journals (Sweden)

    Melanie Schori

    2013-02-01

    Full Text Available Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase.

  3. PCR in forensic genetics

    DEFF Research Database (Denmark)

    Morling, Niels

    2009-01-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more...... and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics....

  4. Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

    Directory of Open Access Journals (Sweden)

    Danielle C. Leal

    2011-07-01

    Full Text Available Conventional PCR (PCRTeq for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128, but did not differ significantly from the M/PCRTeq-Bc (1:64. In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780 and moderate agreement with N/PCR-Teq (k = 0.562 and M/PCRTeq-Bc (k = 0.488. PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05, and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05. PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

  5. A PCR amplification method without DNA extraction.

    Science.gov (United States)

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  6. Establishment and Optimization of ISSR-PCR Reaction System in Precocious Pyrus pyrifolia%早熟砂梨ISSR-PCR反应体系的建立与优化

    Institute of Scientific and Technical Information of China (English)

    宋燕春; 李永裕; 陈建烟; 黄添毅; 龚理; 吴少华

    2013-01-01

      以我国南方主栽的早熟砂梨品种‘翠冠’Pyrus pyrifolia‘Cuiguan’为材料,对ISSR技术体系中的模板DNA浓度、Taq DNA聚合酶用量、引物浓度、dNTP浓度、Mg2+浓度、退火温度、PCR循环数等7个主要因素进行优化和筛选,建立了适合早熟砂梨的ISSR-PCR反应体系。最终反应体系为20μL体系中10×PCR buffer (不含Mg2+)2μL,模板 DNA浓度60 ng,TaqDNA聚合酶0.75 U,引物浓度1μmol/L,dNTP浓度90μmol/L, Mg2+浓度2.25 mmol/L。扩增程序为:预变性94℃5 m in,变性94℃45 s,退火45 s,72℃延伸1 min,共42个循环,然后72℃再延伸10 min,4℃保存,用1.5%琼脂糖凝胶电泳检测多态性。%  To obtain 7 factors (DNA template dosage, Taq DNA polymerase dosage, primer concentration, dNTP concentration, Mg2+concentration, annealing temperature and PCR cycles ) of ISSR-PCR Reaction System in Pyrus pyrifolia ‘Cuiguan’, a precocious cultivar of southern China was been used as template. An optimum reaction system was been established. The final total volume was 20 μL, contains 2 μL 10×PCR buffer (Mg2+ free), 60 ng DNA template, 0.5 U Taq DNA polymerase, 1 μmol/L of primer concentration, 90 μmol/L of dNTP concentration, 2.25 mmol/L of Mg2+ concentration. Amplication procedure was as follows: initial-denaturing of 5 min at 94 ℃, followed by 42 cycles of 45 s for denaturing at 94℃, at annealing temperature for 45 s and 1 min at 72 ℃, after that 10 min extending at 72 ℃, and then keep at 4 ℃. Use 1.5% of agarose gel electrophoresis to test the genetic polymorphism.

  7. Optimization of PCR Reaction System for Adh Gene in Pseudoroegneria(Poaceae: Triticeae)%小麦族拟鹅观草属Adh基因PCR扩增条件优化

    Institute of Scientific and Technical Information of China (English)

    张宁宁; 刘全兰; 李磊

    2009-01-01

    拟开发适用于小麦族拟鹅观草属植物系统发育研究的低拷贝核基因,即Adh基因.通过探索模板DNA、Mg2+、dNTP和引物的浓度,以及退火温度等因素对Adh基因扩增的影响,建立最优PCR体系.在25 μL最优 PCR 反应体系中包括如下成分:2.5 μL 10 × PCR 缓冲液,0.6 μL模板DNA(约15 ng),1.5 mmol Mg2+,0.15 mmol dNTP 混合液,0.12 μmol 正向和反向引物,0.75 U Ex Taq 酶;退火温度为55℃.优化后的体系得到清晰目的条带,可用于后续研究.

  8. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

    Science.gov (United States)

    Kralik, Petr; Ricchi, Matteo

    2017-01-01

    Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety. Although the concept of PCR is relatively simple, there are specific issues in qPCR that developers and users of this technology must bear in mind. These include the use of correct terminology and definitions, understanding of the principle of PCR, difficulties with interpretation and presentation of data, the limitations of qPCR in different areas of microbial diagnostics and parameters important for the description of qPCR performance. It is not our intention in this review to describe every single aspect of qPCR design, optimization, and validation; however, it is our hope that this basic guide will help to orient beginners and users of qPCR in the use of this powerful technique. PMID:28210243

  9. Otimização da técnica de PCR para a detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão Optimization of PCR technique for detection of Xanthomonas axonopodis pv. phaseoli in bean seeds

    Directory of Open Access Journals (Sweden)

    Franklin Cordeiro Silva

    2013-03-01

    Full Text Available O crestamento bacteriano comum, causado por Xanthomonas axonopodis pv. phaseoli (Xap, é a principal bacteriose do feijoeiro no Brasil, sendo transmitida principalmente por sementes. O presente trabalho teve como objetivo aperfeiçoar uma técnica, por meio de diferentes métodos de preparação do extrato, para a detecção de Xap, bem como sua detecção simultânea com Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff nos extratos de sementes de feijão,via PCR. A partir de amostras de sementes de feijão inoculadas artificialmente com Xap e lotes comerciais, foram avaliados: extrato bruto obtido diretamente das sementes, extrato concentrado por filtração em membrana de 0,22µM de diâmetro, extrato concentrado por centrifugação e extrato plaqueado em meio semi-seletivo XCP1 com e sem antibióticos (BIO-PCR. Avaliou-se a presença simultânea de Xap e Cff em 10 lotes comerciais de sementes de feijão através da reação multiplex, utilizandos-se os primers X4c, X4e, CffFOR2 e CffREV4. A partir do extrato bruto, do extrato concentrado por centrifugação e por filtragem em membrana Millipore® não foi possível a detecção de Xap nas sementes de feijão artificialmente contaminadas nem nos 47 lotes comerciais de sementes/ grãos de feijão. A técnica de BIO-PCR permitiu a detecção de Xap a partir de extratos de sementes de feijão artificialmente contaminadas e em 18 dos 47 lotes comerciais. A técnica de detecção simultânea de Xap e Cff no mesmo gel é viável, por amplificar fragmentos de DNA típicos de cada fitobactéria. O uso do meio de cultura XCP1 sem adição de antibióticos permitiu detectar Xap com período de incubação menor em um dia em comparação à detecção utilizando-se o meio de cultura com antibióticosCommon bacterial blight, caused by Xanthomonas axonopodis pv. phaseoli (Xap, is the major bacteriosis affecting bean plants in Brazil and is mainly transmitted by seeds. This study aimed to improve a

  10. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

    Science.gov (United States)

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-05-18

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  11. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

    Directory of Open Access Journals (Sweden)

    Bavo Verhaegen

    2016-05-01

    Full Text Available Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR, which has its specific limitations. Droplet digital PCR (ddPCR has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014 for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan® Environmental Master Mix 2.0; UMM: TaqMan® Universal PCR Master Mix were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  12. PCR, exit stage left ...

    CERN Multimedia

    2004-01-01

    The Prevessin Control Room during LEP's start up in 1989. The Prévessin Control Room (PCR) was recently engulfed in a wave of nostalgia. The PCR, scene of some of the greatest moments in CERN's history, is being dismantled to prepare for a complete overhaul. In February 2006, a new combined control centre for all the accelerators will open its doors on the same site, together with a new building currently under construction (see Bulletin issue 27/2004 of 28 June 2004). This marks the end of an important chapter in CERN's history. The Prévessin Control Room saw its first momentous event 28 years ago when the 400 GeV beam for the SPS was commissioned in the presence of Project Leader John Adams. It was also here that the first proton-antiproton collisions were observed, in 1981. Eight years later, in 1989, operators and directors alike jumped for joy at the announcement of the first electron-positron collisions at the start up of LEP, the biggest accelerator in the world. Today the 80 terminals and PCs have b...

  13. Diagnosis of Cutaneous Leishmaniasis by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    M Heiat

    2010-07-01

    Full Text Available Introduction: Annually, more than 14 million people are reported to be infected with Leishmaniasis all over the world. In Iran, this disease is seen in the form of cutaneous and visceral leishmaniasis, of which the cutaneous form is more wide spread. In recent years, cutaneous leishmaniaisis is diagnosed by PCR utilizing specific primers in order to amplify different parasite genes including ribosomal RNA genes, kinetoplast DNA or tandem repeating sequences. The aim of this research was to detect early stage cutaneous leishmaniasis using Multiplex-PCR technique. Methods: In this study, 67 samples were prepared from patients with cutaneous leishmaniasis. DNA was extracted with phenolchloroform. Each specimen was analyzed using two different pairs of PCR primers. The sensitivity of each PCR was optimized on pure Leishmania DNA prior to use for diagnosis. Two standard parasites L. major and L. tropica were used as positive control. Results: DNA amplification fragments were two 115 bp and 683 bp for AB and UL primers, respectively. The sensitivity of two primers was not equal for detection of L. major and L. tropica. The sensivity of PCR with AB primer was 35 cells, while that for UL primer was 40 cells. Conclusion: The results of this study indicate that PCR is a sensitive diagnostic assay for cutaneous leishmaniasis and could be employed as the new standard for routine diagnosis when species identification is not required. However, the ability to identify species is especially important in prognosis of the disease and in deciding appropriate therapy, especially in regions where more than one type of species and disease are seen by clinicians.

  14. Otimização de metodologia PCR-SSP para identificação de polimorfismos genéticos de TNF e IL2 Optimization of the PCR-SSP methodology in the identification of TNF and IL2 genetic polymorphisms

    Directory of Open Access Journals (Sweden)

    Danilo A. S. Franceschi

    2009-08-01

    Full Text Available A análise de polimorfismos únicos de nucleotídeos (SNPs de citocinas pode ser útil em estudos de frequências alélicas e genotípicas em populações saudáveis de diversas regiões, em estudos de associação com doenças infecciosas ou autoimunes, em estudos antropológicos e na evolução pós-transplante. Estes SNPs podem ser avaliados por diferentes métodos moleculares. O objetivo deste estudo foi aperfeiçoar uma metodologia PCR-SSP simples e rápida para a genotipagem de três SNPs de citocinas usando um único teste laboratorial. Para a identificação de IL2-330T/G e IL2+166G/T foram utilizados dois procedimentos na mesma genotipagem, cada um baseado no uso de quatro iniciadores. Para a detecção de TNF-238G/A foram utilizados dois iniciadores que amplificam a guanina e adenina na posição -238. Este estudo permitiu aperfeiçoar um método simples e rápido para identificar três SNPs de citocinas num único teste, podendo ser utilizado em qualquer laboratório de biologia molecular, como alternativa ao uso de kits de alto custo.The analysis of cytokine single nucleotide polymorphisms (SNPs can be useful in studies of allelic and genotypic frequencies in healthy populations from different regions of Brazil, in association studies of infectious or auto-immune diseases, in anthropological studies and in studies on post-transplant evolution. These SNPs can be assessed by different molecular methods. The objective of this study was to improve a simple and fast methodology, PCR-SSP, for the genotyping of three cytokine SNPs using a single laboratorial test. To identify IL2-330T/G and IL2+166G/T, two procedures were used in the same genotyping assay, each one based on the use of 4 primers. To detect TNF-238G/A, two primers were used that amplify guanine and adenine at position -238. This study enabled the improvement of a simple and fast method for identifying three cytokine SNPs in a single test, which can be adopted in any Molecular

  15. 东海原甲藻细胞色素b基因实时荧光定量PCR检测体系优化%Optimization of real-time quantitative fluorescence PCR system in detecting cytochrome b gene of Prorocentrum donghaiense Lu

    Institute of Scientific and Technical Information of China (English)

    王曲圆; 甄毓; 袁健; 米铁柱; 于志刚

    2013-01-01

    To quantitatively detect the cytochrome b ( Cyt b) gene of Prorocentrum donghaiense Lu in the filed samples, a specific primer was designed, and the quantities of the RNA templates added into the reaction system for reverse transcription as well as the reaction conditions of real-time fluorescent quantitative PCR ( RFQ-PCR) were optimized. The results illustrated that the designed primer had good specificity, being able to be used to differentiate different algal species effectively. In detecting the filed samples, the suitable qualities of the templates for the 20 μX reverse transcription system were 50-200 ng. 10-fold diluting the templates or adding the bovine serum albumin (BSA) with a final concentration 0. 2 μg·μL-1 into the RFQ-PCR system could effectively decrease the inhibitory effect of the inhibitors in the filed samples, and thus, decrease the interferences. The established real-time fluorescent quantitative PCR (RFQ-PCR) assay would facilitate us to study the intrinsic mechanisms of P.donghaiense outbreak and extinction at molecular level.%为定量检测现场样品中东海原甲藻的细胞色素b(cytochrome b,Cytb)基因,本研究设计了该基因的特异性引物,并对现场样品反转录反应体系中加入的模板数量和定量PCR反应条件进行了优化.结果表明:设计的引物具有较好的特异性,可有效区分不同的藻类;针对现场样品,在20μL的反转录体系中,适宜加入的RNA模板的量为50 ~ 200 ng;PCR模板稀释10倍或向定量PCR反应体系中加入终浓度为0.2μg·μL-1的牛血清蛋白(BSA)均能有效降低现场样品中抑制物的抑制作用,减小干扰.该方法的建立对从分子水平探讨东海原甲藻暴发和消亡的内在机制具有重要意义.

  16. 无患子基因组DNA提取及ISSR-PCR反应体系的建立与优化%DNA Extraction and Optimization of ISSR-PCR Reaction System for Sapindus Mukorossi

    Institute of Scientific and Technical Information of China (English)

    姜翠翠; 卢新坤; 方智振; 郭有枝; 姜业先; 刘献勇; 叶新福

    2013-01-01

    应用改良CTAB法提取了无患子基因组DNA,该方法具有操作过程简单、耗时少、能有效降低无患子基因组DNA提取过程中酚类化合物、多糖等杂质的污染.正交试验研究了dNTP浓度、引物浓度、Taq DNA聚合酶这3个因素对ISSR-PCR反应的影响,建立并优化了适合于无患子ISSR-PCR的反应体系.优化的反应总体系20μl含1 ×PCR Buffer,0.4 mmol· L-1 dNTP,0.8μmol· L-1引物,0.75 U Taq DNA聚合酶,20 ng DNA模板.使用此ISSR扩增反应体系,获得了部分不同种质无患子DNA的清晰条带,验证了该体系的稳定性.优化的反应体系为后续无患子遗传多样性的研究奠定了基础.

  17. DNA isolation, optimization of ISSR-PCR system and primers screening of Aralia cordata Thunb%九眼独活基因组DNA提取、ISSR反应体系优化及引物筛选

    Institute of Scientific and Technical Information of China (English)

    王博; 侯大斌; 徐敏; 刘向鸿

    2010-01-01

    目的 探讨九眼独活基因组DNA提取方法 、ISSR反应体系优化及引物筛选,为研究九眼独活居群遗传多样性及原药材DNA鉴定奠定基础.方法 采用改良的CTAB法与常规CTAB法对九眼独活的基因组进行提取,通过紫外、电泳、PCR-ISSR检测方法 进行比较.结果实验表明,改良的CTAB法得到的DNA浓度和纯度较高,并可很好地应用于ISSR分子标记分析;以Mg2+、dNTP、引物和聚合酶建立L9(34)正交设计,并同时考察退火温度和模板浓度,建立适宜的25 μL ISSR-PCR体系为:模板 30 ng,Mg2+ 3.5 mmol·L-1,dNTP 0.6 mmol·L-1,引物0.4 μmol·L-1,Taq酶1 U.结论 以此体系为基础进行引物筛选,在40条ISSR引物中筛选出13 条扩增条带清晰、多态性较高、重复性好的引物.

  18. 均匀设计在候选内参基因RT-qPCR实验条件优化中的应用%Application of uniform design to optimize quantitative real-time RT-qPCR conditions in potential internal control genes

    Institute of Scientific and Technical Information of China (English)

    曹锡梅; 梁俊红; 张潮; 万东方; 蒙晓平; 郭大玮

    2012-01-01

    Objective To investigate the application of the uniform design to optimize quantitative real-time RT-PCR conditions, and apply it for assessment of potential internal control genes in THP-1 cells under different conditions. Methods We obtained from cDNA THP-1 cells. The cDNA template quantity, primer quantity and annealing temperature were studied as 3 key factors for the RT-qPCR conditions. By using the uniform design, 8 levels for each factor were optimized. The standard of score and a standard curve of 5-fold dilution series (1 : 101 ,11 102,1:103,1:104 and 1:105) of template cDNA were used to estimate the amplification condition. Results By using the uniform design, RT-qPCR conditions for assessment of potential internal control genes were obtained from 8 tests in one experiment. The optimized conditions were cDNA template concentration of 0. 5μg, each primer concentration of 200μmol/L in the final reaction, and annealing temperature at 55℃. Conclusion The uniform design is a highly efficient method to optimize the RT-qPCR conditions for potential internal control genes in this study. Furthermore, the uniform design is suitable for the multi-factor and multi-level test conditions.%目的 探讨均匀设计在候选内参基因RT-qPCR实验条件优化中的应用,有助于后期实验筛选不同状态下THP-1细胞的稳定内参基因.方法 以THP 1细胞cDNA为模板,cDNA模板量、引物浓度和退火温度3个因素为影响因素,应用均匀设计3因素8水平,探索影响候选内参基因RT-qPCR的扩增条件,检测不同实验条件下的扩增效果,在最优扩增条件下建立候选内参基因定量扩增的相对标准曲线,并检测扩增效率.结果 通过均匀设计,完成对cDNA模板量、引物浓度和退火温度3因素8水平的实验条件优化,建立候选内参基因RT-qPCR检测的最佳组合为cDNA模板量0.5μg、引物浓度200μmol/L、退火温度55℃.结论 本实验选用均匀设计优化RT-q

  19. High-throughput STR analysis for DNA database using direct PCR.

    Science.gov (United States)

    Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

    2013-07-01

    Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner.

  20. An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications.

    Directory of Open Access Journals (Sweden)

    Sophie Champlot

    Full Text Available BACKGROUND: PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i sample contamination, (ii laboratory surface contamination, (iii carry-over contamination, and (iv contamination of reagents. METHODOLOGY/PRINCIPAL FINDINGS: Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. CONCLUSIONS/SIGNIFICANCE: There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.

  1. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  2. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  3. PCR performance of a thermostable heterodimeric archaeal DNA polymerase.

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  4. Rapid PCR thermocycling using microscale thermal convection.

    Science.gov (United States)

    Muddu, Radha; Hassan, Yassin A; Ugaz, Victor M

    2011-03-05

    temperature distributions in microscale convective thermocyclers(12). Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

  5. Ten-minute purification of PCR products by continuous elution electrophoresis.

    Science.gov (United States)

    Sadakane, Yutaka; Nakagomi, Kazuya; Hatanaka, Yasumaru

    2008-10-01

    We optimized continuous elution electrophoresis (CEE) for rapid purification of PCR products. After PCR amplification, the reaction mixture is applied directly to CEE, and then the PCR products in the size range from 200 to 1500 bp are purified within nearly 10 min. CEE is able to separate two DNA fragments differing in length by 50 bp. As judged by ligation efficiency, the quality of PCR products separated by CEE is equal to that purified by extraction from the melting gels. CEE reduces operational time because purification of the PCR products is a repetitional procedure in recombinant DNA techniques.

  6. Real Time PCR: Principles and Application

    Directory of Open Access Journals (Sweden)

    Safie Amini

    2005-09-01

    especially designated tubes. Both have optimized heatingcooling characteristic. A complete amplification protocol can be performed in 30-45 minutes.The Smart Cycler® is a combination of 16 individual, one tube real time PCR units. It is capable of performing a different PCR program on each of 16 reaction tubes. This is very useful for a rapid optimization of the assay as many variables can be tested at the same time. The Bio-Rad I-cycler IQ® instrument can perform real time amplification with a temperature gradient for specific PCR steps, allowing the optimization of real time PCR assay. The spectrofluorometers in the thermal cycler have a number of differences. Laserbased systems are tuned to excite each fluorophore at a specific wavelength and provide maximum efficiency. Lamp-based systems provide a broad excitation range that can be filtered to work with a number of fluorophores. The laser source not only gives brighter illumination to the fluorophore signal, but also produces less background noise. In conclusion, real time PCR is a powerful advancement of the basic PCR technique. The important steps in deciding which particular assay format to use are related to the type of data required. The requirement for a research laboratory is quite distinct from those of a diagnostic laboratory. For the latter, probe confirmation of the PCR product is an essential part of the assay, whereas SYBR green detection may be sufficient for many other applications such as quantifying expression of a gene. All of the real-time PCR machines analyzed are capable of detecting PCR product in real time and a specific assay can be made optionally on every system. However, there are some decisions to be made when selecting among different formats. The choice of system is dependent on individual laboratory needs.Considering diagnostic applications, the Light Cycler® or Smart Cycler® may obtain faster results for urgent assays. This could reduce the time of analysis to result from 3-4 hours to

  7. Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

    Science.gov (United States)

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-09-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  8. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Roman Wölfel

    2012-08-01

    Full Text Available Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  9. Digital PCR: A brief history

    OpenAIRE

    2014-01-01

    Digital PCR for quantification of a target of interest has been independently developed several times, being described in 1990 and 1991 using the term “limiting dilution PCR” and in 1999 using the term “digital PCR”. It came into use in the decade following its first development but its use was cut short by the description of real-time PCR in 1996. However digital PCR has now had a renaissance due to the recent development of new instruments and chemistry which have made it a much simpler and...

  10. Rapid and simple method of qPCR primer design.

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2015-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized "real-time" using a computer. In qPCR, a reporter dye system is used which intercalates with DNA's region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®). However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest(®), Unafold(®), and Beacon designer(®)) to design qPCR primers.

  11. 抗氮营养阻遏黄孢原毛平革菌pcR5305产酶条件优化%Optimizing Enzyme-production Conditions of Phanerochaete chrysosporium pcR5305 which Can be Resistant to Nitrogen Nutritional Repression

    Institute of Scientific and Technical Information of China (English)

    叶友贤

    2014-01-01

    We optimized the enzyme-production nutritional medium and environment condition of the breeding strain of Phanerochaete chrysosporium pcR5305 which can be resistant to nitrogen nutritional repression. Results show that pcR5305 had the best enzyme activity when it was grown at the following condition:the medium was mixed with ammoni-um tartrate 2.2g/L, glucose 20g/L, Mn2+0.2g/L and Tween 80 1g/L, and adding benzyl alcohol 5.2mmol/L after 24h, the cul-turing temperature was 37℃and the initial pH was 4.5. After 6d static cultured, the LiP activity could reached 2013.59U/L, and the MnP activity could reach 353.42U/L, increased respectively by 23.41% and 27.97% against the un-optimized cul-tured strains.%对选育的抗营养阻遏产酶黄孢原毛平革菌pcR5305菌株的产酶营养基质与环境条件进行了优化。实验结果表明pcR5305在37℃、初始pH4.5条件下,以酒石酸铵(富氮2.2g/L),葡萄糖(20g/L),Mn2+(0.2g/L),Tween 80(1g/L)为主要成分的优质培养基中培养24h后添加5.2mmol/L苯甲醇,pcR5305的产酶活性最高。上述条件下,该菌接种后静置培养6 d,LiP活力可达到2013.59U/L,MnP活力达到353.42U/L,分别提高了23.41%和27.97%。

  12. Primer design versus PCR bias in methylation independent PCR amplifications.

    Science.gov (United States)

    Wojdacz, Tomasz K; Borgbo, Tanni; Hansen, Lise Lotte

    2009-05-16

    Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten "CpG-free" primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the "CpG-free" primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1-0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.

  13. A multiplex PCR for detection of six viruses in ducks.

    Science.gov (United States)

    Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

    2017-10-01

    In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10(2) copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

  14. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    Energy Technology Data Exchange (ETDEWEB)

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  15. Development and Validation of a HPV-32 Specific PCR Assay

    Directory of Open Access Journals (Sweden)

    Leigh Janet

    2009-06-01

    Full Text Available Abstract Background Human Papillomavirus-32 (HPV-32 has traditionally been associated with focal-epithelial-hyperplasia (FEH. It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR assay. Materials and methods An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects. Results The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. Conclusion Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

  16. Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

    Science.gov (United States)

    White, P Lewis; Barnes, Rosemary A; Springer, Jan; Klingspor, Lena; Cuenca-Estrella, Manuel; Morton, C Oliver; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem J G; Mengoli, Carlo; Donnelly, J Peter; Heinz, Werner J; Loeffler, Juergen

    2015-09-01

    Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.

  17. Specific PCR product primer design using memetic algorithm.

    Science.gov (United States)

    Yang, Cheng-Hong; Cheng, Yu-Huei; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2009-01-01

    To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150-300 bps and 500-800 bps, and two different methods of calculating T(m) for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near-optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma-pd/. 2009 American Institute of Chemical Engineers

  18. Validation of real time PCR assays for use in routine diagnostics of pig diarrhoea

    DEFF Research Database (Denmark)

    Ståhl, Marie; Hjulsager, Charlotte Kristiane; Breum, Solvej Østergaard

    At the National Veterinary Institute in Denmark we want to optimize routine diagnostic analyses by screening samples simultaneously for several agents by real time PCR. Here we present the validation of real time PCR assays for E. coli F4. E coli F18 and Lawsonia intracellularis2 in pig feces...

  19. A rapid and direct real time PCR-based method for identification of Salmonella spp

    DEFF Research Database (Denmark)

    Rodriguez-Lazaro, D.; Hernández, Marta; Esteve, T.

    2003-01-01

    The aim of this work was the validation of a rapid, real-time PCR assay based on TaqMan((R)) technology for the unequivocal identification of Salmonella spp. to be used directly on an agar-grown colony. A real-time PCR system targeting at the Salmonella spp. invA gene was optimized and validated ...

  20. Primer design using Primer Express® for SYBR Green-based quantitative PCR.

    Science.gov (United States)

    Singh, Amarjeet; Pandey, Girdhar K

    2015-01-01

    To quantitate the gene expression, real-time RT-PCR or quantitative PCR (qPCR) is one of the most sensitive, reliable, and commonly used methods in molecular biology. The reliability and success of a real-time PCR assay depend on the optimal experiment design. Primers are the most important constituents of real-time PCR experiments such as in SYBR Green-based detection assays. Designing of an appropriate and specific primer pair is extremely crucial for correct estimation of transcript abundance of any gene in a given sample. Here, we are presenting a quick, easy, and reliable method for designing target-specific primers using Primer Express(®) software for real-time PCR (qPCR) experiments.

  1. Optimization of Quantitative PCR Methods for Enteropathogen Detection.

    Science.gov (United States)

    Liu, Jie; Gratz, Jean; Amour, Caroline; Nshama, Rosemary; Walongo, Thomas; Maro, Athanasia; Mduma, Esto; Platts-Mills, James; Boisen, Nadia; Nataro, James; Haverstick, Doris M; Kabir, Furqan; Lertsethtakarn, Paphavee; Silapong, Sasikorn; Jeamwattanalert, Pimmada; Bodhidatta, Ladaporn; Mason, Carl; Begum, Sharmin; Haque, Rashidul; Praharaj, Ira; Kang, Gagandeep; Houpt, Eric R

    2016-01-01

    Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

  2. Methylation-Specific PCR Unraveled

    Directory of Open Access Journals (Sweden)

    Sarah Derks

    2004-01-01

    Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

  3. MPprimer: a program for reliable multiplex PCR primer design.

    Science.gov (United States)

    Shen, Zhiyong; Qu, Wubin; Wang, Wen; Lu, Yiming; Wu, Yonghong; Li, Zhifeng; Hang, Xingyi; Wang, Xiaolei; Zhao, Dongsheng; Zhang, Chenggang

    2010-03-18

    Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2x to 5x plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20x, 20x, 20x, 14x, and 5x plex PCR reactions in five tubes to detect underlying exon deletions. MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

  4. MPprimer: a program for reliable multiplex PCR primer design

    Directory of Open Access Journals (Sweden)

    Wang Xiaolei

    2010-03-01

    Full Text Available Abstract Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy, which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

  5. Optimization of TRAP-PCR System and A Primary Study on Genetic Variation of Genes Related to Lignocellulose Enzyme of Three Wood-rotting Fungi%3种木腐菌木质纤维素相关酶基因的TRAP标记体系优化与遗传变异初探

    Institute of Scientific and Technical Information of China (English)

    王玉英; 吕世翔; 王秋玉

    2012-01-01

    Collecting three typical wood-rotting fungi from Maoer Mountain Forest Farm of Northeast Forestry University as the experimental materials, a preliminary study on polymorphism of the encoding genes of five lignocellulose enzyme, including lip, mnp, lac, cbh II and cdh, have been done. At the same time, the reaction system and process of TRAP-PCR of three wood-rotting fungi have been optimized firstly, and then 11 pair primers were selected from 32 pair primers, including 3 pair primers of MnP encoding gene, 3 pair of Lae encoding gene, 3 pair of CBH encoding gene, as well as 2 pair of CDH encoding gene. The result shows that three wood-rotting fungi generated 265 bands by TRAP-PCR, among which 206 bands are polymorphic bands with 77.74% in potymorphic rate ranging from 60% in minimum and 100% in maximum. This experiment proved the possibility of genetic analysis of wood- rotting fungus by using TRAP marker.%以黑龙江省东北林业大学帽儿山实验林场的3种典型木材腐朽菌为试验材料,采用TRAP分子标记的手段,针对5种木质素与纤雏素相关酶基因LiP、MnP、Lac、CBHⅡ、CDH设计引物,初步分析这些编码基因的多态性。试验确定了3种木腐菌TRAP分子标记的反应体系和反应程序。经过对32对引物进行筛选试验,共确定11对引物,包括3对标记MnP编码基因的引物、3对标记Lac编码基因的引物、3对标记CBH编码基因的引物,以及2对标记CDH编码基因的引物。结果表明:3种木腐菌的TRAP.PCR标记共产生了265条条带,其中多态性条带206条,占总条带的77.74%,多态性最高为100%,最低为60%,证明了TRAP分子标记可以应用于木腐菌的遗传分析。

  6. PCR

    African Journals Online (AJOL)

    ELO

    2012-01-05

    mail: ... Plates transferred to the laboratory were incubated at 25°C for 48 h for growing the .... Use of a probiotic to control lactococcosis and streptococcosis in ... Streptococcus difficile: two new streptococcal species causing a.

  7. Optimally Stopped Optimization

    Science.gov (United States)

    Vinci, Walter; Lidar, Daniel A.

    2016-11-01

    We combine the fields of heuristic optimization and optimal stopping. We propose a strategy for benchmarking randomized optimization algorithms that minimizes the expected total cost for obtaining a good solution with an optimal number of calls to the solver. To do so, rather than letting the objective function alone define a cost to be minimized, we introduce a further cost-per-call of the algorithm. We show that this problem can be formulated using optimal stopping theory. The expected cost is a flexible figure of merit for benchmarking probabilistic solvers that can be computed when the optimal solution is not known and that avoids the biases and arbitrariness that affect other measures. The optimal stopping formulation of benchmarking directly leads to a real-time optimal-utilization strategy for probabilistic optimizers with practical impact. We apply our formulation to benchmark simulated annealing on a class of maximum-2-satisfiability (MAX2SAT) problems. We also compare the performance of a D-Wave 2X quantum annealer to the Hamze-Freitas-Selby (HFS) solver, a specialized classical heuristic algorithm designed for low-tree-width graphs. On a set of frustrated-loop instances with planted solutions defined on up to N =1098 variables, the D-Wave device is 2 orders of magnitude faster than the HFS solver, and, modulo known caveats related to suboptimal annealing times, exhibits identical scaling with problem size.

  8. Optimization and Optimal Control

    CERN Document Server

    Chinchuluun, Altannar; Enkhbat, Rentsen; Tseveendorj, Ider

    2010-01-01

    During the last four decades there has been a remarkable development in optimization and optimal control. Due to its wide variety of applications, many scientists and researchers have paid attention to fields of optimization and optimal control. A huge number of new theoretical, algorithmic, and computational results have been observed in the last few years. This book gives the latest advances, and due to the rapid development of these fields, there are no other recent publications on the same topics. Key features: Provides a collection of selected contributions giving a state-of-the-art accou

  9. Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR

    DEFF Research Database (Denmark)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas

    2017-01-01

    /N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical......Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order...... 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S...

  10. Identification of neotropical felid faeces using RCP-PCR.

    Science.gov (United States)

    Roques, S; Adrados, B; Chavez, C; Keller, C; Magnusson, W E; Palomares, F; Godoy, J A

    2011-01-01

    Faeces similarity among sympatric felid species has generally hampered their use in distributional, demographic and dietary studies. Here, we present a new and simple approach based on a set of species-specific primers, for the unambiguous identification of faeces from sympatric neotropical felids (i.e. puma, jaguar, jaguarundi and ocelot/ margay). This method, referred to as rapid classificatory protocol-PCR (RCP-PCR), consists of a single-tube multiplex PCR yielding species-specific banding patterns on agarose gel. The method was optimized with samples of known origin (14 blood and 15 fresh faeces) and validated in faecal samples of unknown origin (n = 138), for some of which (n = 40) we also obtained species identification based on mtDNA sequencing. This approach proved reliable and provides high identification success rates from faeces. Its simplicity and cost effectiveness should facilitate its application for routine surveys of presence and abundance of these species. © 2010 Blackwell Publishing Ltd.

  11. [Application of rapid PCR to authenticate medicinal snakes].

    Science.gov (United States)

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

    2014-10-01

    To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

  12. 产耐热木聚糖酶细菌的分离鉴定及酶易错PCR致突变条件优化%Isolation & Identification of a Heat-Resistant Xylanase-Producing Bacterial Strain & Optimization of the Enzyme Error-Prone PCR Mutagenic Conditions

    Institute of Scientific and Technical Information of China (English)

    赵超; 张宁宁; 梅凡; 艾超; 阮灵伟; 黄一帆; 刘斌

    2013-01-01

    从福建省永泰县温泉采集样品中筛选到1株产耐热木聚糖酶嗜热菌株TC-W7,并获得该木聚糖酶基因。在此基础上,采用易错PCR技术在木聚糖酶基因中引入突变,研究Mg2+浓度、Mn2+浓度、dTTP/dCTP浓度等条件对突变率的影响。通过形态特征、生理生化试验及16S rRNA序列相似性比对分析,初步鉴定菌株TC-W7为土壤芽胞杆菌(Geobacillus),菌株TC-W7在最适温度75℃和 pH 8.2条件下,其木聚糖酶活力为215.83 U/mL,Triton X-100和DDT能显著增强该酶的活性。在 Mg2+浓度为20μmol/L,Mn2+浓度为0.80μmol/L,dTTP/dCTP浓度为0.30 mmol/L的致突变条件下,碱基突变率为0.98%。 Geobacillus sp. TC-W7产木聚糖酶具有较好的耐热和耐碱等工业应用特性,对该酶易错PCR致突变条件优化结果,可用于后续木聚糖酶的耐热定向进化。%A heat-resistant xylanase-producing bacterial strain TC-W7 from samples collected in a hot spring in Yong-tai County, Fujian Province was screened and obtained xylanase gene of the strain. Based on these an error-prone PCR ( Ep-PCR) technique was adopted to introduce mutation in the xylanase gene, to study the effects of the concentration such as Mg2+, Mn2+ and dTTP/dCTP and other conditions on the mutation rate. It was initially identified that strain TC-W7 belonged to Geobacillus through morphology features, physiological and biochemical tests as well as 16S rRNA sequence comparative analysis. Under the most suitable temperature 75℃ and pH 8. 2, the activity of xylanase was at 215. 83 U/mL, Triton X-100 and DDT could remarkably increase the activity of xylanase. The base mutation rate was at 0. 98% under the mutagenic conditions of 20. 0 μmol/L Mg2+, 0. 80 μmol/L Mn2+ and 0. 30 mmol/L dTTP/dCTP. The xylanase-producing Geobacillus sp. TC-W7 had a fine heat and alkali resistance and other industry appli-cable features. The results of Ep-PCR mutagenic conditions optimization of the enzyme can be used for

  13. LUX real-time PCR assay for the detection of porcine circovirus type 2.

    Science.gov (United States)

    Vilcek, Stefan; Vlasakova, Michaela; Jackova, Anna

    2010-05-01

    Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR.

  14. Selective control of primer usage in multiplex one-step reverse transcription PCR

    Directory of Open Access Journals (Sweden)

    Paul Natasha

    2009-12-01

    Full Text Available Abstract Background Multiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step. Results Herein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats. Conclusions The need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.

  15. Detection of a putative virulence cadF gene of Campylobacter jejuni obtained from different sources using a microfabricated PCR chip

    DEFF Research Database (Denmark)

    Poulsen, Claus Riber; El-Ali, Jamil; Perch-Nielsen, Ivan R.

    2005-01-01

    A microfabricated polymerase chain reaction (PCR) chip made of epoxy-based photoresist (SU-8) was recently designed and developed. In this study, we tested whether the PCR chip could be used for rapid detection of a potential virulence determinant, the cadF gene of Campylobacter jejuni. PCR...... was performed using published PCR conditions and primers for the C. jejuni cadF gene. DNA isolated from a C. jejuni reference strain CCUG 11284, C. jejuni isolates obtained from different sources (chicken and human), and Campylobacter whole cells were used as templates in the PCR tests. Conventional PCR in tube...... was used as the control. After optimization of the PCR chip, PCR positives on the chip were obtained from 91.0% (10/11) of the tested chips. A fast transition time was achieved with the PCR chip, and therefore a faster cycling time and a shorter PCR program were obtained. Using the PCR chip, the cadF gene...

  16. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus

    2015-01-01

    distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were......High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates...... are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial...

  17. Inhibitory effect of common microfluidic materials on PCR outcome

    KAUST Repository

    Kodzius, Rimantas

    2012-02-20

    Microfluidic chips have a variety of applications in the biological sciences and medicine. In contrast with traditional experimental approaches, microfluidics entails lower sample and reagent consumption, allows faster reactions and enables efficient separation. Additionally microfluidics offers other advantages accruing from the fluids’ various distinct behaviors, such as energy dissipation, fluidic resistance, laminar flow, and surface tension. Biological molecules suspended in fluid and transported through microfluidics channels interact with the channel-wall material. This interaction is even stronger in high surface-area-to-volume ratio (SAVR) microfluidic channels. Adsorption and inhibition of biomolecules occur when these materials come in contact with biomolecular reaction components. Polymerase chain reaction (PCR) is a thermal cycling procedure for amplifying target DNA. The PCR compatibility of silicon, silicon dioxide (SiO2) and other surfaces have been studied; however the results are inconclusive. Usually for protein-surface interaction measurements, bulky and expensive equipment is used, such as Atomic Force Microscopy (AFM), Scanning or Transmission Electron Microscopy (SEM, TEM), spectrophotometric protein concentration measurement, Fourier transform infrared spectroscopy (FTIR) or X-Ray photoelectron spectroscopy (XPS). \\tThe PCR reaction components include the DNA template, primers, DNA polymerase (the main component), dNTPs, a buffer, divalent ions (MgCl2), and KCl. \\tWe designed a simple, relatively quick measurement that only requires a PCR cycler; thus it mimics actual conditions in PCR cycling. In our study, we evaluated the inhibitory affect of different materials on PCR, which is one of the most frequently used enzymatic reactions in microfluidics. PCR reaction optimization through choice of surface materials is of the upmost importance, as it enables and improves enzymatic reaction in microfluidics. Our assessment of the PCR

  18. Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

    Science.gov (United States)

    Sanchez, J. Aquiles; Pierce, Kenneth E.; Rice, John E.; Wangh, Lawrence J.

    2004-01-01

    Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFΔ508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells. PMID:14769930

  19. Development of internal controls for PCR detection of Bacillus anthracis.

    Science.gov (United States)

    Brightwell, G; Pearce, M; Leslie, D

    1998-12-01

    This work describes the development and evaluation of a multiplex polymerase chain reaction (PCR) for the detection of Bacillus anthracis strains harbouring plasmid pX02. The multiplex also incorporated an internal control (IC) to avoid false negative reactions. Internal controls consisted of plasmids containing modified PCR target sequences, corresponding to the capC and BA813 genes of B. anthracis, which were then co-amplified with the original target sequences using the same set of amplimers. The initial IC construct comprised of an internally deleted form of the genomic target sequence cloned into pUC19. A series of nested DNA fragments corresponding to the 23S rRNA sequences of Bacillus cereus were then subcloned into the point of deletion, producing a number of IC constructs with similar sequences but increasing product size on PCR amplification. Neither the presence of IC DNA template or IC PCR product size affected the specificity or non-specific cross-reactivity of the original PCR assay. The concentration of IC was critical, too much IC DNA template would out compete the genomic DNA template, thus giving a false negative result. However, when the concentration of IC was optimal assay sensitivity was not compromised.

  20. Simultaneous detection of HBV and HCV by multiplex PCR normalization

    Institute of Scientific and Technical Information of China (English)

    Ning Wang; Xue-Qin Gao; Jin-Xiang Han

    2004-01-01

    AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV.METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method.RESULTS: The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%,respectively for both HBsAg and anti-HCV positive patients,and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165%and 83.3%, respectively for anti-HCV positive patients.CONCLUSION: The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.

  1. Codon optimizing for increased membrane protein production

    DEFF Research Database (Denmark)

    Mirzadeh, K.; Toddo, S.; Nørholm, Morten

    2016-01-01

    . As demonstrated with two membrane-embedded transporters in Escherichia coli, the method was more effective than optimizing the entire coding sequence. The method we present is PCR based and requires three simple steps: (1) the design of two PCR primers, one of which is degenerate; (2) the amplification...

  2. Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

    Institute of Scientific and Technical Information of China (English)

    LI Haitao; LIU Dongming; GAO Jiguo

    2011-01-01

    16S rDNA and ERIC (Enterobacteia Repetitive Intergenic Consensus Sequences) based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus" strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn't have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.

  3. DNA polymerase preference determines PCR priming efficiency

    Science.gov (United States)

    2014-01-01

    Background Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3’ hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Results Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3’ end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. Conclusions DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification

  4. A standard curve based method for relative real time PCR data processing

    Directory of Open Access Journals (Sweden)

    Krause Andreas

    2005-03-01

    Full Text Available Abstract Background Currently real time PCR is the most precise method by which to measure gene expression. The method generates a large amount of raw numerical data and processing may notably influence final results. The data processing is based either on standard curves or on PCR efficiency assessment. At the moment, the PCR efficiency approach is preferred in relative PCR whilst the standard curve is often used for absolute PCR. However, there are no barriers to employ standard curves for relative PCR. This article provides an implementation of the standard curve method and discusses its advantages and limitations in relative real time PCR. Results We designed a procedure for data processing in relative real time PCR. The procedure completely avoids PCR efficiency assessment, minimizes operator involvement and provides a statistical assessment of intra-assay variation. The procedure includes the following steps. (I Noise is filtered from raw fluorescence readings by smoothing, baseline subtraction and amplitude normalization. (II The optimal threshold is selected automatically from regression parameters of the standard curve. (III Crossing points (CPs are derived directly from coordinates of points where the threshold line crosses fluorescence plots obtained after the noise filtering. (IV The means and their variances are calculated for CPs in PCR replicas. (V The final results are derived from the CPs' means. The CPs' variances are traced to results by the law of error propagation. A detailed description and analysis of this data processing is provided. The limitations associated with the use of parametric statistical methods and amplitude normalization are specifically analyzed and found fit to the routine laboratory practice. Different options are discussed for aggregation of data obtained from multiple reference genes. Conclusion A standard curve based procedure for PCR data processing has been compiled and validated. It illustrates that

  5. PCR deduction of invasive and colonizing pneumococcal serotypes from Venezuela: a critical appraisal.

    Science.gov (United States)

    Bello Gonzalez, Teresita; Rivera-Olivero, Ismar Alejandra; Sisco, María Carolina; Spadola, Enza; Hermans, Peter W; de Waard, Jacobus H

    2014-04-15

    Serotype surveillance of Streptococcus pneumoniae is indispensable for evaluating the potential impact of pneumococcal conjugate vaccines. Serotyping by the standard Quellung reaction is technically demanding, time consuming, and expensive. A simple and economical strategy is multiplex PCR-based serotyping. We evaluated the cost effectiveness of a modified serial multiplex PCR (mPCR), resolving 24 serotypes in four PCR reactions and optimally targeting the most prevalent invasive and colonizing pneumococcal serotypes found in Venezuela. A total of 223 pneumococcal isolates, 140 invasive and 83 carriage isolates, previously serotyped by the Quellung reaction and representing the 18 most common serotypes/groups identified in Venezuela, were serotyped with the adapted mPCR. The mPCR serotyped 76% of all the strains in the first two PCR reactions and 91% after four reactions, correctly identifying 17 serotypes/groups. An isolate could be serotyped with mPCR in less than 2 minutes versus 15 minutes for the Quellung reaction, considerably lowering labor costs. A restrictive weakness of mPCR was found for the detection of 19F strains. Most Venezuelan 19F strains were not typeable using the mPCR, and two 19F cps serotype variants were identified. The mPCR assay is an accurate, rapid, and economical method for the identification of the vast majority of the serotypes from Venezuela and can be used in place of the standard Quellung reaction. An exception is the identification of serotype 19F. In this setting, most 19F strains were not detectable with mPCR, demonstrating a need of serology-based quality control for PCR-based serotyping.

  6. Real-time PCR in Food Science: PCR Diagnostics.

    Science.gov (United States)

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  7. Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.

    Science.gov (United States)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Hung, Tran Quang; Wolff, Anders; Bang, Dang Duong

    2017-04-01

    Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 10(11) molecules/mm(2). In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

  8. Polymerase chain reaction (PCR assay for rapid diagnosis and its role in prevention of human Brucellosis in Punjab, India

    Directory of Open Access Journals (Sweden)

    Moti Yohannes Gemechu

    2011-01-01

    Conclusions: Early-case reporting is possible by rapid tests like PCR. Thus, PCR is a promising diagnostic tool for routine investigation and surveillance of brucellosis which is the key element for management of prevention and control programmes. But patient condition before testing, optimal clinical specimen, sample volume used, simple and efficient DNA extraction protocol are the points of concern for PCR to be used as a routine test in clinical laboratory practice.

  9. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  10. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  11. Detection of large expansions in myotonic dystrophy type 1 using triplet primed PCR

    Directory of Open Access Journals (Sweden)

    Susmita eSingh

    2014-04-01

    Full Text Available Myotonic dystrophy type 1 (DM1 is an autosomal dominant neuromuscular disease caused by expansion of a CTG trinucleotide repeat in the DMPK gene. Methodology for genetic testing of DM1 is currently not optimal, in particular for the early-onset patients in pediatric populations where large expanded (CTGn alleles are usually common. Individuals who are homozygous for a normal allele and individuals who are heterozygous for one normal and one large expanded allele are indistinguishable by conventional PCR, as both generate a single product of the normal allele. Thus, reflex Southern blot has often been needed to distinguish these cases. With the aim to decrease the need for reflex Southern blot tests, a novel, single-tube CTG repeat primed PCR technology was designed to distinguish the true homozygous patients from the individuals whose large alleles are missed by conventional PCR. The method utilizes two gene-specific primers that flank the triplet repeat region and a third primer set complementary to the repeated region to detect the large alleles. Compared to traditional PCR, this novel Triplet-repeat Primed PCR can detect the presence of large expanded alleles with demonstrating a ladder pattern. Using this single-step protocol, 45 specimens were tested. The alleles with sizes ≤ 85 repeats were determined by the gene specific primers. 13 abnormal alleles, which missed by the conventional PCR, were successfully detected by the Triplet-repeat Primed PCR. All the abnormal alleles were confirmed and measured by Southern Blot analysis. In summary, optimized TP-PCR can accurately detect the presence of the large expanded alleles. With the ability to distinguish the true homozygous patients from the false negative homozygous individuals, the application of the optimized TP-PCR can significantly reduce the need of Southern Blot tests.

  12. Evaluation of PCR methods for detection of Brucella strains from culture and tissues.

    Science.gov (United States)

    Çiftci, Alper; İça, Tuba; Savaşan, Serap; Sareyyüpoğlu, Barış; Akan, Mehmet; Diker, Kadir Serdar

    2017-03-03

    The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

  13. Mechanism of the interaction between Au nano- particles and polymerase in nanoparticle PCR

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nanoparticle PCR is a novel method to optimize DNA amplification. It performs well in improving specificity, enhancing sensitivity and speed. Several mechanisms were proposed in previous studies: one was based on the interaction between gold nanoparticles (AuNPs) and DNA while the other was attributed to the heat transfer property of AuNPs. In this paper, we propose that the interaction between AuNPs and DNA polymerase can significantly influence PCR. First, the addition of DNA polymerase can eliminate the inhibitory effects of excess AuNPs. Second, the addition of AuNPs will increase yield of the desired PCR product and make the optimum concentration of DNA polymerase move to higher value. Third, while excess polymerase might inhibit amplification efficiency, AuNPs can reverse this process and the yield of PCR amplification. Based on these results we propose a possible mechanism that AuNPs might modulate the activity of polymerase and improve PCR amplification.

  14. Anchored PCR (A-PCR):A new method for chromosome walking

    Institute of Scientific and Technical Information of China (English)

    CHEN Bojun; SUN Chao; WANG Yong; HU Yuanlei; LIN Zhongping

    2004-01-01

    @@ PCR-based techniques are most popular methods for isolation of DNA sequences flanking a known region.Such techniques published to date mainly include three types: inverse PCR (IPCR)[1-3], ligation-mediated PCR (LM-PCR)[4-9] and randomly primed PCR (RP-PCR)[10-12].IPCR was the first method developed for this kind of purpose. However, it is now rarely used because of the difficulty in finding suitable restriction sites in the target region or poor circularization of the template molecule.LM-PCR and RP-PCR are more frequently used nowadays, yet they also have some limitations. For example,LM-PCR depends on restriction sites within a reasonable distance in the flanking regions, while the amplified products of RP-PCR are generally small (<1 kb). Moreover, both methods often result in excessive amplification of non-specific molecules, which greatly reduces their efficiencies in obtaining sequences of interest. To resolve these problems, some new strategies have emerged in the past few years, such as Vectorette-PCR[6], biotin-capture PCR[7], TAIL-PCR[l2] and T-linker PCR[9]. These improved methods are more efficient than their old versions;however, most of them are still limited by restriction digestion or ligation. Although the intervening steps are avoided in TAIL-PCR, the amplified fragments are often small because of the use of random primers.

  15. Chemical surface management for micro PCR in silicon chip thermocyclers

    Science.gov (United States)

    Felbel, Jana; Bieber, Ivonne; Koehler, Johann M.

    2002-11-01

    Silicon, silicon dioxide, glass and other key materials of micro system technology show an inhibiting effect on PCR. This negative influence becomes seriously, if devices are miniaturized, particularly in case of flow-through devices due to their high surface to volume ratio. In contrast, alkyl-substituted surfaces do not inhibit the reaction. Although the silanization improves the compatibility, the suppression of inhibition by wall surface treatment was not stable over longer time intervals. Therefore, the stability of chemical surface modifications was studied in dependence of silanization, material, pH, temperature and buffer composition. The efficiency of surface covering by molecular substitution was characterized by wetting experiments as well as by PCR test runs. The results show that the surface treatment can be optimized by the choice of silanization agents and the concentration of surface active additives.

  16. MultiPLX: automatic grouping and evaluation of PCR primers.

    Science.gov (United States)

    Kaplinski, Lauris; Remm, Maido

    2015-01-01

    In this chapter we describe MultiPLX-a tool for automatic grouping of PCR primers for multiplexed PCR. Both generic working principle and step-by-step practical procedures with examples are presented. MultiPLX performs grouping by calculating many important interaction levels between the different primer pairs and then distributes primer pairs to groups so that the strength of unwanted interactions is kept below user-defined compatibility level. In addition it can be used to select optimal primer pairs for multiplexing from list of candidates. MultiPLX can be downloaded from http://bioinfo.ut.ee/?page_id=167. Graphical web-based interface to most functions of MultiPLX is available at http://bioinfo.ut.ee/multiplx/.

  17. Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

    Science.gov (United States)

    Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

    2017-02-02

    The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loadsPCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Restriction site-dependent PCR: an efficient technique for fast cloning of new genes of microorganisms.

    Science.gov (United States)

    Jiang, Yu; Pei, Jianjun; Song, Xin; Shao, Weilan

    2007-12-31

    New bioactive proteins need to be screened from various microorganisms for the increasing need for industrial and pharmaceutical peptide, proteins, or enzymes. A novel polymerase chain reaction (PCR) method, restriction site-dependent PCR (RSD-PCR), was designed for rapid new genes cloning from genomic DNA. RSD-PCR strategy is based on these principles: (i) restriction sites disperse throughout genomes are candidacy for universal pairing; (ii) a universal primer is a combination of a 3'-end of selected restriction sites, and a 5'-end of degenerated sequence. A two-round PCR protocol was designed and optimized for the RSD-PCR: amplify the single strand target template from genomic DNA by a specific primer and amplify the target gene by using the specific primer and one of the universal RSD-primers. The optimized RSD-PCR was successfully applied in chromosome walking using specific internal primers, and cloning of new genes using degenerated primers derived from NH2-terminal amino acid sequence of protein.

  19. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix. PMID:22975959

  20. Restriction endonucleases digesting DNA in PCR buffer

    Institute of Scientific and Technical Information of China (English)

    LIU Xue-dong; ZHENG Dong; ZHOU Yan-na; MAO Wei-wei; MA Jian-zhang

    2005-01-01

    Six commonly used restriction endonucleases (Res) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for additional magnesium supplemented as activator, Res, except EcoR I appeared star activity, completely digested unmethylated lambda DNA after overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all Res tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed directly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of Res in PCR buffer. This simplified method for RE digestion of PCR products could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymorphism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.

  1. Real-time PCR in microfluidic devices

    Science.gov (United States)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  2. Detection of Eperythrozoon wenyoni by PCR assay

    Institute of Scientific and Technical Information of China (English)

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO

    2009-01-01

    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  3. optimal control

    Directory of Open Access Journals (Sweden)

    L. I. Rozonoer

    1999-01-01

    Full Text Available Necessary and sufficient conditions for existence of optimal control for all initial data are proved for LQ-optimization problem. If these conditions are fulfilled, necessary and sufficient conditions of optimality are formulated. Basing on the results, some general hypotheses on optimal control in terms of Pontryagin's maximum condition and Bellman's equation are proposed.

  4. Absolute quantification by droplet digital PCR versus analog real-time PCR

    Science.gov (United States)

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  5. Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp.

    Science.gov (United States)

    Rodríguez, Islay; Burri, Caroline; Noda, Angel A; Douet, Véronique; Gern, Lise

    2015-01-01

    Ticks transmit a great variety of pathogenic microorganisms to humans and animals. The detection of tick-borne pathogens (TBP) is mainly by molecular techniques based on polymerase chain reactions (PCR). To design and evaluate a multiplex PCR for the molecular screening of zoonotic TBP for exploratory studies. Control DNA from reference strains, DNA from experimentally-infected biological specimens, and from Rhipicephalus sanguineus ticks collected from domestic and homeless dogs were used. A multiplex PCR assay to detect the presence of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. was designed and optimized using primers previously reported for B. burgdorferi sensu lato and Anaplasma spp., while for Babesia spp. they were designed in silico. The multiplex PCR was evaluated on the DNA from biological samples. A new set of specific primers for Babesia spp. was designed. Adjustment of the master mix reactive concentrations and amplification conditions for the multiplex PCR allowed the successful amplification of the specific amplicons for each microbial group from the control DNA and experimentally-infected biological specimens. The efficiency of the multiplex PCR amplifying three DNA targets was confirmed. Individual and co-infection of Anaplasma spp. and Babesia spp. were detected in the R. sanguineus ticks from dogs. A multiplex PCR assay for the screening of three TBP is available. By using it, B. burgdorferi sensu lato, Anaplasma spp. and Babesia spp. can be detected accurately in one PCR reaction.

  6. Direct PCR: Alternative Diagnostic Method for Diagnosis of Diphtheria Rapidly, Easily and Cost Effective

    Directory of Open Access Journals (Sweden)

    Sunarno Sunarno

    2013-12-01

    Full Text Available Some diseases require immediate and appropriate treatment to decrease the fatality risk patients incident, for example diphtheria. Time to help patients is very crucial since delay of therapy may increase the mortality cases up to 20 times. In other hands, conventional diagnostic methods (the gold standard for diagnosis of diphtheria is time consuming and laborious. Therefore, an alternative diagnostic method which is rapid, easy and inexpensive is needed. In this case, direct PCR has been proved to reduce time and cost in laboratory examination. This study aimed to develop direct PCR as alternative diagnostic method for diagnosis of diphtheria rapidly, easily, and inexpensive. Fifteen samples include 10 isolates of Corynebacterium diphtheriae (toxigenic and 3 isolates of Corynebacterium non- diphtheriae (nontoxigenic and 2 clinical specimens (throat swab was examined by performing direct PCR method and a standard PCR method was used for optimizing the protocols. Result showed that direct PCR can be used to amplify target genes correctly as well as standard PCR. All of C. diphtheriae samples showed bands at 168 bp (dtxR gene marker and 551 bp (tox gene marker while no band appeared in others. Direct PCR detected at least 71 CFU/uL of bacterial cells in samples. We concluded that direct PCR can be used for alternative diagnostic method for diagnosis of diphtheria which is rapid, easy and cost effective.

  7. Pre-PCR processing in bioterrorism preparedness: improved diagnostic capabilities for laboratory response networks.

    Science.gov (United States)

    Hedman, Johannes; Knutsson, Rickard; Ansell, Ricky; Rådström, Peter; Rasmusson, Birgitta

    2013-09-01

    Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification--that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis--so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents.

  8. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    Science.gov (United States)

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  9. Digital PCR dynamic range is approaching that of real-time quantitative PCR.

    Science.gov (United States)

    Jones, Gerwyn M; Busby, Eloise; Garson, Jeremy A; Grant, Paul R; Nastouli, Eleni; Devonshire, Alison S; Whale, Alexandra S

    2016-12-01

    Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

  10. Digital PCR for detection of citrus pathogens

    Science.gov (United States)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  11. Testing for Genetically Modified Foods Using PCR

    Science.gov (United States)

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  12. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

    Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to veri...

  13. Testing for Genetically Modified Foods Using PCR

    Science.gov (United States)

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  14. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    Science.gov (United States)

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

  15. COLD-PCR: Applications and Advantages.

    Science.gov (United States)

    Zuo, Zhuang; Jabbar, Kausar J

    2016-01-01

    Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a single-step amplification method that results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position. This method is based on exploitation of the critical temperature, Tc, at which mutation-containing DNA is preferentially melted over wild type. COLD-PCR can be a good strategy for mutation detection in specimens with high nonneoplastic cell content, small specimens in which neoplastic cells are difficult to micro-dissect and therefore enrich, and whenever a mutation is suspected to be present but is undetectable using conventional PCR and sequencing methods. We describe in this chapter our COLD-PCR-based pyrosequencing method for KRAS mutation detection in various clinical samples using DNA extracted from either fresh or fixed paraffin-embedded tissue specimens.

  16. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

  17. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, J.F.; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the adva

  18. [PCR-based diagnosis of mucormycosis in tissue samples].

    Science.gov (United States)

    Bialek, R; Zelck, U E

    2013-11-01

    Mucormycosis is characterized by a rapid, often fatal progression. Early diagnosis of invasive mucormycosis is the key for timely therapeutic intervention and improved survival. Contrary to the more prevalent aspergillosis, effective antifungal therapy of mucormycosis is mainly limited to amphotericin B. Given the importance to guide the timely initiation of amphotericin B and possible surgical intervention, rapid and specific identification of fungal hyphae is essential. Conventional histopathology depends on abundance and morphology of the fungi as well as on the skills of the personnel, and usually shows an accuracy of 80 %. PCR assays targeting fungal ribosomal genes to identify Mucorales at least at genus level increase sensitivity, allow a rapid identification as well as detection of double mold infections. Thus, PCR assays are beneficial to complement existing approaches. They are recommended to rapidly specify tissue diagnosis and accurate identification of fungi. This will help to guide effective therapy and thereby, survival will increase. Retrospective analyses of mucormycosis by PCR help to evaluate therapeutic interventions and will optimize treatment options.

  19. A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

    DEFF Research Database (Denmark)

    She, Qunxin; Confalonieri, F.; Zivanovic, Y.;

    2000-01-01

    -productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...... screening. The PCR approaches included a novel chromosome walking method termed “paired-PCR”. 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were...... selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half...

  20. Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing.

    Science.gov (United States)

    Chen, Zhiyao; Liu, Yunlong; Duan, Wenbang; Ye, Hui; Wu, Haiping; Li, Jinheng; Zhou, Guohua

    2014-06-01

    Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.

  1. New multiplex PCR methods for rapid screening of genetically modified organisms in foods

    Directory of Open Access Journals (Sweden)

    Nelly eDatukishvili

    2015-07-01

    Full Text Available We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs. New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

  2. New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

    Science.gov (United States)

    Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

    2015-01-01

    We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

  3. Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    De Vos Daniel

    2009-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. Results In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. Conclusion In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.

  4. Dynamic Optimization

    Science.gov (United States)

    Laird, Philip

    1992-01-01

    We distinguish static and dynamic optimization of programs: whereas static optimization modifies a program before runtime and is based only on its syntactical structure, dynamic optimization is based on the statistical properties of the input source and examples of program execution. Explanation-based generalization is a commonly used dynamic optimization method, but its effectiveness as a speedup-learning method is limited, in part because it fails to separate the learning process from the program transformation process. This paper describes a dynamic optimization technique called a learn-optimize cycle that first uses a learning element to uncover predictable patterns in the program execution and then uses an optimization algorithm to map these patterns into beneficial transformations. The technique has been used successfully for dynamic optimization of pure Prolog.

  5. Standardization and validation of simple PCR, duplex PCR and RAPD in comparison to blood smear examination for diagnosing bovine tropical theileriosis.

    Science.gov (United States)

    Sudan, Vikrant; Shanker, Daya; Jaiswal, Amit; Singh, Amit; Pandey, Vijay

    2017-03-01

    Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  6. Quantitative multiplex real-time PCR assay for shrimp allergen: comparison of commercial master mixes and PCR platforms in rapid cycling.

    Science.gov (United States)

    Eischeid, Anne C; Kasko, Sasha M

    2015-01-01

    Real-time PCR has been used widely in numerous fields. In food safety, it has been applied to detection of microbes and other contaminants, including food allergens. Interest in rapid (fast) cycling real-time PCR has grown because it yields results in less time than does conventional cycling. However, fast cycling can adversely affect assay performance. Here we report on tests of commercial master mixes specifically designed for fast real-time PCR using a shrimp allergen assay we previously developed and validated. The objective of this work was to determine whether specialized commercial master mixes lead to improved assay performance in rapid cycling. Real-time PCR assays were carried out using four different master mixes and two different rapid cycling protocols. Results indicated that specialized master mixes did yield quality results. In many cases, linear ranges spanned up to 7 orders of magnitude, R(2) values were at least 0.95, and reaction efficiencies were within or near the optimal range of 90 to 110%. In the faster of the two rapid cycling protocols tested, assay performance and PCR amplification were markedly better for the shorter PCR product. In conclusion, specialized commercial master mixes were effective as part of rapid cycling protocols, but conventional cycling as used in our previous work is more reliable for the shrimp assay tested.

  7. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    Science.gov (United States)

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  8. Stochastic optimization

    CERN Document Server

    Schneider, Johannes J

    2007-01-01

    This book addresses stochastic optimization procedures in a broad manner. The first part offers an overview of relevant optimization philosophies; the second deals with benchmark problems in depth, by applying a selection of optimization procedures. Written primarily with scientists and students from the physical and engineering sciences in mind, this book addresses a larger community of all who wish to learn about stochastic optimization techniques and how to use them.

  9. Selective Optimization

    Science.gov (United States)

    2015-07-06

    consider a probabilistically-constrained portfolio optimization problem [16] to determine a minimum cost distribution of a unit investment among n assets...present a branching technique (Section 5). Through computational experiments on the probabilistic portfolio optimization problem (3) and an optimal ...at one. 15 DISTRIBUTION A: Distribution approved for public release. 6.2 Probabilistic Portfolio Optimization The first class of instances we test

  10. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas

    2010-04-23

    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  11. Multiplex PCR for the concurrent detection and differentiation of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium

    Science.gov (United States)

    Pui, Chai Fung; Wong, Woan Chwen; Chai, Lay Ching; Lee, Hai Yen; Noorlis, Ahmad; Zainazor, Tuan Chilek Tuan; Tang, John Yew Huat; Ghazali, Farinazleen Mohamad; Cheah, Yoke Kqueen; Nakaguchi, Yoshitsugu; Nishibuchi, Mitsuaki; Radu, Son

    2011-01-01

    Salmonellosis outbreaks involving typhoid fever and human gastroenteritis are important diseases in tropical countries where hygienic conditions are often not maintained. A rapid and sensitive method to detect Salmonella spp., Salmonella Typhi and Salmonella Typhimurium is needed to improve control and surveillance of typhoid fever and Salmonella gastroenteritis. Our objective was the concurrent detection and differentiation of these food-borne pathogens using a multiplex PCR. We therefore designed and optimized a multiplex PCR using three specific PCR primer pairs for the simultaneous detection of these pathogens. The concentration of each of the primer pairs, magnesium chloride concentration, and primer annealing temperature were optimized before verification of the specificity of the primer pairs. The target genes produced amplicons at 429 bp, 300 bp and 620 bp which were shown to be 100% specific to each target bacterium, Salmonella spp., Salmonella Typhi and Salmonella Typhimurium, respectively. PMID:22028607

  12. A new approach to primer design for the control of PCR bias in methylation studies

    Directory of Open Access Journals (Sweden)

    Hansen Lise

    2008-07-01

    Full Text Available Abstract Primer design for PCR-based methylation analysis following bisulfite conversion of DNA is considerably more complex than primer design for regular PCR. The choice of the optimal primer set is critical to the performance and correct interpretation of the results. Most methodologies in methylation analysis utilize primers that theoretically amplify methylated and unmethylated templates at the same time. The proportional amplification of all templates is critical but difficult to achieve due to PCR bias favouring the amplification of the unmethylated template. The focus of this brief communication is to point out the important criteria needed for the successful choice of primers that will enable the control of PCR bias in bisulfite based methylation-screening protocols.

  13. Development of an improved species specific PCR test for detection of Haemophilus parasuis

    DEFF Research Database (Denmark)

    Angen, Øystein; Oliveira, Simone; Ahrens, Peter

    2007-01-01

    with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published......A PCR test for identification of Haemophilus parasuis was optimized using the 16S rDNA sequences of the 15 serotype reference strains of H. parasuis. The test was evaluated on a collection of 218 Danish field isolates as well as on 81 representatives of 27 other species, including genetically...... affiliated species within Pasteurellaceae. In addition, DNA preparations from 56 H. parasuis isolates from North America were included. To obtain a test that was specific for H. parasuis, a multiplex PCR using 3 different primers was developed. The PCR test produced an amplicon of approximately 1090 bp only...

  14. Establishment of PCR-ELISA for Detecting Glyphosate Resistant Transgenic Soybean

    Institute of Scientific and Technical Information of China (English)

    Yuan Qiang; Wei Yun-min; Fu Ming-ming; Qiu You-wen; Wen Hong-tao; Zhang Ming-hui; Liu Ying; Ao Jin-xia

    2016-01-01

    A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows: the concentration of digoxin tag probe was 0.5 μmol• L-1, the time of hybridization reaction was 15 min and the chromogenic reaction should last for 30 min. The sensitivity and the repeatability of our PCR-ELISA method were evaluated, and the results showed that it could be detected when the concentration of DNA template from transgenic soybean samples was 0.01% or higher, and the coefficient of variation of this method was less than 5% in our research condition. These results suggested that PCR-ELISA method establishment in this study had good repeatability and high precision for detecting the transgenic soybean samples.

  15. Localizzazione e valutazione dell’espressione di Chlamydophila pneumoniae mediante RT-PCR in situ

    Directory of Open Access Journals (Sweden)

    Stefania Cazzavillan

    2005-12-01

    Full Text Available Chlamydophila pneumoniae, an obligate intracellular gram negative bacterium, is involved in a wide spectrum of symptomatic respiratory tract diseases. However more recently it has been reported to be a pathogenic agent in the mechanism leading to atherosclerosis. In the present study the presence of Chlamydophila pneumoniae was assessed, using nested PCR and in situ PCR, while the viability of the microorganism was investigated using RT in situ PCR.The results obtained demonstrated that Chlamydophila pneumoniae was present and alive in the tissues examined.The global concordance of results in the three techniques used was 100%. RT in situ PCR can be considered a precious tool to detect bacterial mRNA in formalin fixed paraffin embedded samples provided an optimal standardization of the key variables is achieved.

  16. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    Science.gov (United States)

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks.

  17. Chronic Chagas disease: PCR-xenodiagnosis without previous microscopic observation is a useful tool to detect viable Trypanosoma cruzi.

    Science.gov (United States)

    Saavedra, Miguel; Zulantay, Inés; Apt, Werner; Martínez, Gabriela; Rojas, Antonio; Rodríguez, Jorge

    2013-01-01

    We evaluate the elimination of the microscopic stage of conventional xenodiagnosis (XD) to optimize the parasitological diagnosis of Trypanosoma cruzi in chronic Chagas disease. To this purpose we applied under informed consent two XD cages to 150 Chilean chronic chagasic patients. The fecal samples (FS) of the triatomines at 30, 60 and 90 days post feeding were divided into two parts: in one a microscopic search for mobile trypomastigote and/or epimastigote forms was performed. In the other part, DNA extraction-purification for PCR directed to the conserved region of kDNA minicircles of trypanosomes (PCR-XD), without previous microscopic observation was done. An XD was considered positive when at least one mobile T. cruzi parasite in any one of three periods of incubation was observed, whereas PCR-XD was considered positive when the 330 bp band specific for T. cruzi was detected. 25 of 26 cases with positive conventional XD were PCR-XD positive (concordance 96.2%), whereas 85 of 124 cases with negative conventional XD were positive by PCR-XD (68.5%). Human chromosome 12 detected by Real-time PCR used as exogenous internal control of PCR-XD reaction allowed to discounting of PCR inhibition and false negative in 40 cases with negative PCR-XD. PCR-XD performed without previous microscopic observation is a useful tool for detection of viable parasites with higher efficiency then conventional XD.

  18. Quantitative DNA Analysis Using Droplet Digital PCR.

    Science.gov (United States)

    Vossen, Rolf H A M; White, Stefan J

    2017-01-01

    Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

  19. Evaluation of real-time PCR coupled with immunomagnetic separation or centrifugation for the detection of healthy and sanitizer-injured Salmonella spp. on mung bean sprouts.

    Science.gov (United States)

    Zheng, Qianwang; Mikš-Krajnik, Marta; Yang, Yishan; Lee, Sang-Myun; Lee, Seung-Cheol; Yuk, Hyun-Gyun

    2016-04-02

    Fresh mung bean sprouts have been identified as a source of many Salmonella outbreaks worldwide. The aim of this study was to develop a rapid and accurate detection methodology for low levels of healthy and sanitizer-injured Salmonella on mung bean sprouts using real-time PCR coupled with either immunomagnetic separation (PCR-IMS) or centrifugation (PCR-cen). Initially, three parameters of IMS; specificity/sensitivity, bacterial concentration and bead incubation time were optimized. Secondly, limit of detection (LOD) was determined for the optimized PCR-IMS and PCR-cen. These two methods were compared against PCR alone (PCR) and the standard culture method (ISO) for their ability to detect Salmonella using inoculated and uninoculated sprouts. Under optimum IMS conditions (10(5)CFU/ml for 30 min), capture efficiency of Salmonella in sprout suspensions was lower than 40%, most probably due to the non-specific binding of the background microbiota. PCR-IMS and PCR-cen had a similar LOD at 10(3)CFU/ml, which was one log unit lower than PCR. Enrichment of 10h was sufficient to detect 100% of the inoculated sprouts with both PCR-IMS and PCR-cen, which was significantly faster compared to PCR and the ISO method. Moreover, the validation study using uninoculated sprouts revealed that PCR-IMS and PCR-cen were equally effective on Salmonella detection, showing 98.3% accuracy. These results suggest that PCR-cen would be the effective and less costly method for the detection of both healthy and sanitizer-injured Salmonella on mung bean sprouts.

  20. Rapid Detection of Different Serovares of Salmonella entrica by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    A Karami

    2007-08-01

    Full Text Available Background: Typhoid fever is still one of the serious public health problems in many geographic areas and is endemic in most countries. Aim of current study was to evaluate a shortened time –Multiplex PCR for rapid detection of different Sal¬monella enterica serovars. Methods: The PCR primers for three target genes tyv, prt and invA were subjected for amplification by PCR. By using sim¬ple DNA extraction method, rapid PCR cycles and rapid electrophoresis procedure with simple and very cheap buffer were utilized in 200 to 300 volts for 15 minutes to separate the PCR products. Results: The results showed that all reference and clinical isolates of S. enterica were accurately identified by this as¬say with no cross reaction with other enterobacterial strains tested. Detection limit of the reaction was to be fewer than 10-1 colony forming unit. Conclusion: These data indicate that the optimized rapid cycle multiplex PCR is a potentially valuable tool for rapid diagno¬sis of S. enterica using a conventional thermal cycler. This method reduced the reaction time of PCR from 3.5 h to less than 1 h.

  1. Competitive PCR-ELISA protocols for the quantitative and the standardized detection of viral genomes.

    Science.gov (United States)

    Musiani, Monica; Gallinella, Giorgio; Venturoli, Simona; Zerbini, Marialuisa

    2007-01-01

    Competitive PCR-ELISA combines competitive PCR with an ELISA to allow quantitative detection of PCR products. It is based on the inclusion of an internal standard competitor molecule that is designed to differ from the target by a short sequence of nucleotides. Once such a competitor molecule has been designed and constructed, target and competitor sequences are concurrently PCR-amplified, before hybridization to two different specific probes and determination of their respective OD values by ELISA. The target can be quantified in relation to a titration curve of different dilutions of the competitor. The competitor can alternatively be used at a unique optimal concentration to allow for standardized detection of the target sequence. PCR-ELISA can be performed in 1 d in laboratories without access to a real-time PCR thermocycler. This technique is applied in diagnostics to monitor the course of infections and drug efficacy. Competitive PCR-ELISA protocols for the quantitative and for the standardized detection of parvovirus B19 are detailed here as an example of the technique.

  2. Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

    Science.gov (United States)

    Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

    2016-03-01

    Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

  3. PCR inhibitor removal using the NucleoSpin® DNA Clean-Up XS kit.

    Science.gov (United States)

    Faber, Korie L; Person, Eric C; Hudlow, William R

    2013-01-01

    Forensic evidence samples are collected from an unlimited variety of substrates, which may contain compounds known to inhibit the polymerase chain reaction (PCR). These PCR inhibitors are co-extracted with the DNA sample and can negatively affect the DNA typing results, which can range from partial to complete inhibition of the short tandem repeat (STR) PCR. One potential solution is to remove the PCR inhibitors from the extracts prior to the STR PCR with the NucleoSpin(®) DNA Clean-Up XS kit. The kit contains a NucleoSpin(®) XS silica column that has a special funnel design of thrust rings along with a very small silica membrane, which allows for sample elution in a small volume that is appropriate for use with current STR typing kits. The NucleoSpin(®) DNA Clean-Up XS kit was optimized for the best possible DNA recovery and then evaluated for its ability to remove eight commonly encountered PCR inhibitors including: bile salt, collagen, hematin, humic acid, indigo, melanin, tannic acid and urea. Each of these PCR inhibitors was effectively removed by the NucleoSpin(®) DNA Clean-Up XS kit as demonstrated by generating more complete STR profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  4. Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR

    Institute of Scientific and Technical Information of China (English)

    Reza Mirnejad; Reza Hosseini Doust; Reza Kachuei; Seied Mojtaba Mortazavi; Mehdi Khoobdel; Ali Ahamadi

    2012-01-01

    Objective:To evaluate simultaneous detection and differentiates of Brucella abortus(B. abortus) and Brucella melitensis (B. melitensis) through the combinatorial PCR method. Methods:This study was designed using three primers that could simultaneously identify and differentiate two major species of pathogenic Brucella in humans and animals. Identification and differentiation of each species using the size of the PCR product were determined. To determine the specificity of the method, bacteria close to the genus Brucella were used. Finally, to confirm PCR products, In addition to the products sequence, RFLP was performed on PCR products using restriction enzymes. Results:The method of optimized combinatorial PCR in this study could simultaneously detect and differentiate B. abortus and B. melitensis with high specificity and sensitivity in clinical samples. Differentiation of species is based on the resulting bands;therefore, the band 494 bp for B. abortus and 733 bp for B. melitensis were obtained. RFLP and sequencing results confirmed PCR results. Conclusions:The results of this study shows that without routine diagnostic methods such as culture and serology tests, using the molecular method of combinatorial PCR, important species of Brucella can be simultaneously identified and differentiated in clinical samples.

  5. Real time PCR. Application in dengue studies

    Directory of Open Access Journals (Sweden)

    Jeanette Prada-Arismendy

    2011-06-01

    Full Text Available PCR (polymerase chain reaction is a routinely used tool in every diagnostic and research laboratory. This technique has been used in detection of mutations and pathogens, forensic investigation, and even is the base tool for human genome sequencing. A modification of PCR technique, real time PCR, allows the quantification of nucleic acids with higher sensibility, specificity and reproducibility. This article is intended to clarify the foundations of real-time PCR, using an application model for virology. In the actual work, it was quantified the viral load of dengue virus serotype 2 produced from infected murine macrophages; the obtained results in this work established that murine strain BALB/c presents a greater susceptibility to dengue virus infection, which establishes BALB/c murine strain as a best model of study for investigation of dengue virus infection physiopathology.

  6. Fundamentals of multiplexing with digital PCR.

    Science.gov (United States)

    Whale, Alexandra S; Huggett, Jim F; Tzonev, Svilen

    2016-12-01

    Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe 'higher order multiplexing' that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.

  7. The potential advantages of digital PCR for clinical virology diagnostics.

    Science.gov (United States)

    Hall Sedlak, Ruth; Jerome, Keith R

    2014-05-01

    Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

  8. Comparison PCR Method and Rapid Urease Test to Detect Helicobacter Pylori in the Gastric Biopsy Tissue Samples

    Directory of Open Access Journals (Sweden)

    Parastoo Chamanrokh

    2013-09-01

    Full Text Available Background & Objective: Helicobacter pylori has been recognized as a major risk factor in gastric and duodenum ulcers and gastric cancer. Some laboratory tests, such as culture, are not entirely satisfying. The aim of this study is to compare RUT with PCR in identifying H. pylori in the gastric biopsy tissue samples.Materials & Methods: First, the standard H.pylori sample (N:oC30 was provided. primers or the glmM (ureC gene were used In PCR method The PCR test was optimized by sensitivity and specificity methods. Then 100 clinical samples composed of stomach tissue biopsy were prepared. The rapid urease test was conducted on all obtained samples to identify H.pylori. DNA was extracted from samples using DNG plus technique. Then, samples were studied using PCR method.  Results: The 294 bp product was amplified in the optimized PCR test. The PCR test sensitivity was obtained at 10 CFU. Any unwanted products were not seen in the specificity test with the exception of H. pylori DNA samples. Of 100 stomach biopsy samples, 64% were reported as positive by RUT and 76% by the PCR test.Conclusion: Given that the PCR test has higher sensitivity and specificity to detect H.pylori comparing rapid ureaas test, therefore this method could be used to detect H.pylori.  

  9. Transgene detection by digital droplet PCR.

    Directory of Open Access Journals (Sweden)

    Dirk A Moser

    Full Text Available Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR protocol for Insulin-Like Growth Factor 1 (IGF1 detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1 and Erythropoietin (EPO transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

  10. Universally Primed PCR (UP-PCR) and its applications for taxonomy in Trichoderma

    Institute of Scientific and Technical Information of China (English)

    Mette Lübeck

    2004-01-01

    @@ Universally Primed PCR (UP-PCR) is a PCR fingerprinting method that has demonstrated its applicability in different aspects of mycology. These applications constitute analysis of genome structures, identification of species, analysis of population and species diversity, revealing of genetic relatedness at infra-and inter-species level, and identification of UP-PCR markers at different taxonomic levels (strain, group and/or species) . A further development of the UP-PCR technique is an UP-PCR product cross hybridisation assay that facilitates investigation of sequence similarity (homology) of UP-PCR products and grouping of strains into UP-PCR hybridisation groups. This separates the strains into entities with high genetic similarity (DNA homology) . UP-PCR has been used as an aid in taxonomy and species delineation, and to monitor biocontrol strains following their release into the environment by fingerprint characterisation of pure cultures and through direct detection in soil by amplification of UP-PCR-derived SCAR markers. The technique has been applied to Trichoderma strains in particularly with the aims of strain recognition and classification.

  11. A naked-eye colorimetric "PCR developer"

    Science.gov (United States)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  12. Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

    Directory of Open Access Journals (Sweden)

    Jensen Jørgen

    2002-07-01

    Full Text Available Abstract Background Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods. Results We compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumoniae. The PCR methods were tested on reference samples containing C. pneumoniae DNA and on 136 nasopharyngeal samples from patients with a chronic cough. We found the same detection limit for the two methods and that clinical performance was equal for the real-time PCR and for the conventional PCR method, although only three samples tested positive. To investigate whether the low prevalence of C. pneumoniae among patients with a chronic cough was caused by suboptimal PCR efficiency in the samples, PCR efficiency was determined based on the real-time PCR. Seventeen of twenty randomly selected clinical samples had a similar PCR efficiency to samples containing pure genomic C. pneumoniae DNA. Conclusions These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but that needs to be confirmed further. Real-time PCR can be used to investigate the PCR efficiency which gives a rough estimate of how well the real-time PCR assay works in a specific sample type. Suboptimal PCR efficiency of PCR is not a likely explanation for the low positivity rate of C. pneumoniae in patients with a chronic cough.

  13. Nonlinear optimization

    CERN Document Server

    Ruszczynski, Andrzej

    2011-01-01

    Optimization is one of the most important areas of modern applied mathematics, with applications in fields from engineering and economics to finance, statistics, management science, and medicine. While many books have addressed its various aspects, Nonlinear Optimization is the first comprehensive treatment that will allow graduate students and researchers to understand its modern ideas, principles, and methods within a reasonable time, but without sacrificing mathematical precision. Andrzej Ruszczynski, a leading expert in the optimization of nonlinear stochastic systems, integrates t

  14. Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

    Science.gov (United States)

    Jawhari, Maan; Abrahamian, Peter; Sater, Ali Abdel; Sobh, Hana; Tawidian, Patil; Abou-Jawdah, Yusuf

    2015-02-01

    Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

  15. Optimal semelparity.

    Directory of Open Access Journals (Sweden)

    James W Vaupel

    Full Text Available Semelparous organisms have a simple life cycle characterized by immediate death after reproduction. We assume that semelparous life histories can be separated into a juvenile non-reproductive period followed by an adult period during which reproduction is possible. We derive formulae for the optimal age and size at reproduction and for the optimal size of the offspring (e.g., seeds. Our main contribution is to determine the conditions under which the optimal size of the offspring does not depend on the optimal size at reproduction and vice versa.

  16. Website Optimization

    CERN Document Server

    King, Andrew

    2008-01-01

    Remember when an optimized website was one that merely didn't take all day to appear? Times have changed. Today, website optimization can spell the difference between enterprise success and failure, and it takes a lot more know-how to achieve success. This book is a comprehensive guide to the tips, techniques, secrets, standards, and methods of website optimization. From increasing site traffic to maximizing leads, from revving up responsiveness to increasing navigability, from prospect retention to closing more sales, the world of 21st century website optimization is explored, exemplified a

  17. Signal and noise in bridging PCR

    Directory of Open Access Journals (Sweden)

    Thaler David S

    2002-07-01

    Full Text Available Abstract Background In a variant of the standard PCR reaction termed bridging, or jumping, PCR the primer-bound sequences are originally on separate template molecules. Bridging can occur if, and only if, the templates contain a region of sequence similarity. A 3' end of synthesis in one round of synthesis that terminates in this region of similarity can prime on the other. In principle, Bridging PCR (BPCR can detect a subpopulation of one template that terminates synthesis in the region of sequence shared by the other template. This study considers the sensitivity and noise of BPCR as a quantitative assay for backbone interruptions. Bridging synthesis is also important to some methods for computing with DNA. Results In this study, BPCR was tested over a 328 base pair segment of the E. coli lac operon and a signal to noise ratio (S/N of approximately 10 was obtained under normal PCR conditions with Taq polymerase. With special precautions in the case of Taq or by using the Stoffel fragment the S/N was improved to 100, i.e. 1 part of cut input DNA yielded the same output as 100 parts of intact input DNA. Conclusions In the E. coli lac operator region studied here, depending on details of protocol, between 3 and 30% per kilobase of final PCR product resulted from bridging. Other systems are expected to differ in the proportion of product that is bridged consequent to PCR protocol and the sequence analyzed. In many cases physical bridging during PCR will have no informational consequence because the bridged templates are of identical sequence, but in a number of special cases bridging creates, or, destroys, information.

  18. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    Science.gov (United States)

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

  19. Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS.

    Science.gov (United States)

    Oberacher, Herbert; Niederstätter, Harald; Casetta, Bruno; Parson, Walther

    2006-02-01

    Ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization mass spectrometry (ICEMS) represents an efficient method for the characterization of nucleic acids amplified by polymerase chain reaction (PCR). Since sample preparation is limited to PCR, the optimization of its solution conditions is of utmost importance for efficient mass spectrometric detection. The compatibility of a number of different commercially available PCR components including DNA polymerases, deoxynucleotide triphosphates, bovine serum albumin, enhancer, and ionic buffers was evaluated. These experiments revealed that higher concentration of enhancer and detergents such as Tween-20 or Nonidet P-40 impairs the mass spectrometric detection of nucleic acids and should be avoided within the PCR mixture. The optimized analytical platform was applied to the characterization of PCR products covering parts of the first hypervariable region of the noncoding mitochondrial control region. Truncated amplicons were detected attributable to the use of low quality primers. Furthermore, due to the proofreading activity of the applied polymerase system, mismatches between the primer and the target sequence located at the last or the second last base at the 3'-end of primers were corrected and detected within the corresponding amplicons.

  20. Detection of Brettanomyces spp. in red wines using real-time PCR.

    Science.gov (United States)

    Tofalo, Rosanna; Schirone, Maria; Corsetti, Aldo; Suzzi, Giovanna

    2012-09-01

    The question if the "Brett character" is a favorable wine attribute is one of the most controversial issues and it is currently addressed by many researches. Actually, the presence of Brettanomyces/Dekkera in wine during barrel aging is often associated to detrimental organoleptic characteristics depending on the release of volatile phenols (for example, 4-ethylphenol and 4-ethylguaiacol); for that reason the possibility to rapidly detect the yeast at the early stage of wine production could allow preventive actions to reduce wine spoilage. In this work, 25 and 5 samples from conventional and organic vineyards, respectively, all suspected to be spoiled by Brettanomyces/Dekkera spp., were analyzed using both culture-dependent and culture-independent techniques. In particular, a DNA extraction protocol and a real-time quantitative PCR (qPCR) assay to directly detect and quantify B. bruxellensis were optimized. Results showed that B. bruxellensis was present in 22 of 30 samples, ranging from 10 to 10(4) CFU/mL, lower values being found in organic wines (10 to 10(2) CFU/mL). Overall, qPCR was proved to be a useful and valuable wine control system, since 12 samples were recorded as positive for yeast presence when analyzed by qPCR and negative in case of plate count analyses. Brettanomyces cells were detected using a qPCR method, optimized in this study, which allows to obtain results quickly. © 2012 Institute of Food Technologists®

  1. Determination of Viable Salmonella Typhimurium Cells in Heat Treated Milk By PMA/Real-Time PCR Method

    Directory of Open Access Journals (Sweden)

    Zülal Kesmen

    2017-06-01

    Full Text Available Applying different technological processes during the production of food has a lethal effect on the bacteria but DNA of these bacterial strains may cause false positive results when detected by real time PCR technique because they preserve their existence for a certain period of time. To overcome this shortcoming of the real time PCR technique, a new method has been developed in recent years, based on the removal of dead cell DNA from the medium by treatment with Propodium Monoazide (PMA before DNA extraction. In this study, real-time PCR method was combined with PMA application for the detection of live cells of Salmonella Typhimurium in heat treated milk samples. For this purpose, milk samples inoculated with S. Tyhimurium were heat treated at different temperatures (60, 65, 70 and 75°C and times (15, 60, 300, 900 sec and number of live bacteria was determined comparatively by direct real-time PCR, PMA/real-time PCR and conventional cultural method. As a result, unlike the direct real time PCR technique, PMA/real-time PCR method prevents to a certain extent of false positive results from dead cells at all tested temperatures and times but higher results were obtained from PMA/real-time PCR method when compared to conventional cultural results. Therefore, further studies should be carried out to optimize the conditions of the PMA application in order to eliminate the high positive results detected by the PMA / real-time PCR method

  2. Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    Boboli Hedwige

    2010-09-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients. Results In this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86% remained P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10% were positive by both culture and qPCR. Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa positive both by culture and qPCR. Conclusion Based on the results of this study, it can be concluded that qPCR may have a predictive value for impending P. aeruginosa infection for only a limited number of patients.

  3. Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing.

    Science.gov (United States)

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-05-01

    With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test. Copyright © 2017 American Society for Microbiology.

  4. Comparison of Diagnostic Accuracy of PCR Targeting the 47-Kilodalton Protein Membrane Gene of Treponema pallidum and PCR Targeting the DNA Polymerase I Gene: Systematic Review and Meta-analysis.

    Science.gov (United States)

    Gayet-Ageron, Angèle; Combescure, Christophe; Lautenschlager, Stephan; Ninet, Béatrice; Perneger, Thomas V

    2015-11-01

    Treponema pallidum PCR (Tp-PCR) testing now is recommended as a valid tool for the diagnosis of primary or secondary syphilis. The objectives were to systematically review and determine the optimal specific target gene to be used for Tp-PCR. Comparisons of the performance of the two main targets are tpp47 and polA genes were done using meta-analysis. Three electronic bibliographic databases, representing abstract books from five conferences specialized in infectious diseases from January 1990 to March 2015, were searched. Search keywords included ("syphilis" OR "Treponema pallidum" OR "neurosyphilis") AND ("PCR" OR "PCR" OR "molecular amplification"). We included diagnostic studies assessing the performance of Tp-PCR targeting tpp47 (tpp47-Tp-PCR) or the polA gene (polA-Tp-PCR) in ulcers from early syphilis. All studies were assessed against quality criteria using the QUADAS-2 tool. Of 37 studies identified, 62.2% were judged at low risk of bias or applicability. Most used the U.S. Centers for Disease Control and Prevention (CDC) case definitions for primary or secondary (early) syphilis (89.2%; n = 33); 15 (40.5%) used darkfield microscopy (DFM). We did not find differences in sensitivity and specificity between the two Tp-PCR methods in the subgroup of studies using adequate reference tests. Among studies using DFM as the reference test, sensitivities were 79.8% (95% confidence intervals [CI], 72.7 to 85.4%) and 71.4% (46.0 to 88.0%) for tpp47-Tp-PCR and polA-Tp-PCR (P = 0.217), respectively; respective specificities were 95.3% (93.5 to 96.6%) and 93.7% (91.8 to 95.2%) (P = 0.304). Our findings suggest that the two Tp-PCR methods have similar accuracy and could be used interchangeably.

  5. PCR+ In Diesel Fuels and Emissions Research

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  6. PCR+ In Diesel Fuels and Emissions Research

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  7. Medicinsk Optimering

    DEFF Research Database (Denmark)

    Birkholm, Klavs

    2010-01-01

    En undersøgelse af anvendelsen af medicin til optimering af koncentration, hukommelse og følelsestonus. Efterfulgt af etiske overvejelser og anbefalinger til det politiske system......En undersøgelse af anvendelsen af medicin til optimering af koncentration, hukommelse og følelsestonus. Efterfulgt af etiske overvejelser og anbefalinger til det politiske system...

  8. A novel temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device

    DEFF Research Database (Denmark)

    Bu, Minqiang; R. Perch-Nielsen, Ivan; Sørensen, Karen Skotte

    We present a new temperature control method capable of effectively shortening the thermal cycling time of polymerase chain reaction (PCR) in a disposable polymer microfluidic device with external heater and temperature sensor. The method employs optimized temperature overshooting and undershooting...

  9. Direct Chromatin PCR (DC-PCR): Hypotonic Conditions Allow Differentiation of Chromatin States during Thermal Cycling

    Science.gov (United States)

    Vatolin, Sergei; Khan, Shahper N.; Reu, Frederic J.

    2012-01-01

    Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90°C, 41 of 61 tested 5′ sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR. PMID:22984542

  10. Direct chromatin PCR (DC-PCR: hypotonic conditions allow differentiation of chromatin states during thermal cycling.

    Directory of Open Access Journals (Sweden)

    Sergei Vatolin

    Full Text Available Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34 were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR.

  11. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

    OpenAIRE

    Tuo, Decai; Shen, Wentao; Yang, Yong; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-01-01

    Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplif...

  12. Topology Optimization

    DEFF Research Database (Denmark)

    A. Kristensen, Anders Schmidt; Damkilde, Lars

    2007-01-01

    . A way to solve the initial design problem namely finding a form can be solved by so-called topology optimization. The idea is to define a design region and an amount of material. The loads and supports are also fidefined, and the algorithm finds the optimal material distribution. The objective function...... dictates the form, and the designer can choose e.g. maximum stiness, maximum allowable stresses or maximum lowest eigenfrequency. The result of the topology optimization is a relatively coarse map of material layout. This design can be transferred to a CAD system and given the necessary geometrically...... refinements, and then remeshed and reanalysed in other to secure that the design requirements are met correctly. The output of standard topology optimization has seldom well-defined, sharp contours leaving the designer with a tedious interpretation, which often results in less optimal structures. In the paper...

  13. Dispositional Optimism

    Science.gov (United States)

    Carver, Charles S.; Scheier, Michael F.

    2014-01-01

    Optimism is a cognitive construct (expectancies regarding future outcomes) that also relates to motivation: optimistic people exert effort, whereas pessimistic people disengage from effort. Study of optimism began largely in health contexts, finding positive associations between optimism and markers of better psychological and physical health. Physical health effects likely occur through differences in both health-promoting behaviors and physiological concomitants of coping. Recently, the scientific study of optimism has extended to the realm of social relations: new evidence indicates that optimists have better social connections, partly because they work harder at them. In this review, we examine the myriad ways this trait can benefit an individual, and our current understanding of the biological basis of optimism. PMID:24630971

  14. A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta.

    Science.gov (United States)

    Biswas, C; Dey, P; Satpathy, S

    2014-04-01

    We have developed a simple method of direct PCR (dPCR) without time-consuming procedures of DNA extraction by directly using the leaf bits for rapid detection of begomoviruses in jute and mesta. The leaf bits were treated with a lysis buffer for 35 min, and the lysate was used as PCR template. Different components and their concentration in lysis buffer systems were optimized and the optimal buffer system composed of 20 mmol l(-1) tris (hydroxymethyl aminomethane (Tris)-Cl (pH 8·0), 1·5 mmol l(-1) ethylenediaminetetraacetic acid (EDTA) (pH 8·0), 1·4 mol l(-1) NaCl and 200 μg/mL Proteinase K. Further, 3% PVP (w/v) and β-marcaptoethanol (1% v/v) were additionally added into the buffer in case of jute. Under optimized PCR conditions, both viral DNA as well as plant (jute and mesta) genomic DNA were amplified from the lysate. dPCR required fewer reagents and less incubation time reducing both time and cost of detection. Identification of begomoviruses by serology is not suitable due to difficulty in preparing high titre and specific antisera. Begomoviruses are routinely detected by PCR-based techniques using universal or specific primers. However, it is a prerequisite to isolate pure DNA from the samples before PCR. DNA extraction from some plants such as jute, mesta is very difficult due to the presence of mucilage and other impurities. Therefore, we have developed a method of direct PCR without DNA extraction for detection of begomoviruses from these crops. It is the first report of a direct PCR method in jute and mesta. © 2013 The Society for Applied Microbiology.

  15. Validation of a sensitive PCR assay for the detection of Chelonid fibropapilloma-associated herpesvirus in latent turtle infections.

    Science.gov (United States)

    Alfaro-Núñez, Alonzo; Gilbert, M Thomas P

    2014-09-01

    The Chelonid fibropapilloma-associated herpesvirus (CFPHV) is hypothesized to be the causative agent of fibropapillomatosis, a neoplastic disease in sea turtles, given its consistent detection by PCR in fibropapilloma tumours. CFPHV has also been detected recently by PCR in tissue samples from clinically healthy (non exhibiting fibropapilloma tumours) turtles, thus representing presumably latent infections of the pathogen. Given that template copy numbers of viruses in latent infections can be very low, extremely sensitive PCR assays are needed to optimize detection efficiency. In this study, efficiency of several PCR assays designed for CFPHV detection is explored and compared to a method published previously. The results show that adoption of a triplet set of singleplex PCR assays outperforms other methods, with an approximately 3-fold increase in detection success in comparison to the standard assay. Thus, a new assay for the detection of CFPHV DNA markers is presented, and adoption of its methodology is recommended in future CFPHV screens among sea turtles.

  16. Early diagnosis of typhoid fever by nested PCR for flagellin gene of Salmonella enterica serotype Typhi.

    Science.gov (United States)

    Khan, S; Harish, B N; Menezes, G A; Acharya, N S; Parija, S C

    2012-11-01

    Typhoid fever caused by Salmonella Typhi continues to be a major health problem in spite of the use of antibiotics and the development of newer antibacterial drugs. Inability to make an early laboratory diagnosis and resort to empirical therapy, often lead to increased morbidity and mortality in cases of typhoid fever. This study was aimed to optimize a nested PCR for early diagnosis of typhoid fever and using it as a diagnostic tool in culture negative cases of suspected typhoid fever. Eighty patients with clinical diagnosis of typhoid fever and 40 controls were included in the study. The blood samples collected were subjected to culture, Widal and nested PCR targeting the flagellin gene of S. Typhi. The sensitivity of PCR on blood was found to be 100 per cent whereas the specificity was 76.9 per cent. The positive predictive value (PPV) of PCR was calculated to be 76.9 per cent with an accuracy of 86 per cent. None of the 40 control samples gave a positive PCR. Due to its high sensitivity and specificity nested PCR can be used as a useful tool to diagnose clinically suspected, culture negative cases of typhoid fever.

  17. A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies.

    Science.gov (United States)

    Bach, H-J; Jessen, I; Schloter, M; Munch, J C

    2003-01-01

    Real-time TaqMan-PCR assays were developed for detection, differentiation and absolute quantification of the pathogenic subspecies of Clavibacter michiganensis (Cm) in one single PCR run. The designed primer pair, targeting intergenic sequences of the rRNA operon (ITS) common in all subspecies, was suitable for the amplification of the expected 223-nt DNA fragments of all subspecies. Closely related bacteria were completely discriminated, except of Rathayibacter iranicus, from which weak PCR product bands appeared on agarose gel after 35 PCR cycles. Sufficient specificity of PCR detection was reached by introduction of the additional subspecies specific probes used in TaqMan-PCR. Only Cm species were detected and there was clear differentiation among the subspecies C. michiganensis sepedonicus (Cms), C. michiganensis michiganensis (Cmm), C. michiganensis nebraskensis (Cmn), C. michiganensis insidiosus (Cmi) and C. michiganensis tessellarius (Cmt). The TaqMan assays were optimized to enable a simultaneous quantification of each subspecies. Validity is shown by comparison with cell counts.

  18. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot.

    Science.gov (United States)

    Albers, Eliene; Sbroggiò, Mauro; Martin-Gonzalez, Javier; Avram, Alexandra; Munk, Stephanie; Lopez-Contreras, Andres J

    2017-01-19

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.

  19. Comparison of phenotypic and PCR methods for detection of carbapenemases production by Enterobacteriaceae

    Directory of Open Access Journals (Sweden)

    Maryam AlTamimi

    2017-01-01

    Full Text Available Dissemination of carbapenem resistance via Enterobacteriaceae, particularly among Klebsiella pneumoniae and Escherichia coli, is a major public health concern. Rapid methods for determining antimicrobial susceptibility are important to ensure adequate and appropriate use of antimicrobial agents and to limit the spread of these bacteria. In the current study, we compared the rapidity, sensitivity and specificity of traditional methods and molecular-based Xpert Carba-R PCR assay to identify sixty isolates, (26 E. coli and 34 K. pneumoniae. The specificity of MicroScan was 100% while sensitivity to ertapenem (ERT, imipenem (IMI, and meropenem (MER was 93%, 68.9%, and 55.17%, respectively. For the modified Hodge test, the specificity was 96.77% and sensitivity was 89.65%. Although some results of phenotypic assays matched with the definite PCR identification, some results were misleading. Out of the 29 positive PCR samples, three samples of K. pneumoniae were negative for the MHT and one E. coli sample was MHT positive but negative for the PCR. Nine samples were positive for the PCR but were determined as carbapenem sensitive by MicroScan. While MicroScan and MHT requires several hours and multi-steps to obtain results, Xpert Carba-R PCR assay takes less than an hour. Therefore, we recommend using Gene xpert Carba-R assay for the optimal carbapenemnase detection with reducing material, manpower and cost. Also it is important to know the type of carbapenemase is present.

  20. How to evaluate PCR assays for the detection of low-level DNA

    DEFF Research Database (Denmark)

    Banch-Clausen, Frederik; Urhammer, Emil; Rieneck, Klaus

    2015-01-01

    are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial...... not significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also...

  1. Improved sensitivity of circulating tumor DNA measurement using short PCR amplicons

    DEFF Research Database (Denmark)

    Andersen, Rikke Fredslund; Spindler, Karen-Lise Garm; Brandslund, Ivan

    2015-01-01

    , however, presents a number of challenges that require attention. The amount of DNA is low and highly fragmented and analyses need to be optimized accordingly. KRAS ARMS-qPCR assays with amplicon lengths of 120 and 85 base pairs, respectively, were compared using positive control material (PCR fragments......) and plasma samples from 46 colorectal cancer patients known to harbor a tumor KRAS mutation. KRAS mutated DNA was detected in significantly more clinical samples using the short amplicon assays compared to the long amplicon assays (74% vs. 61%, p=0.03). The level of mutated DNA in plasma was on average three...

  2. Asymmetric PCR method in generation of HBV ssDNA for pyrosequencing

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50∶1, 100∶1) and concentrations (13.0 pmol/25μL and 0.14 pmol/25μL, 19.5 pmol/25μL and 0.21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio ...

  3. Multiplex PCR testing for nine different sexually transmitted infections.

    Science.gov (United States)

    Kriesel, John D; Bhatia, Amiteshwar S; Barrus, Cammie; Vaughn, Mike; Gardner, Jordan; Crisp, Robert J

    2016-12-01

    Current sexually transmitted infection (STI) testing is not optimal due to delays in reporting or missed diagnoses due to a lack of comprehensive testing. The FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) is a user-friendly, fully automated, multiplex PCR system that is being developed for rapid point-of-care use. A research-use-only STI panel including multiple PCR primer sets for each organism was designed to detect Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus ducreyi, and herpes simplex virus (HSV) types 1 and 2. Standard clinical testing included Gram stain, nucleic acid amplification, wet mount examination, herpes simplex virus culture, and syphilis IgG. Standard clinical tests were not available for all the organisms tested by the FilmArray STI panel. Two hundred and ninety-five clinical specimens from 190 subjects were directly compared to standard testing. Urine (n = 146), urethral/cervical swabs (31), oral swabs (60), rectal swabs (43), and ulcer swabs (15) were tested. Among the tested samples, FilmArray detected C. trachomatis in 39 (13%), N. gonorrhoeae in 20 (7%), T. vaginalis in nine (3%), HSV 1 in five (2%), HSV 2 in five (2%), U. urealyticum in 36 (12%), M. genitalium in eight (3%), and T. pallidum in 11 (4%). Concordance between the FilmArray STI panel and standard nucleic acid amplification testing for C. trachomatis was 98% and for N. gonorrhoeae was 97%. Multiplex PCR STI testing has the potential to improve public health by providing rapid, sensitive, and reliable results within the clinic or nearby laboratory.

  4. Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

    Science.gov (United States)

    Wuyts, Véronique; Mattheus, Wesley; Roosens, Nancy H C; Marchal, Kathleen; Bertrand, Sophie; De Keersmaecker, Sigrid C J

    2017-01-01

    A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

  5. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman J.F.; Pierik, A.

    2012-01-01

    Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques,

  6. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman J.F.; Pierik, A.

    2012-01-01

    Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques,

  7. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    Science.gov (United States)

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  8. Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

    Directory of Open Access Journals (Sweden)

    Van Esbroeck Marjan

    2011-03-01

    Full Text Available Abstract Background This study describes the use of malaria rapid diagnostic tests (RDTs as a source of DNA for Plasmodium species-specific real-time PCR. Methods First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples. Results Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60, Plasmodium vivax (n = 10, Plasmodium ovale (n = 10 and Plasmodium malariae (n = 10. Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20 gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests. Conclusions RDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the

  9. Sex determination in 6 bovid species by duplex PCR.

    Science.gov (United States)

    Prashant; Gour, Digpal S; Dubey, Prem P; Jain, Anubhav; Gupta, Subhash C; Joshi, Balwinder K; Kumar, Dinesh

    2008-01-01

    Sex determination in domestic animals is of potential value to livestock breeding programs. The aim of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to 6 major domesticated species of the family Bovidae, viz. Bos frontalis, B. grunniens, B. indicus, Bubalus bubalis, Capra hircus, and Ovis aries. In silico analysis was done to identify conserved DNA sequence in the HMG box region of the sex-determining region of the Y-chromosome (SRY gene) across the bovids. Duplex PCR assay, including the SRY gene and the GAPDH housekeeping gene, was optimized by using genomic DNA extracted from blood samples of known sex. It was possible to identify the sex of animals by amplifying both gender-specific (SRY) and autosomal (GAPDH) genes simultaneously in the duplex reaction, with the male yielding two bands and the female one band. The protocol was subjected to a blind test that showed a 100 percent specificity and accuracy, thus it can be used in sex determination in livestock breeding programs.

  10. PCR detection of groundwater bacteria associated with colloidal transport

    Energy Technology Data Exchange (ETDEWEB)

    Cruz-Perez, P.; Stetzenbach, L.D.; Alvarez, A.J.

    1996-02-29

    Colloidal transport may increase the amount of contaminant material than that which could be transported by water flow alone. The role of colloids in groundwater contaminant transport is complicated and may involve many different processes, including sorption of elements onto colloidal particles, coagulation/dissolution, adsorption onto solid surfaces, filtration, and migration. Bacteria are known to concentrate minerals and influence the transport of compounds in aqueous environments and may also serve as organic colloids, thereby influencing subsurface transport of radionuclides and other contaminants. The initial phase of the project consisted of assembling a list of bacteria capable of sequestering or facilitating mineral transport. The development and optimization of the PCR amplification assay for the detection of the organisms of interest, and the examination of regional groundwaters for those organisms, are presented for subsequent research.

  11. In Silico PCR Tools for a Fast Primer, Probe, and Advanced Searching.

    Science.gov (United States)

    Kalendar, Ruslan; Muterko, Alexandr; Shamekova, Malika; Zhambakin, Kabyl

    2017-01-01

    The polymerase chain reaction (PCR) is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. The principle of this technique has been further used and applied in plenty of other simple or complex nucleic acid amplification technologies (NAAT). In parallel to laboratory "wet bench" experiments for nucleic acid amplification technologies, in silico or virtual (bioinformatics) approaches have been developed, among which in silico PCR analysis. In silico NAAT analysis is a useful and efficient complementary method to ensure the specificity of primers or probes for an extensive range of PCR applications from homology gene discovery, molecular diagnosis, DNA fingerprinting, and repeat searching. Predicting sensitivity and specificity of primers and probes requires a search to determine whether they match a database with an optimal number of mismatches, similarity, and stability. In the development of in silico bioinformatics tools for nucleic acid amplification technologies, the prospects for the development of new NAAT or similar approaches should be taken into account, including forward-looking and comprehensive analysis that is not limited to only one PCR technique variant. The software FastPCR and the online Java web tool are integrated tools for in silico PCR of linear and circular DNA, multiple primer or probe searches in large or small databases and for advanced search. These tools are suitable for processing of batch files that are essential for automation when working with large amounts of data. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and the online Java version at http://primerdigital.com/tools/pcr.html .

  12. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples.

    Science.gov (United States)

    Madico, G; Quinn, T C; Rompalo, A; McKee, K T; Gaydos, C A

    1998-11-01

    Trichomonas vaginalis infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of T. vaginalis was developed to add T. vaginalis infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of T. vaginalis. All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from Trichomonas tenax, Trichomonas gallinae, Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Chilomastix sulcatus, Dientamoeba fragilis, and Entamoeba histolytica. An optimal analytical sensitivity of one T. vaginalis organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify T. vaginalis. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for T. vaginalis. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the T. vaginalis genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of T. vaginalis infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.

  13. Validation of Reference Genes for Quantitative Real-Time PCR in Laodelphax striatellus

    Institute of Scientific and Technical Information of China (English)

    HE Xiu-ting; LIU Cheng-cheng; LI Zhao-qun; ZHANG Zan; LI Guo-qing; LI Fei; DONG Shuang-lin

    2014-01-01

    The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, ifve new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable thanβ-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantiifcation. These results were further conifrmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantiifcation by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 orβ-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replaceβ-actin as the reference genes for qPCR in L. striatellus.

  14. Application of restriction display PCR technique in the preparation of cDNA microarray probes

    Institute of Scientific and Technical Information of China (English)

    Zhao-Hui Sun; Wen-Li Ma; Bao Zhang; Yi-Fei Peng; Wen-Ling Zheng

    2005-01-01

    AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes.METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template.The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis.RESULTS: A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced,which were the specific gene fragments of HCV. These fragments could be further used as probes in microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility,and linearity in detecting HCV RNA were satisfactory.CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.

  15. Optimized Adaptor Polymerase Chain Reaction Method for Efficient Genomic Walking

    Institute of Scientific and Technical Information of China (English)

    Peng XU; Rui-Ying HU; Xiao-Yan DING

    2006-01-01

    Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.

  16. PCR clonality detection in Hodgkin lymphoma.

    NARCIS (Netherlands)

    Hebeda, K.M.; Altena, M.C. van; Rombout, P.D.M.; Krieken, J.H.J.M. van; Groenen, P.J.T.A.

    2009-01-01

    B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (

  17. The Power of Real-Time PCR

    Science.gov (United States)

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  18. Aspergillus PCR: one step closer to standardization.

    NARCIS (Netherlands)

    White, P.L.; Bretagne, S.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Schulz, B.; Finnstrom, N.; Mengoli, C.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2010-01-01

    PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus

  19. Inverse PCR for Point Mutation Introduction.

    Science.gov (United States)

    Silva, Diogo; Santos, Gustavo; Barroca, Mário; Collins, Tony

    2017-01-01

    Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.

  20. Aspergillus PCR: one step closer to standardization.

    NARCIS (Netherlands)

    White, P.L.; Bretagne, S.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Schulz, B.; Finnstrom, N.; Mengoli, C.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2010-01-01

    PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus P

  1. Portfolio Optimization

    OpenAIRE

    Frajtova-Michalikova, Katarina; Spuchľakova, Erika; Misankova, Maria

    2015-01-01

    In this paper Portfolio Optimization techniques were used to determine the most favorable investment portfolio. In particular, stock indices of three companies, namely Microsoft Corporation, Christian Dior Fashion House and Shevron Corporation were evaluated. Using this data the amounts invested in each asset when a portfolio is chosen on the efficient frontier were calculated. In addition, the Portfolio with minimum variance, tangency portfolio and optimal Markowitz portfolio are presented.

  2. Real-time PCR: Advanced technologies and applications

    Science.gov (United States)

    This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

  3. Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings.

    Science.gov (United States)

    Zheng, Qianwang; Mikš-Krajnik, Marta; Yang, Yishan; Xu, Wang; Yuk, Hyun-Gyun

    2014-09-01

    Conventional culture detection methods are time consuming and labor-intensive. For this reason, an alternative rapid method combining real-time PCR and immunomagnetic separation (IMS) was investigated in this study to detect both healthy and heat-injured Salmonella Typhimurium on raw duck wings. Firstly, the IMS method was optimized by determining the capture efficiency of Dynabeads(®) on Salmonella cells on raw duck wings with different bead incubation (10, 30 and 60 min) and magnetic separation (3, 10 and 30 min) times. Secondly, three Taqman primer sets, Sal, invA and ttr, were evaluated to optimize the real-time PCR protocol by comparing five parameters: inclusivity, exclusivity, PCR efficiency, detection probability and limit of detection (LOD). Thirdly, the optimized real-time PCR, in combination with IMS (PCR-IMS) assay, was compared with a standard ISO and a real-time PCR (PCR) method by analyzing artificially inoculated raw duck wings with healthy and heat-injured Salmonella cells at 10(1) and 10(0) CFU/25 g. Finally, the optimized PCR-IMS assay was validated for Salmonella detection in naturally contaminated raw duck wing samples. Under optimal IMS conditions (30 min bead incubation and 3 min magnetic separation times), approximately 85 and 64% of S. Typhimurium cells were captured by Dynabeads® from pure culture and inoculated raw duck wings, respectively. Although Sal and ttr primers exhibited 100% inclusivity and exclusivity for 16 Salmonella spp. and 36 non-Salmonella strains, the Sal primer showed lower LOD (10(3) CFU/ml) and higher PCR efficiency (94.1%) than the invA and ttr primers. Moreover, for Sal and invA primers, 100% detection probability on raw duck wings suspension was observed at 10(3) and 10(4) CFU/ml with and without IMS, respectively. Thus, the Sal primer was chosen for further experiments. The optimized PCR-IMS method was significantly (P=0.0011) better at detecting healthy Salmonella cells after 7-h enrichment than traditional PCR

  4. Clinical Identification of Common Species of Dermatophytes by PCR and PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    丁娟; 李家文; 刘志香; 谭志建

    2004-01-01

    To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase Ⅱ gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc Ⅱ. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans.From the restriction profiles of Hinc Ⅱ , 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCRRFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase Ⅱ gene is rapid and efficient.

  5. Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water

    Directory of Open Access Journals (Sweden)

    Lucrecia Acosta Soto

    2017-01-01

    Full Text Available The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR and digital PCR (dPCR were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks.

  6. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    Science.gov (United States)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  7. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification.

    Science.gov (United States)

    Ruelle, Jean; Yfantis, Vasilieios; Duquenne, Armelle; Goubau, Patrick

    2014-01-01

    Low or undetectable plasma viral load (VL) using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1). A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines (2). Inter-run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3)) using 46 clinical samples and the NIBSC international HIV-2 RNA standard. The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5'FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods (respective means of 10,612 and 2

  8. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification

    Directory of Open Access Journals (Sweden)

    Jean Ruelle

    2014-11-01

    Full Text Available Introduction: Low or undetectable plasma viral load (VL using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1. Materials and Methods: A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA in an accredited environment (ISO15189:2012 norm. Parameters tested were in line with the digital MIQE guidelines (2. Inter-run coefficient of variation (CV was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3 using 46 clinical samples and the NIBSC international HIV-2 RNA standard. Results: The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5’FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168 were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999 and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5. Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were

  9. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    DEFF Research Database (Denmark)

    Balcells, Ingrid; Cirera Salicio, Susanna; Busk, Peter K.

    2011-01-01

    be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological...... samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision...... settings. RESULTS: We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could...

  10. Simulation and design of a self-heating continuous-flow PCR chip

    Institute of Scientific and Technical Information of China (English)

    CHEN Wei-ping; TIAN Li; LI Ming-jiang; LIU Xiao-wei

    2007-01-01

    A novel continuous-flow PCR chip adopting self-heating, passive-cooling mode to realize the DNA fragments amplification was presented. Using the ANSYS finite element analysis, the temperature distribution of the chip is simulated and analyzed. The optimal size of the chip is 30 × 22 mm2, the roundabout micro-channel is the 90 μm width, 40 μm depth. Two micro-heater with the nickel-chrome alloy material film are formed on the side of silicon belonging to denaturation and renaturation zones needed for PCR reaction, and two adiabatic structures with groove on side of silicon by anisotropy etching. By the mede of heating local zones at single side,three wider constant temperature zones could be formed, which are 60 ℃ ,72 ℃ ,95 ℃ and suitable for PCR,and the temperature-difference could be restricted in less than 5 ℃.

  11. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    DEFF Research Database (Denmark)

    Balcells, Ingrid; Cirera, Susanna; Busk, Peter Kamp

    2011-01-01

    be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological...... settings. Results We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could...... samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision...

  12. Universal Probe Library based real-time PCR for rapid detection of bacterial pathogens from positive blood culture bottles.

    Science.gov (United States)

    Zhu, Lingxiang; Shen, Ding-Xia; Zhou, Qiming; Liu, Chao-Jun; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2014-03-01

    A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.

  13. EMA-real-time PCR as a reliable method for detection of viable Salmonella in chicken and eggs.

    Science.gov (United States)

    Wang, Luxin; Mustapha, Azlin

    2010-04-01

    Culture-based Salmonella detection takes at least 4 d to complete. The use of TaqMan probes allows the real-time PCR technique to be a rapid and sensitive way to detect foodborne pathogens. However, unlike RNA-based PCR, DNA-based PCR techniques cannot differentiate between DNA from live and dead cells. Ethidium bromide monoazide (EMA) is a dye that can bind to DNA of dead cells and prevent its amplification by PCR. An EMA staining step prior to PCR allows for the effective inhibition of false positive results from DNA contamination by dead cells. The aim of this study was to design an accurate detection method that can detect only viable Salmonella cells from poultry products. The sensitivity of EMA staining coupled with real-time PCR was compared to that of an RNA-based reverse transcription (RT)-real-time PCR. To prevent false negative results, an internal amplification control was added to the same reaction mixture as the target Salmonella sequences. With an optimized EMA staining step, the detection range of a subsequent real-time PCR was determined to be 10(3) to 10(9) CFU/mL for pure cultures and 10(5) to 10(9) CFU/mL for food samples, which was a wider detection range than for RT-real-time PCR. After a 12-h enrichment step, EMA staining combined with real-time PCR could detect as low as 10 CFU/mL Salmonella from chicken rinses and egg broth. The use of EMA with a DNA-based real-time PCR can successfully prevent false positive results and represents a simple, yet accurate detection tool for enhancing the safety of food.

  14. Development of a novel real-time qPCR assay for the dual detection of canine and phocine distemper virus

    DEFF Research Database (Denmark)

    Nielsen, Linette Buxbom; Hjulsager, Charlotte Kristiane; Larsen, Helene

    In a commercial diagnostic setting streamlining and optimization is an important factor when the goal is to provide high quality diagnostic results while remaining competitive. In the PCR diagnostics unit at DTU National Veterinary Institute part of this optimization programme is to replace...... conventional PCR assays with real-time PCR assays to obtain a uniform assay palette. The present work describes the development of a novel real-time RT-qPCR assay for the dual detection of canine and phocine distemper virus. The assay is relevant for the future detection of outbreaks of canine distemper virus...... bp in the Phosphoprotein (P) gene of the distemper virus genome. The dynamic range and PCR efficiency (E) was experimentally determined using 10-fold dilutions of a specially designed distemper DNA-oligo in addition to extracted RNA from clinical samples. E of the real-time assay was shown to range...

  15. Strategies for the inclusion of an internal amplification control in conventional and real time PCR detection of Campylobacter spp. in chicken fecal samples

    DEFF Research Database (Denmark)

    Lund, Marianne; Madsen, Mogens

    2006-01-01

    To illustrate important issues in optimization of a PCR assay with an internal control four different primer combinations for conventional PCR, two non-competitive and two competitive set-ups for real time PCR were used for detection of Campylobacter spp. in chicken faecal samples....... In the conventional PCR assays the internal control was genomic DNA from Yersinia ruckeri, which is not found in chicken faeces. This internal control was also used in one of the set LIPS in real time PCR. In the three other set-ups different DNA fragments of 109 bp length prepared from two oligos of each 66 bp...... by a simple extension reaction was used. All assays were optimized to avoid loss of target sensitivity due to the presence of the internal control by adjusting the amount of internal control primers in the duplex assays and the amount of internal control in all assays. Furthermore. the assays were tested...

  16. Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

    Directory of Open Access Journals (Sweden)

    Mojtaba Tahmoorespur

    2016-04-01

    fibrisolvens were successfully used for amplifying the specific fragments from this bacteria. The main and important factors for increasing the accuracy of Q-C PCR is the degree of similarity between competitor and target fragment. In this study the competitor fragment was highest homology to target sequences. In this regards it seems obtained results have considerable accuracy. The intensity of bands was evaluated and analyzed using Image J software. The results of band intensity analysis showed linear trend between competitor and target in different serial dilution of competitors. The specific fragment from 16S rDNA region of Butyrivibrio fibriolvents Bacteria was amplified using specific primers and cloned in pTZ57R/T plasmid. After amplifying the competitor fragment and target sequence simultaneously, the two bands were detectable in gel electrophoresis. The range of 10-1 to 10-6 serial dilution from competitor was selected for QC-PCR reaction. The results of this section showed that the considerable linear correlation was exist between competitor and target fragment in QC-PCR reaction (R2=0.985. In this study, Real-time PCR was also used for quantification of Butyrivibrio fibriolvents Bacteria strain. Melting analysis was showed that the reaction in Real-time PCR had appropriate condition for amplifying the target sequence. We used a standard in this study. This standard was designed as a vector contain of competitor fragment. This kind of standard was successfully used for QC-PCR and Real-time PCR analyses. Standard competitor could be used for absolute quantification for this bacterial strain. Overall, our results showed that the designed standard and optimized QC-PCR have considerable potential for Butyrivibrio fibriolvents Bacteria strain. Conclusion In this study, the two methods of quantification of nucleotide acids in biological samples were optimized. These two methods were performed in order to optimize Butyrivibrio fibriolvents Bacteria strain quantification

  17. OPTIMALISASI EKSTRAKSI DNA DAN PCR-RAPD PADA GREVILLEA SPP. (PROTEACEAE

    Directory of Open Access Journals (Sweden)

    MADE PHARMAWATI

    2014-04-01

    Full Text Available Molecular genetic analysis of plants relies on high yield and high purity of DNA as well as optimized condition of molecular reactions. Appropriate methods for DNA extraction and molecular reactions such as PCR are therefore needed. This study aimed to develop protocol for extraction of high molecular weight DNA from Grevillea leaf and to optimize condition of PCR-RAPD. Standard plant DNA extraction of Doyle and Doyle was modified by increasing EDTA concentration to 50 mM and addition of 2% (v/v 2-mercaptoethanol. Moreover, incubation time was prolonged to 14-16 h at 55oC. This method yielded good quality of DNA and consistent results. Amplification of DNA using PCR-RAPD will become efficient and consistent if the amplification reactions are in ideal condition. In Grevillea, clear, reproducible and scorable PCR-RAPD patterns were obtained using 10ng DNA template, 5 pmol primer, 2.5 mM MgCl2 and the number of thermal cycle was 40 x.

  18. Effective detection of human adenovirus in hawaiian waters using enhanced pcr methods

    Directory of Open Access Journals (Sweden)

    Lu Yuanan

    2011-02-01

    Full Text Available Abstract Background The current criteria for recreational water quality evaluation are primarily based on measurements of fecal indicator bacteria growth. However, these criteria often fail to predict the presence of waterborne human pathogenic viruses. To explore the possibility of direct use of human enteric viruses as improved human fecal contamination indicators, human adenovirus (HAdV was tested as a model in this study. Findings In order to establish a highly sensitive protocol for effective detection of HAdV in aquatic environments, sixteen published PCR primer sets were re-optimized and comparatively evaluated. Primer sets nehex3deg/nehex4deg, ADV-F/ADV-R, and nested PCR primer sets hex1deg/hex2deg and nehex3deg/nehex4deg were identified to be the most sensitive ones, with up to 1,000 fold higher detection sensitivity compared to other published assays. These three PCR protocols were successfully employed to detect HAdV in both treated and untreated urban wastewaters, and also in 6 of 16 recreational water samples collected around the island of Oahu, Hawaii. Conclusions Findings from this study support the possible use of enteric viruses for aquatic environmental monitoring, specifically for the essential routine monitoring of Hawaiian beach waters using the optimized PCR protocol to detect HAdV at certain water sites to ensure a safe use of recreational waters.

  19. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    Science.gov (United States)

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%.

  20. Real‐time PCR (qPCR) primer design using free online software

    National Research Council Canada - National Science Library

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    ...‐stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green‐based qPCR primers...

  1. Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

    Directory of Open Access Journals (Sweden)

    Tandale Babasaheb V

    2008-12-01

    Full Text Available Abstract Background Chandipura virus (CHPV, a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR using TaqMan technology was developed for rapid diagnosis. Methods Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice in ovo (eggs, in vitro (Vero E6, PS, RD and Sand fly cell line] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard. Results Real-time one step RT-PCR was optimized using in vitro transcribed (IVT RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99 with maximum Coefficient of variation (CV = 5.91% for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml. Vero and PS cell-lines (1.2 × 103 PFU/ml were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy

  2. Unconstrained Optimization

    DEFF Research Database (Denmark)

    Frandsen, P. E.; Jonasson, K.; Nielsen, Hans Bruun

    1999-01-01

    This lecture note is intended for use in the course 04212 Optimization and Data Fitting at the Technincal University of Denmark. It covers about 25% of the curriculum. Hopefully, the note may be useful also to interested persons not participating in that course. The aim of the note is to give...... an introduction to algorithms for unconstrained optimization. We present Conjugate Gradient, Damped Newton and Quasi Newton methods together with the relevant theoretical background. The reader is assumed to be familiar with algorithms for solving linear and nonlinear system of equations, at a level corresponding...

  3. Unconstrained Optimization

    DEFF Research Database (Denmark)

    Frandsen, P. E.; Jonasson, K.; Nielsen, Hans Bruun

    1999-01-01

    This lecture note is intended for use in the course 04212 Optimization and Data Fitting at the Technincal University of Denmark. It covers about 25% of the curriculum. Hopefully, the note may be useful also to interested persons not participating in that course. The aim of the note is to give...... an introduction to algorithms for unconstrained optimization. We present Conjugate Gradient, Damped Newton and Quasi Newton methods together with the relevant theoretical background. The reader is assumed to be familiar with algorithms for solving linear and nonlinear system of equations, at a level corresponding...

  4. Chicken QTL mapping by multiplex PCR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To facilitate rapid determination of the chromosomal location of quantitative trait loci, the current approaches to gene mapping are improved using a multiplex PCR technique. The high-throughput linkage analysis method described here allows selection of 178 from 328 microsatellite markers through the multiplex PCR method combined with the semi-automatic fluorescence-labeled DNA analysis technology. Those polymorphism markers are distributed on 23 autosomes and one sex chromosome (chromosome Z), covering 3080cM genetic distance. The average marker density is 18cM, dispersed into 30 different sets. These selected polymorphism microsatellite markers segregate with the family members, following the Mendel's heritage laws, and are very useful for chicken linkage map analysis as well as for the research on some important economic quantitative characters of chicken.

  5. PCR-SSCP检测水稻SNPs

    Institute of Scientific and Technical Information of China (English)

    李飞; 郭凌; 余潮

    2006-01-01

    本文选择5个水稻SNPs位点为对象,研究凝胶组成和电泳条件等对PCR-SSCP检测SNPs的影响.发现凝胶浓度、交联度、甘油和电泳温度等均可明显影响DNA单链分子迁移率,且对不同DNA单链分子的影响程度不同.因此,可以通过调节凝胶浓度、交联度、甘油和电泳温度等提高PCR-SSCP检测水稻SNPs的分辨率及效果.在一定条件下,较低交联度和4°电泳条件检测SNPs的效果更好.

  6. Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis.

    Science.gov (United States)

    Kirchmayr, Manuel Reinhart; Segura-Garcia, Luis Eduardo; Flores-Berrios, Ericka Patricia; Gschaedler, Anne

    2011-11-01

    An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. [Molecular identification of Mentha haplocalyx and Mentha spicata with specific primers multi-PCR system].

    Science.gov (United States)

    Cao, Liang; Qin, Shuang-shuang; Yuan, Yuan; Zhu, Xiao-qi

    2014-01-01

    To screen specific SNPs loci of Mentha haplocalyx and Mentha spicata,and then specific primers were designed to identify the two species and their mixture rapidly. PsbA-trnH sequences of Mentha haplocalyx and Mentha spicata were obtained by PCR product sequencing and downloading from GenBank. SNPs in the psbA-trnH sequences of Mentha haplocalyx and Mentha spicata were found by ClustulW program and Bioedit software. Primers for authentication of the two species were designed according to the SNP loci, and PCR reaction system was optimized to identify the original plants. Multi-PCR reaction system was constructed. The 181 bp identification band for Mentha haplocalyx or(and) 288 bp identification band for Mentha spicata could be produced by a single PCR reaction,which showed good identification ability to the two species. The multi-PCR reaction system can be applied to identify Mentha haplocalyx and Mentha spicata as well as their mixture.

  8. Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR.

    Science.gov (United States)

    Lebuhn, M; Effenberger, M; Garcés, G; Gronauer, A; Wilderer, P A

    2005-01-01

    In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

  9. Determination of allele frequency in pooled DNA: comparison of three PCR-based methods.

    Science.gov (United States)

    Wilkening, Stefan; Hemminki, Kari; Thirumaran, Ranjit Kumar; Bermejo, Justo Lorenzo; Bonn, Stefan; Försti, Asta; Kumar, Rajiv

    2005-12-01

    Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).

  10. PCR for the diagnosis of abdominal angiostrongyliasis in formalin-fixed paraffin-embedded human tissue.

    Directory of Open Access Journals (Sweden)

    Rubens Rodriguez

    Full Text Available To date the diagnosis of abdominal angiostrongyliasis (AA depends on the histological identification of Angiostrongylus costaricensis (AC in surgical specimens. However, microscopic evaluation is time consuming and often fails in identifying the parasite. We tested whether PCR might help in the diagnosis of AA by identifying parasite DNA in formalin-fixed paraffin-embedded (FFPE tissue. We used primers based on DNA from Angiostrongilus cantonensis. Four groups of FFPE intestinal tissue were tested: (1 confirmed cases (n = 20, in which AC structures were present in the target tissue; (2 presumptive cases (n = 20, containing changes secondary to AC infection in the absence of AC structures; (3 negative controls (n = 3, consisting of normal colonic tissue; and (4 tissue affected by other parasitoses (n = 7, including strongyloidiasis, ascaridiasis, schistosomiasis, and enterobiasis. Most lesions of confirmed cases were located in small and/or large bowel (90%, as compared with presumptive cases, in which 70% of lesions were in appendix (P = 0.0002. When confronted with cases of other parasitoses, PCR showed sensitivity of 55%, specificity of 100% and positive predictive value of 100%. In presumptive cases PCR was positive in 4 (20%. All specimens from negative controls and other parasitoses were negative. In conclusion, the PCR technique showed intermediate sensitivity and optimal specificity, being clinically relevant when positive for abdominal angiostrongyliasis. It allowed a 20% gain in diagnosis of presumptive cases. PCR might help in the diagnosis of abdominal angiostrongyliasis, particularly when the pathologists are not experienced with such disease.

  11. Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts.

    Science.gov (United States)

    Turton, Keren B; Esnault, Stephane; Delain, Larissa P; Mosher, Deane F

    2016-10-09

    Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (ΔS) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (α) or a truncated transactivation domain with 7 unique residues (β). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (Sα, Sβ, ΔSα and ΔSβ) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

  12. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.

    Science.gov (United States)

    Jahn, Michael; Vorpahl, Carsten; Türkowsky, Dominique; Lindmeyer, Martin; Bühler, Bruno; Harms, Hauke; Müller, Susann

    2014-06-17

    Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

  13. In situ PCR and immunohistochemical studies on p16 gene in pituitary adenomas

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To examine the occurrence of p16 gene deletion and to analyze p16 expression on paraffin-embedded human pituitary adenoma specimens. Efforts were made to optimize the technical conditions for in situ PCR. Methods: In situ PCR techniques and inimuno-histochemistry were used. Results: Immunohistochemically, p16-positive tumor cells were observed in all cases with various proportions. The majority of the stromal cells and part of tumor cells was devoid of p16 immunostaining, but signal of in situ PCR for p16 gene, exon 2, was displayed in these cells. Conclusion: The results implied that p16 gene might not be deleted in these pituitary adenomas. It also indicated that in situ PCR, both direct and indirect methods, proved feasible and informative to the aim of DNA detection. It is critical to overcome non-specific amplification in direct in situ PCR by means of higher annealing temperature, fewer cycle, lower magnesium concentration and stringent washing. A target DNA-deleted sample as the negative control is extremely necessary. For the indirect method, the way to improve the sensitivity is to loosen the conditions for amplification and washing, so that more amplification products are subject to hybridization, and signal detection is facilitated.

  14. Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision.

    Directory of Open Access Journals (Sweden)

    Nivedita Majumdar

    Full Text Available The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po is achieved by targeting ~1.59 mean copies per partition (λ, and that dynamic range (R includes the space spanning one positive (λL to one negative (λU result from the total number of partitions (n, these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.

  15. Mutagenic primer design for mismatch PCR-RFLP SNP genotyping using a genetic algorithm.

    Science.gov (United States)

    Yang, Cheng-Hong; Cheng, Yu-Huei; Yang, Cheng-Huei; Chuang, Li-Yeh

    2012-01-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is useful in small-scale basic research studies of complex genetic diseases that are associated with single nucleotide polymorphism (SNP). Designing a feasible primer pair is an important work before performing PCR-RFLP for SNP genotyping. However, in many cases, restriction enzymes to discriminate the target SNP resulting in the primer design is not applicable. A mutagenic primer is introduced to solve this problem. GA-based Mismatch PCR-RFLP Primers Design (GAMPD) provides a method that uses a genetic algorithm to search for optimal mutagenic primers and available restriction enzymes from REBASE. In order to improve the efficiency of the proposed method, a mutagenic matrix is employed to judge whether a hypothetical mutagenic primer can discriminate the target SNP by digestion with available restriction enzymes. The available restriction enzymes for the target SNP are mined by the updated core of SNP-RFLPing. GAMPD has been used to simulate the SNPs in the human SLC6A4 gene under different parameter settings and compared with SNP Cutter for mismatch PCR-RFLP primer design. The in silico simulation of the proposed GAMPD program showed that it designs mismatch PCR-RFLP primers. The GAMPD program is implemented in JAVA and is freely available at http://bio.kuas.edu.tw/gampd/.

  16. Role of RT-PCR and FISH in diagnosis and monitoring of acute promyelocytic leukemia

    Directory of Open Access Journals (Sweden)

    S Polampalli

    2011-01-01

    Full Text Available Background: Patients with a presence of Promyelocytic Leukemia-Retinoic Acid Receptor Alpha (PML-RARA genes rearrangement predict a favorable response to all-trans retinoic acid (ATRA, and a significant improvement in survival. Therefore, establishing the presence of PML-RARA rearrangement is important for optimal patient management. Aim: The objective of this study is to compare and assess the role of fluorescent in situ hybridization (FISH and reverse transcriptase polymerase chain reaction (RT-PCR in the diagnosis and long-term monitoring of Acute Promyelocytic Leukemia (APL. Materials and Methods: We compared 145 samples received at different interval of times to analyze the sensitivity of RT-PCR and FISH. Results: The failure rate for RT-PCR was 4% at baseline, 13% at induction, and 0% at the end of consolidation. And for FISH it was 8% at baseline, 38% at induction, and 66% at the end of consolidation. The predictive values of relapse in the patients who were positive and negative by RT-PCR, at the end of induction, were 60 % and 3%, respectively, and at end of consolidation it was 67 % and 4%, respectively. On the other hand the predictive values of relapse in patients who were positive and negative by FISH at end of induction were 57 % and 6%, respectively; while at end of consolidation it was 14% who were negative by FISH. Conclusion: Both RT-PCR and FISH are important for the diagnosis of APL cases, as both techniques complement each other in the absence or failure of any one of them. However, RT-PCR is more sensitive than FISH for the detection of minimal residual disease in the long-term monitoring of these patients. The present study shows that the predictive value of relapse is more associated with minimal residual disease (MRD results by RT-PCR than that by FISH.

  17. Droplet digital PCR-aided screening and characterization of Pichia pastoris multiple gene copy strains.

    Science.gov (United States)

    Cámara, Elena; Albiol, Joan; Ferrer, Pau

    2016-07-01

    Pichia (syn. Komagataella) pastoris is a widely used yeast platform for heterologous protein production. Expression cassettes are usually stably integrated into the genome of this host via homologous recombination. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden, particularly for secreted proteins. We have constructed a series of strains harboring different copy numbers of a Rhizopus oryzae lipase gene (ROL), aiming to find the optimum gene dosage for secreted Rol production. In order to accurately determine ROL gene dosage, we implemented a novel protocol based on droplet digital PCR (ddPCR), and cross validated it with conventional real-time PCR. Gene copy number determination based on ddPCR allowed for an accurate ranking of transformants according to their ROL gene dosage. Results indicated that ddPCR was particularly superior at lower gene dosages (one to five copies) over quantitative real-time PCR (qPCR). This facilitated the determination of the optimal ROL gene dosage as low as two copies. The ranking of ROL gene dosage versus Rol yield was consistent at both small scale and bioreactor chemostat cultures, thereby easing clone characterization in terms of gene dosage dependent physiological effects, which could be discriminated even among strains differing by only one ROL copy. A selected two-copy strain showed twofold increase in Rol specific production in a chemostat culture over the single copy strain. Conversely, strains harboring more than two copies of the ROL gene showed decreased product and biomass yields, as well as altered substrate consumption specific rates, compared to the reference (one-copy) strain. Biotechnol. Bioeng. 2016;113: 1542-1551. © 2015 Wiley Periodicals, Inc.

  18. Diagnosis of Caprine Arthritis Encephalitis Virus infection in dairy goats by ELISA, PCR and Viral Culture.

    Science.gov (United States)

    Panneum, S; Rukkwamsuk, T

    2017-03-01

    For preventive and control strategies of Caprine Arthritis Encephalitis Virus (CAEV) infection in dairy goats, performance of the available diagnostic tests was described as one of the most important and necessary aspects. The study aimed at evaluating the diagnostic test performance, including PCR, ELISA and viral culture, for CAEV infection in dairy goats in Thailand. Blood samples of 29 dairy goats from five low- to medium-prevalence herds and one very low-prevalence herd were collected for PCR and ELISA methods. The performance of these two diagnostic methods was evaluated by comparing with cytopathic effects (CPE) in the co-cultivation of CAEV and primary synovial cells. Results indicated that sensitivity, specificity were, respectively, 69.6%, 100%, for PCR; and 95.7%, 83.3% for ELISA. The PCR assay tended to have lower sensitivity and higher specificity than ELISA. When multiple tests were applied, parallel testing provided sensitivity and specificity of 98.7% and 83.3%, while series testing showed sensitivity and specificity of 66.6% and 100% respectively. These results indicated that combination of ELISA and PCR provided some advantages and possibly offered optimal methods to detect CAEV-infected goats. Kappa value of the agreement between PCR and ELISA test was 0.34, indicating fair agreement. Regarding the possibility of antigenic variation between CAEV strains used in both PCR and ELISA assays, the actual circulating CAEV strain should be reviewed in order to develop and enhance the diagnostic tests using the CAE viral antigens derived from specific local strains of Thailand.

  19. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    Science.gov (United States)

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  20. Development of a CMOS-compatible PCR chip: comparison of design and system strategies

    Science.gov (United States)

    Erill, Ivan; Campoy, Susana; Rus, José; Fonseca, Luis; Ivorra, Antoni; Navarro, Zenón; Plaza, José A.; Aguiló, Jordi; Barbé, Jordi

    2004-11-01

    In the last decade research in chips for DNA amplification through the polymerase chain reaction (PCR) has been relatively abundant, but has taken very diverse approaches, leaving little common ground for a straightforward comparison of results. Here we report the development of a line of PCR chips that is fully compatible with complementary-metal-oxide-semiconductor (CMOS) technology and its revealing use as a general platform to test and compare a wide range of experimental parameters involved in PCR-chip design and operation. Peltier-heated and polysilicon thin-film driven PCR chips have been produced and directly compared in terms of efficiency, speed and power consumption, showing that thin-film systems run faster and more efficiently than Peltier-based ones, but yield inferior PCR products. Serpentine-like chamber designs have also been compared with standard rectangular designs and with the here reported rhomboidal chamber shape, showing that serpentine-like chambers do not have detrimental effects in PCR efficiency when using non-flow-through schemes, and that chamber design has a strong impact on sample insertion/extraction yields. With an accurate temperature control (±0.2 °C) we have optimized reaction kinetics to yield sound PCR amplifications of 25 µl mixtures in 20 min and with 24.4 s cycle times, confirming that a titrated amount of bovine albumin serum (BSA, 2.5 µg µl-1) is essential to counteract polymerase adsorption at chip walls. The reported use of a CMOS-compatible technological process paves the way for an easy adaption to foundry requirements and for a scalable integration of electro-optic detection and control circuitry.

  1. Application of PCR techniques in toxicology

    Directory of Open Access Journals (Sweden)

    Maja Kazubek

    2010-10-01

    Full Text Available Molecular biology techniques have become widely used in toxicology, leading to the creation of a new science – molecular toxicology. The goal of molecular toxicology is to detect and study the changes induced by xenobiotics at the molecular level. The research scope of molecular toxicology includes examination of mutations in genomic DNA, differences in mRNA expression and study of genotype indicating individual sensitivity.The processes of activation and detoxification of xenobiotics, drugs and environmental carcinogens involve several enzymes (xenobiotic-metabolizing enzymes – XMEs. Most of the chemicals entering our bodies, regardless of whether they have medical, pathogenic or carcinogenic properties, require metabolic activation by phase I enzymes (cytochrome P-450. In the next process the phase I products are usually detoxified by phase II enzymes, mainly by epoxide hydrolase, glutathione transferase, N-acetyltransferase or sulfotransferase. PCR techniques allow precise study of the effects of xenobiotics on cells and tissues by examining the level of activation of genes coding for phase I and II enzymes, or by testing the activity of other elements of the transcriptome. Studies of sensitivity of individual cells or tissues based on examination of mutation or gene polymorphism presence are also relevant.This paper presents the possibility of using various PCR techniques in toxicology and especially in the study of genetically determined sensitivity to xenobiotics. It also covers the possibilities of applying qPCR and qRT-PCR methods in the search for exposure biomarkers with particular emphasis on individual cytochrome P450 isoforms. Furthermore, it provides information about the possibility of implementing the differential display technique in the identification of new genes activated by toxic agents.

  2. Miniature PCR based portable bioaerosol monitor development.

    Science.gov (United States)

    Agranovski, I E; Usachev, E V; Agranovski, E; Usacheva, O V

    2017-01-01

    A portable bioaerosol monitor is greatly demanded technology in many areas including air quality control, occupational exposure assessment and health risk evaluation, environmental studies and, especially, in defence and bio-terrorism applications. Our recent groundwork allowed us to formulate the concept of a portable bioaerosol monitor, which needs to be light, user friendly, reliable and capable of detecting airborne pathogens within 1-1·5 h on the spot. Conceptually, the event of a bioaerosol concentration burst is determined by triggers to commence the representative air sampling with sequential real-time polymerase chain reaction (PCR) confirmation of the targeted micro-organism present in the air. To minimize reagent consumption and idle running of the technology, an event of a bioaerosol burst is confirmed by three parameters: aerosol particle size, concentration and composition. Only particle sizes above 200 nm attract interest in the bioaerosol. Only an elevated aerosol concentration above the threshold (background aerosol concentration) is a signal to commence the analytical procedure. The combination of our previously developed personal bioaerosol sampler, aerosol particle counter based trigger and portable real-time PCR device formed the basis of the bioaerosol monitoring technology. The portable real-time PCR device was advanced to provide internally controlled detection, significantly reducing false-positive alarms. The technique is capable of detecting selected airborne micro-organisms on the spot within 30-80 min, depending on the genome organization of the particular strain. Due to recent outbreaks of infectious airborne diseases and the continuing threat of intentionally released bioaerosol attacks, investigations into the possibility of the early and reliable detection of pathogenic micro-organisms in the air is becoming increasingly important. The proposed technology consisting of a bioaerosol sampler, technology trigger and PCR device is

  3. Reproducibility of AMPLICOR enterovirus PCR test results.

    OpenAIRE

    1997-01-01

    The reproducibility of AMPLICOR enterovirus PCR test results was determined with clinical samples of cerebrospinal fluid, serum, urine, and throat and rectal swabs. Among 608 samples from which duplicate aliquots were run simultaneously, only seven pairs gave discordant results. Among 104 samples from which duplicate aliquots were run in separate assays, no discordance was seen. Overall, the reproducibility of test kit results was 99% (705 of 712).

  4. Clostridium perfringens isolate typing by multiplex PCR

    Directory of Open Access Journals (Sweden)

    MR Ahsani

    2010-01-01

    Full Text Available Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

  5. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    Directory of Open Access Journals (Sweden)

    Yun Zhao

    Full Text Available Droplet digital polymerase chain reaction (ddPCR is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001. Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications.

  6. Amp-PCR: combining a random unbiased Phi29-amplification with a specific real-time PCR, performed in one tube to increase PCR sensitivity.

    Directory of Open Access Journals (Sweden)

    Lena Erlandsson

    Full Text Available In clinical situations where a diagnostic real-time PCR assay is not sensitive enough, leading to low or falsely negative results, or where detection earlier in a disease progression would benefit the patient, an unbiased pre-amplification prior to the real-time PCR could be beneficial. In Amp-PCR, an unbiased random Phi29 pre-amplification is combined with a specific real-time PCR reaction. The two reactions are separated physically by a wax-layer (AmpliWax® and are run in sequel in the same sealed tube. Amp-PCR can increase the specific PCR signal at least 100×10(6-fold and make it possible to detect positive samples normally under the detection limit of the specific real-time PCR. The risk of contamination is eliminated and Amp-PCR could replace nested-PCR in situations where increased sensitivity is needed e.g. in routine PCR diagnostic analysis. We show Amp-PCR to work on clinical samples containing circular and linear viral dsDNA genomes, but can work well on DNA of any origin, both from non-cellular (virus and cellular sources (bacteria, archae, eukaryotes.

  7. Optimal transport

    CERN Document Server

    Eckmann, B

    2008-01-01

    At the close of the 1980s, the independent contributions of Yann Brenier, Mike Cullen and John Mather launched a revolution in the venerable field of optimal transport founded by G Monge in the 18th century, which has made breathtaking forays into various other domains of mathematics ever since. The author presents a broad overview of this area.

  8. Topology optimization

    DEFF Research Database (Denmark)

    Bendsøe, Martin P.; Sigmund, Ole

    2007-01-01

    Taking as a starting point a design case for a compliant mechanism (a force inverter), the fundamental elements of topology optimization are described. The basis for the developments is a FEM format for this design problem and emphasis is given to the parameterization of design as a raster image...

  9. Optimization of cDNA amplification of Apricot Latent Virus (ApLV) from various plant tissues sources.

    Science.gov (United States)

    Gumus, M; Sipahioğlu, H M; Paylan, I C; Erkan, S

    2007-03-15

    Although the reverse transcriptase polymerase chain reaction (RT-PCR) procedure is basically simple operation, often it is not possible to achieve optimum results without optimizing the protocols. An RT-PCR method targeting a 200 bp sequence of the CP gene of Apricot Latent Virus (ApLV) was used as a model to improve the detection limit and to compare the behavior of three different plant tissues in a RT-PCR assay. A number of factors should be considered when selecting the optimal system for RT-PCR. Important considerations include the optimal concentrations of MgCl2, dNTP, Taq DNA polymerase enzyme, specific primer and the amount of cDNA for the downstream applications. This study therefore discusses a series of critical PCR parameters and feasible strategies for optimization of RT-PCR detection of ApLV.

  10. Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

    Science.gov (United States)

    Laprade, Natacha; Cloutier, Michel; Lapen, David R; Topp, Edward; Wilkes, Graham; Villemur, Richard; Khan, Izhar U H

    2016-05-01

    Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological

  11. Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.

    Science.gov (United States)

    Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

    2016-12-01

    In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30μg/mL the number of PCR-positive living bacteria decreased from 10(6) to 10(5) cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes.

  12. Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.)

    NARCIS (Netherlands)

    Rakus, K.L.; Wiegertjes, G.F.; Adamek, M.; Bekh, V.; Stet, R.J.M.; Irnazarow, I.

    2008-01-01

    A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals

  13. Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

    Science.gov (United States)

    van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

    2016-01-01

    Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.

  14. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

    Science.gov (United States)

    Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

    2017-06-01

    A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

    Science.gov (United States)

    Luan, Huaibiao; Wang, Yixin; Li, Yang; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2016-09-01

    Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers).

  16. 锚定PCR(Anchored PCR):一种新的染色体步行方法

    Institute of Scientific and Technical Information of China (English)

    陈柏君; 孙超; 王勇; 胡鸢雷; 林忠平

    2004-01-01

    基于PCR的技术是克隆已知DNA片段侧翼序列的最常用方法.到目前为止,这些方法大致可以分为3种类型:反向PCR(inverse PCR)、连接介导的PCR(1igation-mediated PCR)和随机引物PCR(randomly primed PCR).反向PCR是使用最早的方法,其原理是用限制性内切酶消化基因组总

  17. Detection and Comparison of Pathogen of Virus Disease in Pumpkin by RT-PCR and IC-PCR

    Institute of Scientific and Technical Information of China (English)

    YANG Guohui; ZHANG Zhongkai; CUI Chongshi

    2006-01-01

    Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased samples gathered in Yunnan Province, while WMV and CMV were detected in diseased samples gathered in Heilongjiang Province. The sensitivity of RT-PCR is higher than that of IC-PCR, but the effect of IC-PCR in the specialization of bonding reaction and requisition for experiment material is better than that of RT-PCR.

  18. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    Science.gov (United States)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

  19. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

    Science.gov (United States)

    Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

    2015-04-15

    Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins.

    Science.gov (United States)

    Ziebell, Kim A; Read, Susan C; Johnson, Roger P; Gyles, Carlton L

    2002-06-01

    This study evaluated two generic polymerase chain reaction (PCR) protocols, and nine subtyping protocols and three PCR-restriction fragment length polymorphism (RFLP) protocols for detection of stx genes. The PCR protocols were evaluated by testing 12 reference isolates and 496 field strains of Shiga toxin-producing Escherichia coli (STEC). Both generic methods detected all stx genes. In tests with the reference isolates, all methods detected stx1 and stx2, seven subtyping methods detected stx2v(EH250), seven detected stx2e and only two detected stx2f. Four of the subtyping protocols identified stx genes in all of the field isolates. The PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested. The observed limitations of the protocols were shown to be due to nucleotide sequence variation in the region of the PCR primers. One subtyping protocol that detected the virulence-related genes, eae and ehxA, and all stx except for the stx2f gene, was modified by newly designed primers so that it identified all stx genes. This modified protocol provides comprehensive characterization of STEC in a single multiplex reaction.

  1. Optimal control

    CERN Document Server

    Aschepkov, Leonid T; Kim, Taekyun; Agarwal, Ravi P

    2016-01-01

    This book is based on lectures from a one-year course at the Far Eastern Federal University (Vladivostok, Russia) as well as on workshops on optimal control offered to students at various mathematical departments at the university level. The main themes of the theory of linear and nonlinear systems are considered, including the basic problem of establishing the necessary and sufficient conditions of optimal processes. In the first part of the course, the theory of linear control systems is constructed on the basis of the separation theorem and the concept of a reachability set. The authors prove the closure of a reachability set in the class of piecewise continuous controls, and the problems of controllability, observability, identification, performance and terminal control are also considered. The second part of the course is devoted to nonlinear control systems. Using the method of variations and the Lagrange multipliers rule of nonlinear problems, the authors prove the Pontryagin maximum principle for prob...

  2. Viral diagnostics in the era of digital PCR

    Science.gov (United States)

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  3. Distributed optimality

    OpenAIRE

    Trommer, Jochen

    2005-01-01

    In dieser Dissertation schlage ich eine Synthese (Distributed Optimality, DO) von Optimalitätstheorie und einem derivationellen, morphologischem Asatz, Distributed Morphology (DM; Halle & Marantz, 1993) vor. Durch die Integration von OT in DM wird es möglich, Phänomene, die in DM durch sprachspezifische Regeln oder Merkmale von lexikalischen Einträge erfasst werden, auf die Interaktion von verletzbaren, universellen Constraints zurückzuführen. Andererseits leistet auch DM zwei substantielle B...

  4. Optimal learning

    CERN Document Server

    Powell, Warren B

    2012-01-01

    Learn the science of collecting information to make effective decisions Everyday decisions are made without the benefit of accurate information. Optimal Learning develops the needed principles for gathering information to make decisions, especially when collecting information is time-consuming and expensive. Designed for readers with an elementary background in probability and statistics, the book presents effective and practical policies illustrated in a wide range of applications, from energy, homeland security, and transportation to engineering, health, and business. This book covers the

  5. Optimization Problems

    Directory of Open Access Journals (Sweden)

    Yaping Hu

    2015-01-01

    the nonsmooth convex optimization problem. First, by using Moreau-Yosida regularization, we convert the original objective function to a continuously differentiable function; then we use approximate function and gradient values of the Moreau-Yosida regularization to substitute the corresponding exact values in the algorithm. The global convergence is proved under suitable assumptions. Numerical experiments are presented to show the effectiveness of this algorithm.

  6. Evaluation of a Probe-Based PCR-ELISA System for Simultaneous Semi Quantitative Detection and Genotyping of Human Cytomegalovirus (HCMV) Infection in Clinical Specimens.

    Science.gov (United States)

    Talkhabifard, Majid; Javid, Naeme; Moradi, Abdolvahab; Ghaemi, Amir; Tabarraei, Alijan

    2017-01-01

    Human cytomegalovirus (HCMV) is a common opportunistic pathogen that causes serious complications in immunosuppressed patients and infected newborns. In this study, PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance. During DIG-labeling PCR, a pair of primers amplifies a fragment of variable region of the glycoprotein B encoding sequence. Under optimized conditions, labeled Target amplicons hybridize to biotinated specific probes and are detected in an ELISA system. PCR-ELISA system showed specific performance with detection limit of approximately 100 copies of CMV DNA. The linear correlation was observed between the PCR-ELISA results (OD) and logarithmic scale of CMV (r=0.979). Repeatability of PCR-ELISA detection system for intra-assay and inter-assay was evaluated for negative and positive samples. In optimized conditions of hybridization, differentiation between genotypes of glycoprotein B was feasible using genotype-specific probes in PCR-ELISA genotyping system. In comparison with sequencing method, genotyping system was confirmed with kappa index of 1. PCR-ELISA is proposed as an applicable and reliable technique for semi-quantitative diagnosis and typing of the infection. This technique is flexible to apply in a variety of molecular fields.

  7. Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR.

    Science.gov (United States)

    Wu, Bin; Liang, Weili; Kan, Biao

    2015-08-01

    The well-known human pathogenic bacterium, Vibrio cholerae, can enter a physiologically viable but non-culturable (VBNC) state under stress conditions. The differentiation of VBNC cells and nonviable cells is essential for both disease prevention and basic research. Among all the methods for detecting viability, propidium monoazide (PMA) combined with real-time PCR is popular because of its specificity, sensitivity, and speed. However, the effect of PMA treatment is not consistent and varies among different species and conditions. In this study, with an initial cell concentration of 1×10(8) CFU/ml, time and dose-effect relationships of different PMA treatments were evaluated via quantitative real-time PCR using live cell suspensions, dead cell suspensions and VBNC cell suspensions of V. cholerae O1 El Tor strain C6706. The results suggested that a PMA treatment of 20 μM PMA for 20 min was optimal under our conditions. This treatment maximized the suppression of the PCR signal from membrane-compromised dead cells but had little effect on the signal from membrane-intact live cells. In addition to the characteristics of PMA treatment itself, the initial concentration of the targeted bacteria showed a significant negative influence on the stability of PMA-PCR assay in this study. We developed a strategy that mimicked a 1×10(8) CFU/ml cell concentration with dead bacteria of a different bacterial species, the DNA of which cannot be amplified using the real time PCR primers. With this strategy, our optimal approach successfully overcame the impact of low cell density and generated stable and reliable results for counting viable cells of V. cholerae in the VBNC state. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Modification of Cre Gene by PCR Method

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Cre/LoxP site-specified recombination system is mainly used for excision,inversion and integration of target gene.Therefore,this system can be used for plant marker free genetic transformation,site-specific transgene expression and so on.However,the application of this system was limited due to its low expression and excision efficiency.In this study,an intron,which can enhance gene expression in plants,was inserted into Cre by using PCR method.And a modified Cre gene,named Crein,was obtained.This gene was ...

  9. Analysis of extracellular RNA by digital PCR

    Directory of Open Access Journals (Sweden)

    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  10. Nanoscale superstructures assembled by polymerase chain reaction (PCR): programmable construction, structural diversity, and emerging applications.

    Science.gov (United States)

    Kuang, Hua; Ma, Wei; Xu, Liguang; Wang, Libing; Xu, Chuanlai

    2013-11-19

    Polymerase chain reaction (PCR) is an essential tool in biotechnology laboratories and is becoming increasingly important in other areas of research. Extensive data obtained over the last 12 years has shown that the combination of PCR with nanoscale dispersions can resolve issues in the preparation DNA-based materials that include both inorganic and organic nanoscale components. Unlike conventional DNA hybridization and antibody-antigen complexes, PCR provides a new, effective assembly platform that both increases the yield of DNA-based nanomaterials and allows researchers to program and control assembly with predesigned parameters including those assisted and automated by computers. As a result, this method allows researchers to optimize to the combinatorial selection of the DNA strands for their nanoparticle conjugates. We have developed a PCR approach for producing various nanoscale assemblies including organic motifs such as small molecules, macromolecules, and inorganic building blocks, such as nanorods (NRs), metal, semiconductor, and magnetic nanoparticles (NPs). We start with a nanoscale primer and then modify that building block using the automated steps of PCR-based assembly including initialization, denaturation, annealing, extension, final elongation, and final hold. The intermediate steps of denaturation, annealing, and extension are cyclic, and we use computer control so that the assembled superstructures reach their predetermined complexity. The structures assembled using a small number of PCR cycles show a lower polydispersity than similar discrete structures obtained by direct hybridization between the nanoscale building blocks. Using different building blocks, we assembled the following structural motifs by PCR: (1) discrete nanostructures (NP dimers, NP multimers including trimers, pyramids, tetramers or hexamers, etc.), (2) branched NP superstructures and heterochains, (3) NP satellite-like superstructures, (4) Y-shaped nanostructures and DNA

  11. Real-Time PCR for Universal Phytoplasma Detection and Quantification

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Nyskjold, Henriette; Nicolaisen, Mogens

    2013-01-01

    Currently, the most efficient detection and precise quantification of phytoplasmas is by real-time PCR. Compared to nested PCR, this method is less sensitive to contamination and is less work intensive. Therefore, a universal real-time PCR method will be valuable in screening programs and in other...

  12. Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

    NARCIS (Netherlands)

    White, P.L.; Mengoli, C.; Bretagne, S.; Cuenca-Estrella, M.; Finnstrom, N.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2011-01-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was

  13. Multiplex polymerase chain reaction (PCR) on a SU-8 chip

    DEFF Research Database (Denmark)

    Christensen, Troels Balmer; Bang, Dang Duong; Wolff, Anders

    2008-01-01

    We present the detection of Campylobacter at species level using multiplex PCR in a micro fabricated PCR chip. The chip is based on the polymer SU-8 that allows integration with different microfluidic components, e.g., sample pre-treatment before PCR, and DNA detection simultaneously with or afte...

  14. Determining Fungi rRNA Copy Number by PCR

    Science.gov (United States)

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  15. Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

    NARCIS (Netherlands)

    White, P.L.; Mengoli, C.; Bretagne, S.; Cuenca-Estrella, M.; Finnstrom, N.; Klingspor, L.; Melchers, W.J.G.; McCulloch, E.; Barnes, R.A.; Donnelly, J.P.; Loeffler, J.

    2011-01-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was re

  16. Determining Fungi rRNA Copy Number by PCR

    Science.gov (United States)

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  17. Application of EMA-qPCR as a complementary tool for the detection and monitoring of Legionella in different water systems.

    Science.gov (United States)

    Qin, Tian; Tian, Zhengan; Ren, Hongyu; Hu, Guangchun; Zhou, Haijian; Lu, Jinxing; Luo, Chengwang; Liu, Zunyu; Shao, Zhujun

    2012-05-01

    Legionella are prevalent in human-made water systems and cause legionellosis in humans. Conventional culturing and polymerase chain reaction (PCR) techniques are not sufficiently accurate for the quantitative analysis of live Legionella bacteria in water samples because of the presence of viable but nonculturable cells and dead cells. Here, we report a rapid detection method for viable Legionella that combines ethidium monoazide (EMA) with quantitative real-time PCR (qPCR) and apply this method to detect Legionella in a large number of water samples from different sources. Results yielded that samples treated with 5 μg/ml EMA for 10 min and subsequently exposed to light irradiation for 5 min were optimal for detecting Legionella. EMA treatment before qPCR could block the signal from approximately 4 log(10) of dead cells. When investigating environmental water samples, the percent-positive rate obtained by EMA-qPCR was significantly higher than conventional PCR and culture methods, and slightly lower than qPCR. The bacterial count of Legionella determined by EMA-qPCR were mostly greater than those determined by culture assays and lower than those determined by qPCR. Acceptable correlations were found between the EMA-qPCR and qPCR results for cooling towers, piped water and hot spring water samples (r = 0.849, P EMA-qPCR and culture results for hot spring water samples (r = 0.698, P EMA-qPCR could be used as a complementary tool for the detection and monitoring of Legionella in water systems, especially in hot spring water samples.

  18. Comparison of a Commercially Available Repetitive-Element PCR System (DiversiLab) with PCR Ribotyping for Typing of Clostridium difficile Strains ▿

    OpenAIRE

    Eckert, C; Van Broeck, J; Spigaglia, P.; Burghoffer, B.; Delmée, M; Mastrantonio, P; Barbut, F

    2011-01-01

    This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

  19. Comparison of a Commercially Available Repetitive-Element PCR System (DiversiLab) with PCR Ribotyping for Typing of Clostridium difficile Strains ▿

    OpenAIRE

    Eckert, C.; Van Broeck, J.; Spigaglia, P.; Burghoffer, B; Delmée, M; Mastrantonio, P; Barbut, F

    2011-01-01

    This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

  20. Identification of periodontopathogen microorganisms by PCR technique

    Directory of Open Access Journals (Sweden)

    Milićević Radovan

    2008-01-01

    Full Text Available INTRODUCTION Periodontitis is an inflammatory disease of the supporting tissues of teeth and is a major cause of tooth loss in adults. The onset and progression of periodontal disease is attributed to the presence of elevated levels of a consortium of pathogenic bacteria. Gram negative bacteria, mainly strict anaerobes, play the major role. OBJECTIVE The present study aimed to assess the presence of the main types of microorganisms involved in the aetiopathogenesis of periodontal disease: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Treponema denticola, Tanerella forsythia and Prevotella intermedia in different samples collected from the oral cavity of 90 patients diagnosed with periodontitis. METHOD Bacterial DNA detection was performed in diverse biological materials, namely in dental plaque, gingival tissue and saliva, by means of multiplex PCR, a technique that allows simultaneous identification of two different bacterial genomes. RESULTS In the dental plaque of the periodontitis patients, Treponema denticola dominated. In the gingival tissue, Tannerella forsythia and Treponema denticola were the microbiota most frequently detected, whilst in saliva Treponema denticola and Eikenella corrodens were found with the highest percentage. CONCLUSION The identification of microorganisms by multiplex PCR is specific and sensitive. Rapid and precise assessment of different types of periodontopathogens is extremely important for early detection of the infection and consequently for the prevention and treatment of periodontal disease. In everyday clinical practice, for routine bacterial evaluation in patients with periodontal disease, the dental plaque is the most suitable biological material, because it is the richest in periodontal bacteria.

  1. In silico PCR primer designing and validation.

    Science.gov (United States)

    Kumar, Anil; Chordia, Nikita

    2015-01-01

    Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like "Primer3" and "Web Primer". For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment.

  2. Applications of real-time PCR in the screening of platelet concentrates for bacterial contamination.

    Science.gov (United States)

    Mohammadi, Tamimount; Savelkoul, Paul H M; Pietersz, Ruby N I; Reesink, Henk W

    2006-11-01

    Although there have been major improvements over the past few decades in detection methods for blood-borne infectious agents, platelet concentrates are still responsible for most cases of transfusion-transmitted bacterial infections. To date, real-time PCR is an indispensable tool in diagnostic laboratories to detect pathogens in a variety of biological samples. In this article, the applications of this powerful technique in the screening of platelet concentrates for bacterial contamination are discussed. Next to pathogen-specific (real-time) PCR assays, particular attention is directed to the recently developed 16S rDNA real-time PCR. This assay has been proven as a convenient way to detect bacterial contamination of platelet concentrates. The assay is sensitive and enables rapid detection of low initial numbers of bacteria in platelet concentrates. The short turnaround time of this assay allows high-throughput screening and reduction of the risk of transfusion of bacterially contaminated units. As with every method, real-time PCR has its advantages and disadvantages. These and especially limitations inherent to generation of false-positive or -negative results are emphasized. The universal nature of detection of the assay may be suitable for generalized bacterial screening of other blood components, such as red blood cells and plasma. Therefore, it is necessary to adapt and optimize detection in red blood cells and plasma with real-time PCR. Further sophistication, miniaturization and standardization of extraction and amplification methods should improve the total performance and robustness of the assay. Hence, real-time PCR is an attractive method in development as a more rapid screening test than currently used culture methods to detect bacterial contamination in blood components.

  3. An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples.

    Science.gov (United States)

    Repetto, S A; Alba Soto, C D; Cazorla, S I; Tayeldin, M L; Cuello, S; Lasala, M B; Tekiel, V S; González Cappa, S M

    2013-05-01

    Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.

  4. Comparison of different protocols for DNA preparation and PCR amplification of mitochondrial genes of tardigrades

    Directory of Open Access Journals (Sweden)

    Ralph O. SCHILL

    2007-09-01

    Full Text Available Phylogenetic relationships and molecular taxonomy within the Tardigrada have been given a lot of attention in recent years. Here I present the first comparison of different protocols for DNA preparation by investigating six commercial available DNA extraction kits and the CTAB method. Successful extraction of DNA from tardigrades depends strongly on the life-stage (embryo, adult, and on the condition of the specimens, respectively on the preservation (anhydrobiotic, ethanol. Although the extraction kits showed differences in the amount of extracted DNA, in all cases fresh tissue of live animals or embryos resulted in the best quality and quantity of DNA. A lesser amount of DNA was extractable from anhydrobiotic animals and embryos and the results of specimens fixed in ethanol were unsatisfactory. All used commercially available DNA extraction kits and PCR cocktails have been focused on vertebrate tissues, blood, cultured cells, bacteria and yeast. However, I used successfully the kits according to the manufacturer’s instruction without changes in the protocols for DNA extraction of tardigrades. Commercial kits provide a simple and convenient way to isolate pure genomic DNA of high-quality from tardigrades. Furthermore I tested eight different Taq polymerase enzymes for PCR amplification of mitochondrial genes of tardigrades. Each of the enzymes resulted in a PCR product, and even if the amount of the PCR products was quite different, it was possible to use it successful for direct sequencing. Summarizing, the successful PCR of the target DNA depends on the purity and quality of the DNA template and for this the species preservation is more critical than the extraction method or the PCR cocktail which can be optimized.

  5. A real-time PCR antibiogram for drug-resistant sepsis.

    Directory of Open Access Journals (Sweden)

    John R Waldeisen

    Full Text Available Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL. Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01. Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24

  6. Comparison of the sensitivity of RT-PCR and general PCR in detecting pathogenic microorganisms%实时荧光PCR和普通PCR方法检测病原微生物的灵敏度比较

    Institute of Scientific and Technical Information of China (English)

    钟岸; 蔡蓁; 王毅

    2013-01-01

    Objective To compare the detecting threshold of two different PCR methods,and to select the accurate and economical way to detect the pathogenic microorganisms.Methods Standard samples were applied to establish the optimal condition for the two kinds of PCR methods.Primer design was based on the targeting sequence of the pathogenic microorganism.The products of the PCR were detected by amplification plot or agarose-gel electrophoresis.The highest detecting sensitivity was set by the lowest concentration which is able to be detected by PCR.By comparison,the optimal method was chosen for the pathogenic microorganism detection.Results Supematant of bacteria lysis solution in real-time PCR after 10-8 dilution can still be observed with amplification curve.The general PCR products after agarose gel electrophoresis staining can be observed only with the concentration of 10-5.The sensitivity of general PCR was significantly lower than that of RT-PCR.Conclusion RT-PCR has higher detecting sensitivity,but general PCR is more economical.%目的 通过比较两种PCR检测的灵敏度,选择不同条件下经济准确的方法检测病原微生物.方法 制备标准样品用以两种PCR扩增条件的确定,通过目的基因片段设计引物,扩增菌体裂解液上清.PCR产物分别通过扩增曲线和琼脂糖电泳检测目的条带的存在,以最低检测的条带浓度作为两种不同PCR检测的最高灵敏度,通过比较,选择在不同的限制条件下较为方便经济的方法检测病原微生物.结果 实时PCR中裂解菌液上清经过10-8倍稀释后仍能观测到扩增曲线的存在.而普通PCR产物经过琼脂糖电泳染色后观测只能观测到10-5浓度的条带,灵敏度远不及RT-PCR.结论 荧光PCR检测灵敏度较高,普通PCR琼脂糖电泳方法只能达到RT-PCR检测灵敏度的一半但比较经济.

  7. Molecular diagnosis of African Swine Fever by a new real-time PCR using universal probe library.

    Science.gov (United States)

    Fernández-Pinero, J; Gallardo, C; Elizalde, M; Robles, A; Gómez, C; Bishop, R; Heath, L; Couacy-Hymann, E; Fasina, F O; Pelayo, V; Soler, A; Arias, M

    2013-02-01

    A highly sensitive and specific real-time PCR method was developed for the reliable and rapid detection of African swine fever virus (ASFV). The method uses a commercial Universal Probe Library (UPL) probe combined with a specifically designed primer set to amplify an ASFV DNA fragment within the VP72 coding genome region. The detection range of the optimized UPL PCR technique was confirmed by analysis of a large panel (n = 46) of ASFV isolates, belonging to 19 of the 22 viral p72 genotypes described. No amplification signal was observed when closely clinically related viruses, such as classical swine fever, or other porcine pathogens were tested by this assay. The detection limit of the UPL PCR method was established below 18 DNA copies. Validation experiments using an extensive collection of field porcine and tick samples (n = 260), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over 10% more positive samples than the real-time TaqMan PCR test recommended in the OIE manual, confirming its superior diagnostic sensitivity. Clinical material collected during experimental infections with different ASFV p72 genotypes was useful for assuring both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. The reliability and robustness of the UPL PCR was finally verified with a panel of ASFV-infected clinical samples which was repeatedly tested at different times. Additionally, an internal control PCR assay was also developed and standardized using UPL probes within the endogenous β-actin gene. Finally, the complete study offers a new validated real-time PCR technique, by means of a standardized commercial probe, providing a simple, rapid and affordable test, which is ready for application in the routine diagnosis of ASF.

  8. Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning.

    Science.gov (United States)

    Qiu, X; Wu, L; Huang, H; McDonel, P E; Palumbo, A V; Tiedje, J M; Zhou, J

    2001-02-01

    To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

  9. Comparison of three different commercial PCR assays for the detection of pathogens in critically ill sepsis patients.

    Science.gov (United States)

    Schreiber, J; Nierhaus, A; Braune, S A; de Heer, G; Kluge, S

    2013-05-01

    The high mortality rate associated with sepsis necessitates a timely identification of the causative organism in order to optimize antimicrobial therapy. PCR assays are increasingly being used for this purpose. The aim of this study was to compare three commercially available PCR systems for the diagnosis of systemic infections. In a prospective observational study, a broad-range (SepsiTest®; Molzym, Bremen, Germany) and two multiplex PCR assays (VYOO®; SIRS-Lab, Jena, Germany and LightCycler® SeptiFast; Roche, Mannheim, Germany) were compared to blood cultures with respect to the clinical course of 50 critically ill patients with sepsis, severe sepsis or septic shock. Pathogens were detected by PCR in 12 % (SepsiTest®), 10 % (VYOO®) and 14 % (LightCycler® SeptiFast) of samples and in 26 % by blood culture. Negative results were obtained using all four methods in 32 samples (64 %) and 3 (6 %) samples were positive in all tests. Upon consideration of additional diagnostic findings and the clinical course, eight (16 %) of the positive blood culture results were deemed clinically relevant. All three PCR assays could also identify the causative organism (or a specific gene thereof) in three of these eight positive blood cultures, whereas for five of the eight, all three PCR assays were negative. In one patient with a negative blood culture, the SepsiTest®, VYOO® and LightCycler® SeptiFast assays were positive for Streptococcus species. The PCR assays appeared to be less susceptible than blood cultures to false-positive results arising from contamination with coagulase-negative staphylococcal organisms. There was some variability between the three PCR assays tested and the corresponding blood cultures with regards to the type of pathogen detected. The three PCR assays appeared to be less susceptible to false-positive results than blood cultures.

  10. Discrete optimization

    CERN Document Server

    Parker, R Gary

    1988-01-01

    This book treats the fundamental issues and algorithmic strategies emerging as the core of the discipline of discrete optimization in a comprehensive and rigorous fashion. Following an introductory chapter on computational complexity, the basic algorithmic results for the two major models of polynomial algorithms are introduced--models using matroids and linear programming. Further chapters treat the major non-polynomial algorithms: branch-and-bound and cutting planes. The text concludes with a chapter on heuristic algorithms.Several appendixes are included which review the fundamental ideas o

  11. Combinatorial Optimization

    CERN Document Server

    Chvátal, V

    2011-01-01

    This book is a collection of six articles arising from the meeting of the NATO Advanced Study Institute (ASI) "Combinatorial Optimization: Methods and Applications," which was held at the University of Montreal in June 2006. This ASI consisted of seven series of five one-hour lectures and one series of four one-hour lectures. It was attended by some sixty students of graduate or postdoctoral level from fifteen countries worldwide. It includes topics such as: integer and mixed integer programming, facility location, branching on split disjunctions, convexity in combinatorial optimizat

  12. A Guide to Using STITCHER for Overlapping Assembly PCR Applications.

    Science.gov (United States)

    O'Halloran, Damien M

    2017-01-01

    Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping assembly PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online.

  13. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

    Science.gov (United States)

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  14. Aircraft Trajectory Optimization Using Parametric Optimization Theory

    OpenAIRE

    Valenzuela Romero, Alfonso

    2012-01-01

    In this thesis, a study of the optimization of aircraft trajectories using parametric optimization theory is presented. To that end, an approach based on the use of predefined trajectory patterns and parametric optimization is proposed. The trajectory pat

  15. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  16. A survey of tools for the analysis of quantitative PCR (qPCR data

    Directory of Open Access Journals (Sweden)

    Stephan Pabinger

    2014-09-01

    Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  17. Browsed twig environmental DNA: diagnostic PCR to identify ungulate species.

    Science.gov (United States)

    Nichols, Ruth V; Königsson, Helena; Danell, Kjell; Spong, Göran

    2012-11-01

    Ungulate browsing can have a strong effect on ecological processes by affecting plant community structure and composition, with cascading effects on nutrient cycling and animal communities. However, in the absence of direct observations of foraging, species-specific foraging behaviours are difficult to quantify. We therefore know relatively little about foraging competition and species-specific browsing patterns in systems with several browsers. However, during browsing, a small amount of saliva containing buccal cells is deposited at the bite site, providing a source of environmental DNA (eDNA) that can be used for species identification. Here, we describe extraction and PCR protocols for a browser species diagnostic kit. Species-specific primers for mitochondrial DNA were optimized and validated using twigs browsed by captive animals. A time series showed that about 50% of the samples will amplify up to 12 weeks after the browsing event and that some samples amplify up to 24 weeks after browsing (12.5%). Applied to samples of natural browsing from an area where moose (Alces alces), roe deer (Capreolus capreolus), fallow deer (Cervus dama) and red deer (Cervus elaphus) are sympatric, amplification success reached 75%. This method promises to greatly improve our understanding of multispecies browsing systems without the need for direct observations.

  18. Molecular Identification of Transgenic Tomato in Iran by P35S PromoterBased PCR

    Directory of Open Access Journals (Sweden)

    Parastoo Khanmohammad

    2016-06-01

    Full Text Available Background: With the increasing population of the world, we should face food shortages in the near future. In this regard, agriculture of transgenic crops becomes very important. However, ensuring of GM products labeling is of great importance. Tomato is one of the most important food crops and for this reason it has undergone many genetic changes. This study aims to introduce a rapid, sensitive, and accurate method to identify non-labeled transgenic tomatoes in Iran market. Results: In this study, after optimization of PCR test based on P35S promoter, the amplicon was cloned in the plasmid PTZ57R in Escherichia coli JM107 to confirm and make positive control. After optimized PCR test of 50 tomatoes it was found that 4% of the Iranian markets’ tomatoes contain P35S promoter and are probably transgenic while none of the samples have been labeled in this regard. Conclusion: PCR technique is a suitable, available, fast and accurate method for screening transgenic products such as tomatoes.

  19. Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction.

    Science.gov (United States)

    Tang, Lixia; Zheng, Kai; Liu, Yu; Zheng, Huayu; Wang, Hu; Song, Chunlei; Zhou, Hong

    2013-01-01

    Although megaprimer PCR mutagenesis has been used routinely in protein directed evolution, users sometimes encounter technical hurdles, particularly inefficiency during amplification when large fragments are used or the template is difficult to be amplified. Instead of methodology development, here we simply overcome the limitation by optimizing megaprimer PCR conditions via orthogonal array design of the four PCR components in three levels of each: template, primer, Mg(2+) , and dNTPs. For this, only nine PCRs need to be performed. The strategy (termed as OptiMega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase HheC, which failed to be constructed previously using the standard QuikChange™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of HheA and HheC. Most importantly, OptiMega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the OptiMega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.

  20. Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples.

    Science.gov (United States)

    Jimenez, L; Smalls, S; Ignar, R

    2000-08-01

    PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.

  1. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies.

    Science.gov (United States)

    Mutter, G L; Boynton, K A

    1995-04-25

    Trinucleotide CAG repeats in the X-linked human androgen receptor gene (HUMARA) have proved a useful means of determining X chromosome haplotypes, and when combined with methylation analysis of nearby cytosine residues permits identification of non-random X inactivation in tumors of women. Co-amplification of two alleles in a heterozygote generates PCR products which differ in the number of CAG units, and thus their melting and secondary structure characteristics. We have shown that under optimal conditions amplification efficiency of two HUMARA alleles is near-equivalent, generating PCR products in a ratio proportional to that of the genomic template. In contrast, reduction of template quantity, damage of template by ultraviolet irradiation or addition of monovalent salts (sodium chloride, sodium acetate or ammonium acetate) produces highly variable imbalances of allelic PCR products, with a strong tendency to preferentially amplify lower molecular weight alleles. Variability and biasing was diminished by substitution of 7-deaza-2'-dGTP for dGTP during amplification, an intervention which reduces stability of intramolecular and intermolecular GC base pairing. We conclude that DNA which is scanty, damaged or salt contaminated may display amplification bias of GC-rich PCR targets, potentially confounding accurate interpretation or reproducibility of assays which require co-amplification of alleles.

  2. A Multiplex PCR-coupled Liquid Bead Array for the Simultaneous Detection of Four Biothreat Agents

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, W J; Erler, A M; Nasarabadi, S L; Skowronski, E W; McCready, P M

    2004-02-04

    We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species -specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high- throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3,000 individual data points within a single 8-hour shift for approximately $1.20 per sample in a 10-plexed assay.

  3. Generic RT-PCR tests for detection and identification of tospoviruses.

    Science.gov (United States)

    Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W

    2016-07-01

    A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.

  4. Sex determination in goat by amplification of the HMG box using duplex PCR.

    Science.gov (United States)

    Shi, Lei; Yue, Wenbin; Ren, Youshe; Lei, Fulin; Zhao, Junxing

    2008-05-01

    The objective of this study was to obtain a fast, accurate and reliable method of determining the sex of goat embryos prior to implantation through amplification of the high-motility-group (HMG) box of the sex-determining region of the Y chromosome (SRY) gene of the goats. Goat specific primers were designed for duplex polymerase chain reaction (PCR). As an internal control gene, the goat beta-action gene sequence was simultaneously amplified together with the HMG box of goat SRY gene. Males showed both 1 SRY band and 1 beta-action band, but only 1 beta-action band was present in the agarose gel electrophoresis of females. The result indicated that the goat HMG-box sequence motif of SRY was male specific. Afterward, the optimized PCR procedure was applied in 30 embryo biopsies and the biopsied embryos were transferred into 30 recipient female goats. The sex of the 13 kids proved anatomically corresponded to the sex determined by PCR (100% accuracy). Thus, this study showed that this duplex PCR method can be applied to sex the goat pre-implantation embryos and to manipulate the sex ratio of offspring in goat breeding programs.

  5. PCR-DGGE analysis of nematode diversity in Cu-contaminated soil

    Institute of Scientific and Technical Information of China (English)

    WANG Shi-Bin; LI Qi; LIANG Wen-Ju; JIANG Yong; JIANG Si-Wei

    2008-01-01

    A wheat pot experiment was conducted under greenhouse conditions to assess the effect of copper contamination on soil nematode diversity by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method and morphological analysis.The soil was treated with CuSO4.5H2O at the following concentrations:0,50,100,200,400,and 800 mg kg-1 dry soil,and the soil samples were collected at wheat jointing and ripening stages.Nematode diversity index (H') from morphological analysis showed no difference between the control and the treated samples in either of the sampling dates.At the wheat ripening stage,nematode diversity obtained by the PCR-DGGE method decreased noticeably in the Cu800 treatment in comparison with the control.With optimization of the method of nematode DNA extraction,PCR-DGGE could give more information on nematode genera,and the intensity of the bands could reflect the abundance of nematode genera in the assemblage.The PCR-DGGE method proved promising in distinguishing nematode diversity in heavy metal contaminated soil.

  6. [PCR-RFLP/Hsp70 for identification and tipification of leishmania from the tropical region].

    Science.gov (United States)

    Margarita Montalvo, Ana; Fraga, Jorge; Aylema Romero, Jaqueline; Monzote, Lianet; Ivon Montano, Ing; Dujardin, Jean Claude

    2006-01-01

    The optimization of the PCR conditions for amplification of the gene coding for the 70 kDa (HSp70) heat shock protein as well as the analysis of the restriction fragment length polymorphism (RFLP) were carried out. DNA from a reference strain of Leishmania mexicana was used as template. Analytical sensitivity and specificity, and reproducibility of PCR using DNA from L. mexicana, Lamazonensis, L. guyanensis and L. lainsoni were determined. A 1.3 kp band was obtained, which confirmed gene amplification. The band patterns derived from Haelll enzyme digestion allowed differentiating several species. L. guyanensis and L. lainsoni were different from each other, while L. mexicana and L. amazonensis, which shared a common pattern, were different from the other two species. Analytical sensitivity and specificity were adequate. The enzymatic restriction of the PCR product made it possible to differentiate Leishmania spp. from T. cruzi. The feasibility of identifying and typifying species from the American continent through PCR-RFLP/Hsp70 and of using enzymatic restriction of amplified product to distinguish Leishmania spp. from Trypanosornma cruzi was shown. This was the first step in implementing these molecular methods in the reference laboratory of the Institute.

  7. A RAPID PCR-QUALITY DNA EXTRACTION METHOD IN FISH

    Institute of Scientific and Technical Information of China (English)

    LI Zhong; LIANG Hong-Wei; ZOU Gui-Wei

    2012-01-01

    PCR has been a general preferred method for biological research in fish, and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories[1-4]. The same problem among these procedures is waiting for tissue digesting for a long time. The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay, especially in large-scale PCR amplification, such as SSR-based genetic-mapping construction [5,6], identification of germ plasm resource[7,8] and evolution research [9,10], etc. In this study, a stable and rapid PCR-quality DNA extraction method was explored, using a modified alkaline lysis protocol. Extracting DNA for PCR only takes approximately 25 minutes. This stable and rapid DNA extraction method could save much laboratory time and promotes.%PCR has been a general preferred method for biological research in fish,and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories [1-4].The same problem among these procedures is waiting for tissue digesting for a long time.The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay,especially in large-scale PCR amplification,such as SSR-based genetic-mapping construction [5,6],identification of germ plasm resource[7,8] and evolution research [9,10],etc.In this study,a stable and rapid PCR-quality DNA extraction method was explored,using a modified alkaline lysis protocol.Extracting DNA for PCR only takes approximately 25 minutes.This stable and rapid DNA extraction method could save much laboratory time and promotes.

  8. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.

    Science.gov (United States)

    Tang, Hui; Cai, Qingchun; Li, Hu; Hu, Peng

    2016-06-16

    Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland-Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.

  9. Novel computational methods for increasing PCR primer design effectiveness in directed sequencing

    Directory of Open Access Journals (Sweden)

    Busam Dana

    2008-04-01

    Full Text Available Abstract Background Polymerase chain reaction (PCR is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. Results We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research. Conclusion The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing

  10. Optimal produktion

    Directory of Open Access Journals (Sweden)

    Öje Danell

    1999-04-01

    Full Text Available Optimal Production in Reindeer Husbandry.There are three ways to optimize the reindeer herd: 1 Adjusting the reindeer number to pasture resources. 2 Keeping as many productive animals in the herd as possible through slaughter. 3 Increasing the herd quality through selection.Renhjorden bör optimeras så att den fungerar som en "skördeapparat" for bete och lämnar största möjliga bidrag till försörjningen för dem som lever av renskötsel. Det finns minst tre sätt att optimera renhjorden, nämligen (1 anpassning av djurantalet till betesresurserna så att djurens kondition och därmed produktiviteten kan bibehållas på hög nivå, (2 utforma renhjordens struktur med hjalp av slaktuttaget så att den innehåller så stor andel produktiva djur som möjligt, och (3 förbättra djurmaterialets produktionsmässiga kvalitet genom urval baserat på registrerad produktion. Betesanpassningen är den mest grundläggande åtgärden och ger den snabbaste effekten. Även hjordstrukturering är en åtgärd som ger relativt snabb effekt och som dessutom kan beslutas och utforas av den enskilde djuraägaren utan att störa den kollektiva renskötseln. Urval är en åtgärd som ger effekt först på längre sikt och därför kräver en konsekvent insats under en längre tid.

  11. Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

    Science.gov (United States)

    Barletta, Francesca; Vandelannoote, Koen; Collantes, Jimena; Evans, Carlton A; Arévalo, Jorge; Rigouts, Leen

    2014-10-01

    Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture.

  12. A multiplex PCR method for detection of Clavibacter michi(g)anensis subsp. michlganensls with co-amplification of its host DNA

    Institute of Scientific and Technical Information of China (English)

    Yan ZHANG; Wenxiang YANG; Yaning LI; Daqun LIU; Ting ZHANG

    2009-01-01

    A multiplex PCR assay system was developed for the detection of Clavibacter michiganensis subsp. Michiganensis (Cmm), which combined two tests in one reaction mixture. Cmm-specific primers PSA-4/PSA-R and Solanum lycopersicum-specific primers NS-7-F/NS-8-R (internal PCR control primer) were combined in one PCR reaction mixture with Cmm and plant DNA as template. The primer sets could amplify the target product successfully. Different combinations and concentrations of primers and annealing temperatures were tested, respec tively. The detection level of the optimized multiplex PCR assay was up to 5×l02cfu-mL-1. To verify the applicability of this system, it was employed to detect Cmm in tomato seeds and plantlet samples. Seeds mixed with Cmm and diseased plantlets were detected successfully. The multiplex PCR system will avoid false-negative results and provide a reliable method for the detection of Cmm.

  13. Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

    Science.gov (United States)

    Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A

    2015-01-01

    Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

  14. Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation.

    Science.gov (United States)

    Knutsson, Rickard; Fontanesi, Massimo; Grage, Halfdan; Rådström, Peter

    2002-02-05

    A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation or = 10(6) (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment. Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10(1) (CFU/ml) Y. enterocolitica in the presence of at least an inoculation concentration of 10(3) (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR.

  15. PROBEmer: A web-based software tool for selecting optimal DNA oligos

    National Research Council Canada - National Science Library

    Emrich, Scott J; Lowe, Mary; Delcher, Arthur L

    2003-01-01

    PROBEmer (http://probemer.cs.loyola.edu) is a web-based software tool that enables a researcher to select optimal oligos for PCR applications and multiplex detection platforms including oligonucleotide microarrays and bead-based arrays...

  16. Development and application of a multiplex PCR method for rapid differential detection of subgroup A, B, and J avian leukosis viruses.

    Science.gov (United States)

    Gao, Qi; Yun, Bingling; Wang, Qi; Jiang, Lili; Zhu, Haibo; Gao, Yanni; Qin, Liting; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei; Gao, Yulong

    2014-01-01

    Avian leukosis virus (ALV) subgroups A, B, and J are very common in poultry flocks and have caused serious economic losses in recent years. A multiplex PCR (mPCR) method for the detection of these three subgroups was developed and optimized in this study. We first designed a common forward primer, PF, and three downstream primers, AR, BR, and JR, which can amplify 715 bp for subgroup A, 515 bp for subgroup B, and 422 bp for subgroup J simultaneously in one reaction. The mPCR method produced neither cross-reactions with other subgroups of ALVs nor nonspecific reactions with other common avian viruses. The detection limit of the mPCR was as low as 1 × 10(3) viral DNA copies of each of the three subgroups. In animal experiments, the mPCR detected ALVs 2 to 4 days earlier than did virus isolation from whole-blood samples and cloaca swabs. Furthermore, a total of 346 clinical samples (including 127 tissue samples, 86 cloaca swabs, 59 albumen samples, and 74 whole-blood samples) from poultry flocks with suspected ALV infection were examined by mPCR, routine PCR, and virus isolation. The positive sample/total sample ratios for ALV-A, ALV-B, and ALV-J were 48% (166/346) as detected by mPCR and 48% (166/346) as detected by routine PCR. However, the positive sample/total sample ratio detected by virus isolation was 40% (138/346). The results of the mPCR and routine PCR were confirmed by sequencing the specific fragments. These results indicate that the mPCR method is rapid, specific, sensitive, and convenient for use in epidemiological studies of ALV, clinical detection of ALV, and ALV eradication programs.

  17. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...... and to accept or reject the results according to existing rules for quality assurance....... and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17......). We conclude that the CURT-PCR method developed is suitable for routine applications such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run...

  18. False negative results from using common PCR reagents

    Directory of Open Access Journals (Sweden)

    Atwood Allison A

    2011-10-01

    Full Text Available Abstract Background The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG, and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results. Findings We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA. Conclusions These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is.

  19. Implications of PCR and ELISA results on the routes of bulk-tank contamination with Mycobacterium avium ssp. paratuberculosis.

    Science.gov (United States)

    Beaver, A; Cazer, C L; Ruegg, P L; Gröhn, Y T; Schukken, Y H

    2016-02-01

    Mycobacterium avium ssp. paratuberculosis (MAP), the etiologic agent of Johne's disease in dairy cattle, may enter the bulk tank via environmental contamination or direct excretion into milk. Traditionally, diagnostics to identify MAP in milk target either MAP antibodies (by ELISA) or the organism itself (by culture or PCR). High ELISA titers may be directly associated with excretion of MAP into milk but only indirectly linked to environmental contamination of the bulk tank. Patterns of bulk-milk ELISA and bulk-milk PCR results could therefore provide insight into the routes of contamination and level of infection or environmental burden. Coupled with questionnaire responses pertaining to management, the results of these diagnostic tests could reveal correlations with herd characteristics or on-farm practices that distinguish herds with high and low environmental bulk-tank MAP contamination. A questionnaire on hygiene, management, and Johne's specific parameters was administered to 292 dairy farms in New York, Oregon, and Wisconsin. Bulk-tank samples were collected from each farm for evaluation by real-time PCR and ELISA. Before DNA extraction and testing of the unknown samples, bulk-milk template preparation was optimized with respect to parameters such as MAP fractionation patterns and lysis. Two regression models were developed to explore the relationships among bulk-tank PCR, ELISA, environmental predictors, and herd characteristics. First, ELISA optical density (OD) was designated as the outcome in a linear regression model. Second, the log odds of being PCR positive in the bulk tank were modeled using binary logistic regression with penalized maximum likelihood. The proportion of PCR-positive bulk tanks was highest for New York and for organic farms, providing a clue as to the geographical patterns of MAP-positive bulk-tank samples and relationship to production type. Bulk-milk PCR positivity was also higher for large relative to small herds. The models

  20. [SIAM conference on optimization

    Energy Technology Data Exchange (ETDEWEB)

    1992-05-10

    Abstracts are presented of 63 papers on the following topics: large-scale optimization, interior-point methods, algorithms for optimization, problems in control, network optimization methods, and parallel algorithms for optimization problems.

  1. Detection of Human Papillomavirus DNA in Cervical Samples: Analysis of the New PGMY-PCR Compared To the Hybrid Capture II and MY-PCR Assays and a Two-Step Nested PCR Assay

    OpenAIRE

    Giovannelli, Lucia; Lama, Anna; Capra, Giuseppina; Giordano, Viviana; Aricò, Pietro; Ammatuna, Pietro

    2004-01-01

    The PGMY-PCR for human papillomavirus (HPV) was evaluated, in parallel with nested PCR (nPCR), in samples with noted Hybrid Capture II (HCII) and MY-PCR results. PGMY-PCR detected HPV DNA in 2.5% of HCII-negative-MY-PCR-negative samples and in 71.7% of HCII-positive-MY-PCR-negative samples; also, it detected the MY-PCR-negative-nPCR-negative types HPV-42, HPV-44, HPV-51, HPV-87, and HPV-89.

  2. IDH1 mutation detection by droplet digital PCR in glioma.

    Science.gov (United States)

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; To, Shing shun Tony; Chen, Zhong-ping

    2015-11-24

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.

  3. Quantitative detection of Clostridium tyrobutyricum in milk by real-time PCR.

    Science.gov (United States)

    López-Enríquez, Lorena; Rodríguez-Lázaro, David; Hernández, Marta

    2007-06-01

    We developed a real-time PCR assay for the quantitative detection of Clostridium tyrobutyricum, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (R(2) > 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300 C. tyrobutyricum spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.

  4. Asymmetric PCR method in generation of HBV ssDNA for pyrosequencing

    Institute of Scientific and Technical Information of China (English)

    Nian-cai Peng; Chun-lin Wang; Li-li Zhang; Mao-li Lu; Zhen-xi Zhang

    2009-01-01

    Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50 : 1, 100 : 1) and concentrations (13. 0 pmol/25μL and 0.14 pmol/25μL, 19. 5 pmol/25μL and 0. 21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio of 50 : 1 provided better yield and concentration of 19. 5 pmol/25μL and 0. 21 pmol//25μL generated a clearer band. Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.

  5. Asymmetric PCR for good quality ssDNA generation towards DNA aptamer production

    Directory of Open Access Journals (Sweden)

    Junji Tominaga4

    2012-04-01

    Full Text Available Aptamers are ssDNA or RNA that binds to wide variety of target molecules with high affinity and specificity producedby systematic evolution of ligands by exponential enrichment (SELEX. Compared to RNA aptamer, DNA aptamer is muchmore stable, favourable to be used in many applications. The most critical step in DNA SELEX experiment is the conversion ofdsDNA to ssDNA. The purpose of this study was to develop an economic and efficient approach of generating ssDNA byusing asymmetric PCR. Our results showed that primer ratio (sense primer:antisense primer of 20:1 and sense primer amountof 10 to 100 pmol, up to 20 PCR cycles using 20 ng of initial template, in combination with polyacrylamide gel electrophoresis,were the optimal conditions for generating good quality and quantity of ssDNA. The generation of ssDNA via this approachcan greatly enhance the success rate of DNA aptamer generation.

  6. A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen, Norway

    Directory of Open Access Journals (Sweden)

    Haanshuus Christel G

    2013-01-01

    distribution; 20 P. falciparum, six Plasmodium vivax, one Plasmodium ovale and one Plasmodium malariae. Conclusions In this study, design of PCR programmes with suitable parameters and optimization resulted in simpler and faster single-round amplification assays. Both sensitivity and specificity of the novel mitochondrial PCR was 100% and proved non-inferior to that of the reference nested PCR. Sequencing of genus-specific mitochondrial PCR products could be used for species determination.

  7. Evaluation of PCR-based quantification techniques to estimate the abundance of atrazine chlorohydrolase gene atzA in rhizosphere soils.

    Science.gov (United States)

    Thompson, Brian M; Lin, Chung-Ho; Hsieh, Hsin-Yeh; Kremer, Robert J; Lerch, Robert N; Garrett, Harold E

    2010-01-01

    There are many challenges in the accurate quantification of bacterial genes, such as the atrazine-degrading enzyme antA from Pseudomonas sp. strain ADP, from soil samples. We compared four quantitative methods for enumeration of atrazine-degrading bacteria in rhizosphere environments and utilized the optimal probe-based real-time polymerase chain reaction (PCR)-based method in an ongoing bioremediation experiment to monitor atzA copy number over time. We compared three quantitative PCR (qPCR) based methods--quantitative competitive PCR and two real-time qPCR methods--to traditional dilution-plate counting techniques. The optimal real-time qPCR assay was then used to monitor atzA copy number over time in the robust atrazine-degrading Pseudomonas sp. strain ADP-spiked rhizosphere environment. The use of sensitive and reliable probe-based real-time qPCRs for the enumeration of bacterial catabolic genes allows for their detection from soil samples and monitoring of potential degradative populations over time. The addition of arrazine-biodegrading bacteria into arrazine-contaminated sites to remove entrapped atrazine is a promising approach for mitigating atrazine pollution and its metabolites. The methodology contained herein will allow for optimal monitoring of atzA in rhizosphere soil with or without the addition of biodegradative Pseudomonas sp. strain ADP of bacteria.

  8. Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

    Science.gov (United States)

    Shirasu, Naoto; Kuroki, Masahide

    2014-01-01

    We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

  9. PCR detection of Salmonella typhimurium in pharmaceutical raw materials and products contaminated with a mixed bacterial culture using the BAX system.

    Science.gov (United States)

    Jimenez, L; Scalici, C; Smalls, S; Bosko, Y; Ignar, R

    2001-01-01

    The BAX system, a PCR-based assay, was evaluated for detecting Salmonella typhimurium in pharmaceutical raw materials and products contaminated with mixed bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhimurium. Artificially contaminated samples were preenriched in lactose broth with and without Tween 20. After preenrichment, samples were analyzed by PCR and standard methods. Ten of 25 samples did not show presence of the specific Salmonella spp. 740-base pair DNA fragment. However, S. typhimurium was isolated and identified by standard methods from all 25 samples. To optimize S. typhimurium detection in PCR negative samples, lactose broth was replaced by buffered peptone water (BPW) as the preenrichment broth. When BPW was used, all 10 samples were PCR positive. BPW enrichments increased S. typhimurium growth resulting in rapid PCR detection. The presence of non-Salmonella bacteria influenced the performance of the PCR-based assay. Optimization of S. typhimurium PCR detection in mixed culture required the use of different preenrichment broths. However, the BAX system detected S. typhimurium within 27 hours while standard methods required 5-7 days.

  10. Comparison of a commercially available rep-PCR system (Diversilab(R)) with PCR-ribotyping for typing of Clostridium difficile strains.

    OpenAIRE

    Eckert, C.; van Broeck, Johan; Spigaglia, P.; Burghoffer, B; Delmée, Michel; Mastrantonio, P; Barbut, F

    2011-01-01

    This study compared a rep-PCR method (DiversiLab® system) to PCR-ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR-ribotype 027, different rep-types could be distinguished. Rep-PCR showed a higher discriminatory power than PCR-ribotyping. Nevertheless, this method requires technical skill and visual interpretation of rep-PCR fingerprint patterns may be difficult.

  11. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    Directory of Open Access Journals (Sweden)

    Tülin GÜVEN GÖKMEN

    Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

  12. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    Science.gov (United States)

    GÖKMEN, Tülin GÜVEN; SOYAL, Ayben; KALAYCI, Yıldız; ÖNLEN, Cansu; KÖKSAL, Fatih

    2016-01-01

    SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis. PMID:27680169

  13. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

    Science.gov (United States)

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  14. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Asami, Yukihiro; Kitamura, Eiko; Saito, Kosuke; Suzuki, Tsukasa; Takahashi, Sachiko Nonaka; Matsumoto, Koichi; Sawada, Shigemasa; Yokoyama, Eise; Takasu, Toshiaki

    2008-05-01

    Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

  15. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

    Science.gov (United States)

    Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

    2017-02-14

    In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

  16. Development of duplex PCR for simultaneous detection of Theileria spp. and Anaplasma spp. in sheep and goats.

    Science.gov (United States)

    Cui, Yanyan; Zhang, Yan; Jian, Fuchun; Zhang, Longxian; Wang, Rongjun; Cao, Shuxuan; Wang, Xiaoxing; Yan, Yaqun; Ning, Changshen

    2017-05-01

    Theileria spp. and Anaplasma spp., which are important tick-borne pathogens (TBPs), impact the health of humans and animals in tropical and subtropical areas. Theileria and Anaplasma co-infections are common in sheep and goats. Following alignment of the relevant DNA sequences, two primer sets were designed to specifically target the Theileria spp. 18S rRNA and Anaplasma spp. 16S rRNA gene sequences. Genomic DNA from the two genera was serially diluted tenfold for testing the sensitivities of detection of the primer sets. The specificities of the primer sets were confirmed when DNA from Anaplasma and Theileria (positive controls), other related hematoparasites (negative controls) and ddH2O were used as templates. Fifty field samples were also used to evaluate the utility of single PCR and duplex PCR assays, and the detection results were compared with those of the PCR methods previously published. An optimized duplex PCR assay was established from the two primer sets based on the relevant genes from the two TBPs, and this assay generated products of 298-bp (Theileria spp.) and 139-bp (Anaplasma spp.). The detection limit of the assay was 29.4 × 10(-3) ng per μl, and there was no cross-reaction with the DNA from other hematoparasites. The results showed that the newly developed duplex PCR assay had an efficiency of detection (P > 0.05) similar to other published PCR methods. In this study, a duplex PCR assay was developed that can simultaneously identify Theileria spp. and Anaplasma spp. in sheep and goats. This duplex PCR is a potentially valuable assay for epidemiological studies of TBPs in that it can detect cases of mixed infections of the pathogens. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Reevaluation of the Correlation between Angiotensinogen Gene M235T Polymorphism and Familial Essential Hypertension Using MS-PCR Technique

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Using mutagenically separated allele-specific polymerase-chain-reaction (MS-PCR) technique to determine the correlation between angiotensinogen gene M235T (Ag TM235T) polymorphism and the onset of familial essential hypertension in the population from Jiangsu and Anhui Provinces. Methods (1) Establish and compare the optimal reaction system of PCR-RFLP and MS-PCR technique to detect AgTM235T polymorphism. (2) All subjects were divided into four groups: 62 patients with both hypertension and familial background (FH), 32 normal persons who had familial background (FNH), 26 persons in control group (N) and 10 patients with hypertension but without familial background (NFH group). The genotype of all subjects was determined by MS-PCR technique.Results (1) The frequency of T allele in PCR-RFLP was 0.5, much lower than 0.95 in MS-PCR, which was demonstrated by DNA sequencing. (2) The TT-genotype and the frequency of T allele (TT/T) in FH and FNH groups were much higher than those in N and NFH groups (0.581/0.766 and 0.563/0.766 vs 0.346/0.577 and 0.40/0.550, P<0.005). (3) Persons developing hypertension in FNH group were much younger than other three groups (28.07±9.72 , P<0.025). Conclusion (1) Compared with PCR-RFLP, MS-PCR is a rapid, simple and reliable technique for detection gene polymorphism of Ag TM25T. (2) In Jiangsu and Anhui area, the present study confirms the observation of a higher frequency of the 235T allele of the angiotensinogen gene in hypertension and identifies individuals with family history. Concerning of the age, we might speculate that the AgTM235T polymorphism is only associated with familial essential hypertension.

  18. Blood grouping based on PCR methods and agarose gel electrophoresis.

    Science.gov (United States)

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  19. Microfluidics-Based PCR for Fusion Transcript Detection.

    Science.gov (United States)

    Chen, Hui

    2016-01-01

    The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

  20. Standardization of diagnostic PCR for the detection of foodborne pathogens

    DEFF Research Database (Denmark)

    Malorny, B.; Tassios, P.T.; Radstrom, P.

    2003-01-01

    In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has...... neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end......-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection...

  1. Template-blocking PCR: an advanced PCR technique for genome walking.

    Science.gov (United States)

    Bae, Jung-Hoon; Sohn, Jung-Hoon

    2010-03-01

    This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3' ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.

  2. PCR for the Diagnosis of Abdominal Angiostrongyliasis in Formalin-Fixed Paraffin-Embedded Human Tissue

    Science.gov (United States)

    Rodriguez, Rubens; da Silva, Ana Cristina Aramburú; Müller, Carla Aristonara; Alves, Silvana Lunardini; Graeff-Teixeira, Carlos; Fornari, Fernando

    2014-01-01

    To date the diagnosis of abdominal angiostrongyliasis (AA) depends on the histological identification of Angiostrongylus costaricensis (AC) in surgical specimens. However, microscopic evaluation is time consuming and often fails in identifying the parasite. We tested whether PCR might help in the diagnosis of AA by identifying parasite DNA in formalin-fixed paraffin-embedded (FFPE) tissue. We used primers based on DNA from Angiostrongilus cantonensis. Four groups of FFPE intestinal tissue were tested: (1) confirmed cases (n = 20), in which AC structures were present in the target tissue; (2) presumptive cases (n = 20), containing changes secondary to AC infection in the absence of AC structures; (3) negative controls (n = 3), consisting of normal colonic tissue; and (4) tissue affected by other parasitoses (n = 7), including strongyloidiasis, ascaridiasis, schistosomiasis, and enterobiasis. Most lesions of confirmed cases were located in small and/or large bowel (90%), as compared with presumptive cases, in which 70% of lesions were in appendix (P = 0.0002). When confronted with cases of other parasitoses, PCR showed sensitivity of 55%, specificity of 100% and positive predictive value of 100%. In presumptive cases PCR was positive in 4 (20%). All specimens from negative controls and other parasitoses were negative. In conclusion, the PCR technique showed intermediate sensitivity and optimal specificity, being clinically relevant when positive for abdominal angiostrongyliasis. It allowed a 20% gain in diagnosis of presumptive cases. PCR might help in the diagnosis of abdominal angiostrongyliasis, particularly when the pathologists are not experienced with such disease. PMID:24705328

  3. Doubling Throughput of a Real-Time PCR

    OpenAIRE

    Christian D. Ahrberg; Pavel Neužil

    2015-01-01

    The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one...

  4. THE IMPROVEMENT OF RT-PCR TECHNIQUE ON DETECTING ROTAVIRUS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To establish a speed and effective method to detect rotavirus. Methods Using ELISA and one step RT-PCR to detect 196 clinic samples from Xi'an area. Results Compared with ELISA method, one step RT PCR was more sensitive and specific (P <0.05). Conclusion One step RT-PCR is a simple, speed, sensitive and spe cific method for clinic and epidemic studies of rotavirus.

  5. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.

    Science.gov (United States)

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-10-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

  6. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

    Science.gov (United States)

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-01-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051

  7. A thermodynamic approach to PCR primer design.

    Science.gov (United States)

    Mann, Tobias; Humbert, Richard; Dorschner, Michael; Stamatoyannopoulos, John; Noble, William Stafford

    2009-07-01

    We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.

  8. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    Science.gov (United States)

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

  9. Validation of a sensitive PCR assay for the detection of Chelonid fibropapilloma-associated herpesvirus in latent turtle infections

    DEFF Research Database (Denmark)

    Alfaro Nuñez, Luis Alonso; Gilbert, M Thomas P

    2014-01-01

    clinically healthy (non exhibiting fibropapilloma tumours) turtles, thus representing presumably latent infections of the pathogen. Given that template copy numbers of viruses in latent infections can be very low, extremely sensitive PCR assays are needed to optimize detection efficiency. In this study......, efficiency of several PCR assays designed for CFPHV detection is explored and compared to a method published previously. The results show that adoption of a triplet set of singleplex PCR assays outperforms other methods, with an approximately 3-fold increase in detection success in comparison to the standard...... assay. Thus, a new assay for the detection of CFPHV DNA markers is presented, and adoption of its methodology is recommended in future CFPHV screens among sea turtles....

  10. Rapid quantitative detection of, Listeria monocytogenes in salmon products: evaluation of pre-real-time PCR strategies.

    Science.gov (United States)

    Rodríguez-Lázaro, David; Jofré, Anna; Aymerich, Teresa; Garriga, Margarita; Pla, Maria

    2005-07-01

    The spread and persistence of Listeria monocytogenes in smoked fish products and seafood processing factories are big concerns. Thus, the corresponding quality assurance programs must include adequate microbiological control measures. We evaluated eight different pre-PCR sample processing strategies to be coupled with a previously developed real-time PCR assay for the quantitative detection of L. monocytogenes in salmon products. The optimal pre-PCR procedure involved filtration and DNA purification with the use of a commercial kit. This strategy could detect 10 CFU of L. monocytogenes per g of smoked salmon and could quantify 1,000 CFU/g with excellent accuracy compared with the standard plate count method. Thus, this method could be a promising alternative for the quantitative detection of L. monocytogenes in smoked fish products and processing factories. This method could also detect the bacterium in raw salmon.

  11. [Analytical performances of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine].

    Science.gov (United States)

    De Monte, Anne; Cannavo, Isabelle; Caramella, Anne; Ollier, Laurence; Giordanengo, Valérie

    2016-01-01

    Congenital cytomegalovirus (CMV) infection is the leading cause of sensoneurinal disability due to infectious congenital disease. The diagnosis of congenital CMV infection is based on the search of CMV in the urine within the first two weeks of life. Viral culture of urine is the gold standard. However, the PCR is highly sensitive and faster. It is becoming an alternative choice. The objective of this study is the validation of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine. Repeatability, reproducibility, detection limit and inter-sample contamination were evaluated. Urine samples from patients (n=141) were collected and analyzed simultaneously in culture and PCR in order to assess the correlation of these two methods. The sensitivity and specificity of PCR were also calculated. The Abbott RealTime CMV PCR in urine is an automated and sensitive method (detection limit 200 UI/mL). Fidelity is very good (standard deviation of repeatability: 0.08 to 0.15 LogUI/mL and reproducibility 0.18 LogUI/mL). We can note a good correlation between culture and Abbott RealTime CMV PCR (kappa 96%). When considering rapid culture as reference, real-time PCR was highly sensitive (100%) and specific (98.2%). The real-time PCR by Abbott RealTime CMV with m2000 is optimal for CMV detection in urine.

  12. Contribution of real-time PCR to Plasmodium species identification and to clinical decisions: a nationwide study in a non-endemic setting.

    Science.gov (United States)

    Grossman, T; Schwartz, E; Vainer, J; Agmon, V; Glazer, Y; Goldmann, D; Marva, E

    2017-04-01

    Treatment choice for patients with malaria in Israeli hospitals is based on microscopy and rapid diagnostic tests (RDTs). Here, we demonstrate the cumulative value of real-time polymerase chain reaction (PCR) in optimizing the treatment of malaria. Between January 2009 and December 2015, 451 samples from 357 patients were tested in our laboratory using a real-time PCR assay. Hospital laboratory results (without real-time PCR) were compared to those obtained in our laboratory. A total of 307 patients had a malaria-positive laboratory finding in the hospital. Out of those, 288 were confirmed positive and 19 negative using real-time PCR. Two negative hospital results were found to be positive by real-time PCR. More specifically, of 153 cases positive for Plasmodium falciparum by real-time PCR, only 138 (90%) had been correctly identified at the hospitals. Similarly, 66 (67%) of 99 cases positive for P. vivax, 2 (11%) of 18 cases positive for P. ovale, and 3 (30%) of 10 cases positive for P. malariae had been correctly identified. Of 10 cases of mixed infection, only one had been identified as such at the hospital. Thus, real-time PCR was required for correct identification in 81 (28%) out of 290 positive cases. In 52 (18%) of those, there was an erroneous categorization of relapsing versus non-relapsing parasites. In a nationwide study, we found that the use of real-time PCR is definitely beneficial and may change the decision regarding the choice of treatment.

  13. Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

    Science.gov (United States)

    El Khattabi, Laïla Allach; Rouillac-Le Sciellour, Christelle; Le Tessier, Dominique; Luscan, Armelle; Coustier, Audrey; Porcher, Raphael; Bhouri, Rakia; Nectoux, Juliette; Sérazin, Valérie; Quibel, Thibaut; Mandelbrot, Laurent; Tsatsaris, Vassilis; Vialard, François; Dupont, Jean-Michel

    2016-01-01

    NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women.

  14. Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

    Directory of Open Access Journals (Sweden)

    Laïla Allach El Khattabi

    Full Text Available NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR assay which allows increasing the number of available targets and thus overcomes statistical obstacles.After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome.The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups.Our re