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Sample records for random polymerase chain

  1. Randomly amplified polymorphic DNA-polymerase chain reaction ...

    Indian Academy of Sciences (India)

    Genetic similarity and diversity of cultured catfish Silurus asotus populations collected from two areas in western Korea were examined using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Out of 20 random primers tested, 5 produced 1344 RAPD bands ranging from 8.2 to 13.6 polymorphic ...

  2. Identification of Species Related to Anopheles (Nyssorhynchus) albitarsis by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (Diptera: Culicidae)

    Science.gov (United States)

    1995-11-01

    plified polymorphic DNA in the population genet- ics and systematics of grasshoppers . Genome 35: 569-574. Galvgo ALA, Damesceno RG 1942. Sobre urn...iynchus) albitarsis by Random Amplified Polymorphic DNA -Polymerase Chain Reaction (Diptera: Culicidae) Richard C Wilkerson/+, Thomas V Caffigan, Jo...Instituto de Biologia do ExCrcito, Rua Francisco Manuel 102, 2091 l-270 Rio de Janeiro, RJ, Brasil Species-specific Random Amplified Polymorphic DNA

  3. Inter- and intraspecific identification of the screwworm, Cochliomyia hominivorax, using random amplified polymorphic DNA-polymerase chain reaction.

    Science.gov (United States)

    Skoda, Steven R; Figarola, James L; Pornkulwat, Saowaluck; Foster, John E

    2013-01-01

    The screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most devastating arthropod pests of livestock in the Western Hemisphere. Early instars are very difficult to distinguish morphologically from several closely related blow fly species. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers were developed for identifying C. hominivorax from other wound inhabiting species. Forty decameric primers were screened; nine showed clear reproducible RAPD profiles suitable for distinguishing all life stages of C. hominivorax from 7 other species, including C. macellaria (Fabricius). The results from RAPD-PCR with field-collected samples of unknown first instars agreed with morphological identification that the samples were not C. hominivorax. Three different primers showed DNA polymorphisms (intraspecific) for samples originating from Mexico, Costa Rica, Panama, Jamaica, and Brazil. Therefore, RAPD-PCR may be useful for determining the geographic origin of C. hominivorax samples. Comparing products from these primers, used with known and unknown screwworm samples from an outbreak in Mexico, clearly showed that the outbreak did not originate from the mass rearing facility. Accurate identification of suspected C. hominivorax samples is possible using RAPD-PCR. Further development to identify the geographic origin of samples would benefit the ongoing surveillance programs against C. hominivorax and the decision process during suspected outbreaks of this important pest.

  4. Red light-controlled polymerase chain reaction.

    Science.gov (United States)

    Meyer, A; Schikora, Margot; Mokhir, A

    2015-09-04

    A 23-mer DNA "caged" at its 3'-terminus with a 9-anthracenyl moiety was prepared. It can be uncaged in the presence of photosensitizer (In(pyropheophorbide-a)chloride)-containing DNAs (9-12 mers) and upon irradiation with red light. This mixture of DNAs was used to design red-light controlled polymerase chain reaction.

  5. Original Research Polymerase chain reaction identification of ...

    African Journals Online (AJOL)

    Polymerase chain reaction identification of Trypanosoma brucei rhodesiensein wild tsetse flies from Nkhotakota. Wildlife Reserve, Malawi northwest and southeast Uganda.4 It is clear that Glossina fuscipes and Glossina morsitans are ubiquitous in their locations, making these species of tsetse flies good vectors for both.

  6. Real-Time Polymerase Chain Reaction

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 4. Real-Time Polymerase Chain Reaction - A Revolution in ... Author Affiliations. Simarjot Singh Pabla1 Sarabjot Singh Pabla1. GH Patel Post Graduate Department of Computer Science and Technology Sardar Patel University Gujarat.

  7. Multiplex polymerase chain reaction for detection and ...

    African Journals Online (AJOL)

    Moreover, 20 strains of E. coli (25%) were isolated from milk samples collected from 80 animals suffering from mastitis and subclinical mastitis. E. coli serovars yielded from bacteriological examination of milk samples were similar to that of fecal samples. Serogroup-specific multiplex polymerase chain reaction (PCR) assay ...

  8. Immunoassay and polymerase chain reaction techniques for ...

    African Journals Online (AJOL)

    Objectives: To compare the Reverse Passive Latex Agglutination (RPLA) and Enzyme Linked Immunosorbent Assay (ELISA) techniques with a Polymerase Chain Reaction (PCR) for detection of enterotoxigenic Bacillus cereus. Design: A cross-sectional study. Setting: The Department of Public Health, Pharmacology and ...

  9. Polymerase chain reaction (PCR) based molecular characterization ...

    African Journals Online (AJOL)

    Polymerase chain reaction (PCR) based molecular characterization of popular wheat varieties of Khyber Pukhtunkhwa (KPK) region of Pakistan. ... Molecular markers used in this study show high rate of genetic diversity that can be used to assist a breeding program for the improvement of wheat in KPK-Pakistan. Key words: ...

  10. Integration of random amplification of polymorphic DNA-polymerase ...

    African Journals Online (AJOL)

    adjusted reaction conditions strain-distinctive PCR-restriction fragment lenght polymorphism (RFLP) can be designed. Key words: Echinococcus granulosus, strain-specificity, random amplification of polymorphic DNA-polymerase chain reaction ...

  11. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Peter McInerney

    2014-01-01

    Full Text Available As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

  12. Bordetella pertussis diagnosed by polymerase chain reaction

    DEFF Research Database (Denmark)

    Birkebaek, N H; Heron, I; Skjødt, K

    1994-01-01

    The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity...... of PCR for the diagnosis of BP, we used known concentrations of BP, Bordetella parapertussis and Bordetella bronchiseptica in aqueous solutions. PCR was furthermore carried out on species of bacteria that might be isolated from the nasopharynx. The applicability of PCR to patient specimens was tested...... in 25 patients in whose nasopharyngeal secretions BP had been demonstrated after 4-7 days of culture. The detection limit of PCR in aqueous solution was 1-2 BP bacteria per reaction tube. PCR was 100% specific for BP, showing no response with other Bordetella species or other bacteria known to colonize...

  13. Dual phase multiplex polymerase chain reaction

    Energy Technology Data Exchange (ETDEWEB)

    Pemov, Alexander [Charlottesville, VA; Bavykin, Sergei [Darien, IL

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  14. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    OpenAIRE

    Peter McInerney; Paul Adams; Masood Z. Hadi

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differe...

  15. Actinobaculum suis Detection Using Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Cristina Román Amigo

    2012-01-01

    Full Text Available Actinobaculum suis is an important agent related to urinary infection in swine females. Due to its fastidious growth characteristics, the isolation of this anaerobic bacterium is difficult, thus impairing the estimation of its prevalence. The purpose of this study was to develop and test a polymerase chain reaction (PCR for the detection and identification of A. suis and then compare these results with traditional isolation methods. Bacterial isolation and PCR were performed on one hundred and ninety-two urine samples from sows and forty-five preputial swabs from boars. The results indicate that this PCR was specific for A. suis, presenting a detection limit between 1.0×101 CFU/mL and 1.0×102 CFU/mL. A. suis frequencies, as measured by PCR, were 8.9% (17/192 in sow urine samples and 82.2% (37/45 in preputial swabs. Assessed using conventional culturing techniques, none of the urine samples were positive for A. suis; however, A. suis was detected in 31.1% (14/45 of the swabs. This PCR technique was shown to be an efficient method for the detection of A. suis in urine and preputial swabs.

  16. Reverse transcriptase-quantitative polymerase chain reaction (RT ...

    African Journals Online (AJOL)

    The reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a highly specific polymerase chain reaction (PCR) method that allows one to detect very low transcription levels of functional gene(s) in soil. RT-qPCR helps us to know the active members of the microbial community, and their activities can be ...

  17. an overview on the application of polymerase chain reaction (pcr)

    African Journals Online (AJOL)

    DR. AMINU

    Correspondence author. ABSTRACT. This overview ... The polymerase chain reaction (PCR) is a technique widely used in Molecular biology. ..... and research centres. Seminars and workshops should be organized in order to update knowledge of.

  18. Robust and inexpensive equipment design for polymerase chain ...

    African Journals Online (AJOL)

    Robust and inexpensive equipment design for polymerase chain reaction detection of sequence mutations Cystic fibrosis in a mother and 2 children analysed. L. R. Purves, R. Rubinstein, J. Maxwell, F. Leisegang, J. Ireland ...

  19. Multiplex polymerase chain reaction (PCR) assay for simultaneous ...

    African Journals Online (AJOL)

    Multiplex polymerase chain reaction (PCR) assay for simultaneous detection of shiga-like toxin ( stx 1 and stx 2), intimin (eae) and invasive plasmid antigen H ( ipaH ) genes in diarrheagenic Escherichia coli.

  20. Simplex and triplex polymerase chain reaction (PCR) for ...

    African Journals Online (AJOL)

    Simplex and triplex polymerase chain reaction (PCR) for identification of three medically important Candida species. Nabil S Harmal, Alireza Khodavandi, Mohammed A Alshawsh, Farida Jamal, Zamberi Sekawi, Ng Kee Peng, Pei Pei Chong ...

  1. Polymerase chain reaction to search for Herpes viruses in uveitic ...

    African Journals Online (AJOL)

    Objective: To analyse aqueous polymerase chain reaction (PCR) results in patients diagnosed with undifferentiated uveitis and determine prevalence of herpesviridae in non-uveitic patients undergoing routine cataract extraction. Design: Retrospective comparative case series and prospective cross-sectional study.

  2. Polymerase Chain Reaction (PCR) provides a superior tool for the ...

    African Journals Online (AJOL)

    Polymerase Chain Reaction (PCR) provides a superior tool for the diagnosis of Pneumococcal Infection in Burkina Faso. Y Chaibou, M Congo/Ouedraogo, I Sanou, H Somlare, K Ouattara, CM Kienou, H Belem, E Sampo, SA Traore, R Traore/Ouedraogo, C Hatcher, L Mayer, X Wang, L Sangare ...

  3. One-stop polymerase chain reaction (PCR): An improved PCR ...

    African Journals Online (AJOL)

    Yomi

    2011-12-21

    Dec 21, 2011 ... 2RNA Group, College of Life Science, Wuhan University, Wuhan 430072, P. R. China. 3Institute of ... The widely used polymerase chain reaction (PCR) protocol requires several post-cycling steps to visualize amplicons ... direct loading and simultaneous staining of PCR products for electrophoresis and ...

  4. Polymerase chain reaction: Theory, practice and application: A review

    African Journals Online (AJOL)

    Polymerase Chain Reaction (PCR) is a rapid procedure for in vitro enzymatic amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. Repetitive cycles involving template denaturation, primer annealing and the extension ...

  5. An overview on the application of Polymerase Chain Reaction (PCR ...

    African Journals Online (AJOL)

    This overview describes the principle of polymerase chain reaction as one of the most important techniques used in the diagnosis of bacterial infections due to its reliability, sensitivity and rapidness. It also identifies some of the limitations of the technique as well as some of the bacterial infections that can be diagnosed ...

  6. Development of a sensitive nested-polymerase chain reaction (PCR ...

    African Journals Online (AJOL)

    A species-specific polymerase chain reaction (PCR) assay was developed for rapid and accurate detection of Ustilago scitaminea, the causal agent of sugarcane smut disease. Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of U. scitaminea, a pair of species-specific primers, SL1 ...

  7. Improvement of specific polymerase chain reaction (PCR) for the ...

    African Journals Online (AJOL)

    A polymerase chain reaction (PCR) test for the identification of Mycoplasma capricolum subsp. capripneumoniae (Mccp) was optimized using the H2 gene sequences of M1601. The test was evaluated on 20 strains including six representative strains of the Mycoplasma mycoides cluster as well as 13 field isolates from ...

  8. A Double Polymerase Chain Reaction Method for Detecting African ...

    African Journals Online (AJOL)

    Purpose: To establish a double polymerase chain reaction (PCR) method for the simultaneous detection of African swine fever virus (ASFV) and swine vesicular disease virus (SVDV). Methods: By using reference sequences of ASFV and SVDV, this study synthesized parts of the genes connected to the 19-T vector which ...

  9. an overview on the application of polymerase chain reaction (pcr)

    African Journals Online (AJOL)

    DR. AMINU

    ABSTRACT. This overview describes the principle of polymerase chain reaction as one of the most important techniques used in the diagnosis of bacterial infections due to its reliability, sensitivity and rapidness. It also identifies some of the limitations of the technique as well as some of the bacterial infections that can be ...

  10. Multiplex polymerase chain reaction (PCR) and fluorescence-based ...

    African Journals Online (AJOL)

    Multiplex polymerase chain reaction (PCR) and fluorescence-based capillary electrophoresis (CE) of blood and tissue samples have been used to distinguish between deer species such as red deer, sika deer, wapiti and reindeer. We constructed 4 species-specific primers by using the D-loop of mitochondrial DNA and ...

  11. TaqMan Real-Time Polymerase Chain Reaction and ...

    African Journals Online (AJOL)

    TaqMan Real-Time Polymerase Chain Reaction and. Pyrosequencing using Single Nucleotide Polymorphism. Protocol for Rapid Determination of ALDH2 *2 in a Chinese. Population. Ju-yi Li1, Jin-hu Wu2, Yan Zhang2, Xiu-fang Wang2, Jie Jin2 and Yi Wang1*. 1Department of Pharmacy, The Central Hospital of Wuhan, ...

  12. Application of polymerase chain reaction to differentiate between ...

    African Journals Online (AJOL)

    A polymerase chain reaction (PCR) assay was used to identify and differentiate between strains of Campylobacter jejuni and Campylobacter coli. Nine Campylobacter reference strains; C. jejuni NCTC 11168, C. jejuni NCTC 11322, C. jejuni NCTC 11828, Campylobacter coli NCTC 12110, C. coli NCTC 11437, C. coli ...

  13. Species specific polymerase chain reaction (PCR) assay for ...

    African Journals Online (AJOL)

    A highly specific single step polymerase chain reaction (PCR) is described for the detection of pig (Sus domesticus) meat. A PCR assay was successfully optimized for amplification of 629 and 322-bp DNA fragment extracted from pig meat using designed species-specific primer pairs based on mitochondrial D-loop and 12S ...

  14. Prevalence and detection of cytomegalovirus by polymerase chain ...

    African Journals Online (AJOL)

    A total of 327 women were screened, amongst them, 7 (2.14%) were cytomegalovirus (CMV) DNA positive by polymerase chain reaction (PCR). Antibodies against toxoplasma were also detected in 106 (32.41%) women, while 54 (16.51%) were anti CMV positive. Eleven (3.36%) and thirteen (3.97%) women were anti HSV ...

  15. Evaluation of Usefulness of Polymerase Chain Reaction in the ...

    African Journals Online (AJOL)

    Methods used included Giemsa staining procedure for estimation of parasite densities and polymerase chain reaction (PCR) to detect the presence of malaria parasite in the whole blood. Using microscopy as reference gold standard, patients comprising 120 males and 330 females with age ranging between less than 1 ...

  16. Role of Polymerase Chain Reaction (PCR) in the detection of ...

    African Journals Online (AJOL)

    Background: Staphylococcus aureus is mainly acquired from hospital infections and demonstrated the ability of developing resistance to many antibiotics. Polymerase Chain Reaction (PCR) was used to identify antibiotic-resistant isolates. This study was conducted in Al-Mujtahed, Al-Mouwasat and the Children Hospitals in ...

  17. (DNA) fragment using two-step polymerase chain reaction (PCR)

    African Journals Online (AJOL)

    Polymerase chain reaction (PCR), an essential tool in many fields such as molecular biology, normally comprises three steps: denaturation at a high temperature, annealing at a low temperature and elongation at a moderate temperature. Here, we report a two-step PCR method which incorporates annealing and elongation ...

  18. Comparison of polymerase chain reaction (PCR) and loop-mediated ...

    African Journals Online (AJOL)

    Comparison of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for diagnosis of Fusarium solani in human immunodeficiency virus (HIV) positive patients. ... The test was carried out in 1 h reaction at 65°C in a heater block. The specificity of the test was 100% and its sensitivity was a ...

  19. Detection of Specific Polymerase Chain Reaction Product by Utilizing the 5' → 3' Exonuclease Activity of Thermus aquaticus DNA Polymerase

    National Research Council Canada - National Science Library

    Pamela M. Holland; Richard D. Abramson; Robert Watson; David H. Gelfand

    1991-01-01

    The 5' → 3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal...

  20. Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

    Directory of Open Access Journals (Sweden)

    R.S. Diaz

    1998-10-01

    Full Text Available For certain applications of the polymerase chain reaction (PCR, it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA™ DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA™ and Taq, respectively, after reading about 3,000 bases each. We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.

  1. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

    Science.gov (United States)

    Nasarabadi, Shanavaz [Livermore, CA

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reaction chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.

  2. Polymerase chain reaction to search for Herpes viruses in uveitic ...

    African Journals Online (AJOL)

    Cite as: Laaks D, Smit DP, Harvey J. Polymerase chain reaction to search for Herpes viruses in uveitic and healthy eyes: a South African perspective. Afri Health Sci. 2015;15(3):748-54. doi: http://dx.doi.org/10.4314/ahs.v15i3.7. Introduction. Uveitis is internationally classified according to the an- atomical locus of infection: ...

  3. Block copolymer nanoparticles as nanobeads for the polymerase chain reaction.

    Science.gov (United States)

    Zhang, Siyan; Prud'homme, Robert K; Link, A James

    2011-04-13

    New sequencing technologies based on massively parallel signature sequencing (MPSS) have been developed to reduce the cost of genome sequencing. In some current MPSS platforms, DNA-modified micrometer-scale beads are used to template the polymerase chain reaction (PCR). Reducing the size of the beads to nanoscale can lead to significant improvements in sequencing throughput. To this end, we have assembled polymeric nanobeads that efficiently template PCR, resulting in DNA-decorated "nanobeads" with a high extent of functionalization.

  4. [Differentiation of capripoxvirus species and strains by polymerase chain reaction].

    Science.gov (United States)

    Orlova, E S; Shcherbakova, A V; Diev, V I; Zakharov, V M

    2006-01-01

    A fast and simple method for capripoxvirus species identification has been developed. The method is based on multiplex polymerase chain reaction (MPCR) with species-specific primers and does not require nucleotide sequencing or restriction analysis of PCR products. To differentiate vaccine stains used in Russia and countries of the former Soviet Union from epizootic isolates of sheep pox virus, a method based on restriction analysis of the ankyrin-repeat protein gene fragment amplified by PCR has been developed. Being highly specific, both methods may be used for routine diagnosis of capripoxvirus-associated diseases.

  5. A specific polymerase chain reaction method to identify Stenotrophomonas maltophilia

    Directory of Open Access Journals (Sweden)

    Stephanie Wagner Gallo

    2013-05-01

    Full Text Available Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that is difficult to identify unequivocally using current methods. Accordingly, because the presence of this microorganism in a patient may directly determine the antimicrobial treatment, conventional polymerase chain reaction (PCR and real-time PCR assays targeting 23S rRNA were developed for the specific identification of S. maltophilia. The PCR protocol showed high specificity when tested against other species of Stenotrophomonas, non-fermentative Gram-negative bacilli and 100 clinical isolates of S. maltophilia previously identified using the Vitek system.

  6. POSSIBILITIES OF THE POLYMERASE CHAIN REACTION IN THE HUMAN IDENTIFICATION

    Directory of Open Access Journals (Sweden)

    Zoran Budimlija

    2001-05-01

    Full Text Available Biological traces can be found in various forms. The basic question to deal withduring the biological traces' analysis is the one about their origin, that is, the questionof identity of the person to whom such samples belong. Up to now the most precisemethod of the DNA is the PCR of Polymerase Chain Reaction.The generalassumptions about applying the method for the purpose of human identification aredescribed, namely, the one that is used in the Institute for Forensic Medicine in NoviSad.

  7. Building block synthesis using the polymerase chain assembly method.

    Science.gov (United States)

    Marchand, Julie A; Peccoud, Jean

    2012-01-01

    De novo gene synthesis allows the creation of custom DNA molecules without the typical constraints of traditional cloning assembly: scars, restriction site incompatibility, and the quest to find all the desired parts to name a few. Moreover, with the help of computer-assisted design, the perfect DNA molecule can be created along with its matching sequence ready to download. The challenge is to build the physical DNA molecules that have been designed with the software. Although there are several DNA assembly methods, this section presents and describes a method using the polymerase chain assembly (PCA).

  8. Effects of Superparamagnetic Nanoparticle Clusters on the Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Toshiaki Higashi

    2012-04-01

    Full Text Available The polymerase chain reaction (PCR method is widely used for the reproduction and amplification of specific DNA segments, and a novel PCR method using nanomaterials such as gold nanoparticles has recently been reported. This paper reports on the effects of superparamagnetic nanoparticles on PCR amplification without an external magnetic field, and clarifies the mechanism behind the effects of superparamagnetic particle clusters on PCR efficiency by estimating the structures of such clusters in PCR. It was found that superparamagnetic nanoparticles tend to inhibit PCR amplification depending on the structure of the magnetic nanoparticle clusters. The paper also clarifies that Taq polymerase is captured in the spaces formed among magnetic nanoparticle clusters, and that it is captured more efficiently as a result of their motion from heat treatment in PCR thermal cycles. Consequently, Taq polymerase that should be used in PCR is reduced in the PCR solution. These outcomes will be applied to novel PCR techniques using magnetic particles in an external magnetic field.

  9. Polymerase chain reaction: A molecular diagnostic tool in periodontology.

    Science.gov (United States)

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

  10. Polymerase chain reaction: A molecular diagnostic tool in periodontology

    Directory of Open Access Journals (Sweden)

    Rajendran Maheaswari

    2016-01-01

    Full Text Available This review discusses the principles of polymerase chain reaction (PCR and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

  11. Real-Time Polymerase Chain Reaction: Applications in Diagnostic Microbiology

    Directory of Open Access Journals (Sweden)

    Kordo B. A. Saeed

    2013-11-01

    Full Text Available The polymerase chain reaction (PCR has revolutionized the detection of DNA and RNA. Real-Time PCR (RT-PCR is becoming the gold standard test for accurate, sensitive and fast diagnosis for a large range of infectious agents. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, high throughout and quantification. RT-PCR assays have advanced the diagnostic abilities of clinical laboratories particularly microbiology and infectious diseases. In this review we would like to briefly discuss RT-PCR in diagnostic microbiology laboratory, beginning with a general introduction to RT-PCR and its principles, setting up an RT PCR, including multiplex systems and the avoidance and remediation of contamination issues. A segment of the review would be devoted to the application of RT-PCR in clinical practice concentrating on its role in the diagnosis and treatment of infectious diseases.

  12. Use of polymerase chain reaction for detection of Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Østergaard, Lars; Birkelund, Svend; Christiansen, Gunna

    1990-01-01

    A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. From the published sequence of the common C. trachomatis plasmid, two primer sets were selected. Detection of amplified sequences was done by agarose gel electrophoresis of cleaved or uncleaved...... amplified sequences, Southern hybridization, or dot blot analysis. The PCR assay was optimized and, after 40 cycles of amplification with primer set II, demonstrated a sensitivity of 10(-17) g of DNA, which corresponds to the detection of one copy of the plasmid. Because of the high sensitivity, we...... developed a closed system in which airborne contamination was minimized. Analysis of 228 clinical samples tested by cell culture, IDEIA enzyme immunosorbent assay (Medico-Nobel, Boots-Celltech Ltd., Berkshire, United Kingdom), and PCR showed a sensitivity of 100%, a specificity of 93% when PCR was compared...

  13. Polymerase chain reaction-based gene removal from plasmids

    Directory of Open Access Journals (Sweden)

    Vishnu Vardhan Krishnamurthy

    2015-09-01

    Full Text Available This data article contains supplementary figures and methods to the research article entitled, “Multiplex gene removal by two-step polymerase chain reactions” (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033, which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116. Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.

  14. Rapid electrochemiluminescence assays of polymerase chain reaction products.

    Science.gov (United States)

    Kenten, J H; Casadei, J; Link, J; Lupold, S; Willey, J; Powell, M; Rees, A; Massey, R

    1991-09-01

    We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

  15. Development of polymerase chain reaction primers to detect Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Artiushin, S; Stipkovits, L; Minion, F C

    1993-10-01

    Mycoplasma hyopneumoniae (Mhp) is the etiologic agent of mycoplasma pneumonia in swine. The purpose of this study was to develop a species-specific polymerase chain reaction (PCR)-based diagnostic reagent for the identification of Mhp. Based upon DNA sequence analysis of a cloned fragment of Mhp DNA, PCR primers were constructed and tested against different strains of Mhp, Mycoplasma flocculare, other mycoplasma species, and non-Mollicute micro-organisms which commonly inhabit the respiratory tracts of swine. A total of 40 field isolates from Mhp and four field isolates of M. flocculare have been examined. Positive signals were obtained in PCR with Mhp reference strains and all 40 Mhp field isolates, but not with other Mollicutes micro-organisms.

  16. Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA.

    Science.gov (United States)

    Chen, Tingjian; Romesberg, Floyd E

    2017-07-26

    There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2'-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2'-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.

  17. Mutagenicity Assessment of Organophosphates using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay

    National Research Council Canada - National Science Library

    Bhinder, Preety; Chaudhry, Asha

    2013-01-01

    In this study we have evaluated the mutagenicity of organophosphate pesticides acephate, chlorpyrifos, and profenofos using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP...

  18. Effects of upconversion nanoparticles on polymerase chain reaction.

    Directory of Open Access Journals (Sweden)

    Sang-Hyun Hwang

    Full Text Available Nanoparticles (NPs are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. Photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. Nucleic acids detection based on upconversion nanoparticles (UCNPs, which display a high signal-to-noise ratio and no photobleaching, has been widely applied. We evaluated whether UCNPs can improve polymerase chain reaction (PCR specificity and affect PCR amplification. The effects of UCNPs with a diameter size of 40, 70, and 250 nm were evaluated using 3 PCR kits (AccuPower PCR PreMix, AmpliTaq Gold 360 Master Mix, and HotStarTaq Plus Master Mix and 3 real-time PCR kits (AccuPower GreenStar qPCR PreMix, SYBR Green PCR Master Mix, and QuantiTect SYBR Green PCR Kit. Quantum dots were used for comparison with the UCNPs. In the presence of an appropriate concentration of UCNPs, PCR specificity was optimized. UCNPs of 40-nm size improved PCR specificity more effectively than did UCNPs sized 70 or 250 nm. As the size and concentrations of the UCNPs were increased, PCR amplification was more severely inhibited. At lower annealing temperatures (25°C-45°C, addition of the 40 nm UCNP (1 µg/µL to the PCR reagent produced specific PCR products without nonspecific sequence amplification. Therefore, UCNPs of different sizes, with different DNA polymerases used in the commercial kits, showed different inhibitory effects on PCR amplification. These results demonstrate that optimization of UCNPs, added to reaction mixtures at appropriate concentrations, can improve PCR specificity. However, the mechanism underlining UCNPs effect on PCR remains unclear and will require further investigation.

  19. An Emulsion System Based on a Chip Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Pinfei Yu

    2008-12-01

    Full Text Available In this paper we describe a novel method for detecting many DNA fragments through efficient amplification by using an emulsion system based on “on-chip” PCR instead of conventional multiplex polymerase chain reaction (PCR. During the preparation of on-chip PCR, a set of primers were immobilized on a slide and other sets were in an emulsion system. Different emulsion phase primers and other related PCR components were dispersed in different droplets of the emulsion system, and then, due to the thermal instability of emulsion droplets, they would be released onto the surface of the slide after preheating in the first PCR step. To test the above method, we used plasma DNAs from pregnant women who was carrying a male fetus for gender identification. Four different Y chromosome DNA fragments were selected. Results showed that different DNA fragments could be simultaneously amplified with satisfactory results. It is suggested that a simple, convenient and inexpensive on-chip PCR method has been developed.

  20. Integrated polymerase chain reaction chips utilizing digital microfluidics.

    Science.gov (United States)

    Chang, Yi-Hsien; Lee, Gwo-Bin; Huang, Fu-Chun; Chen, Yi-Yu; Lin, Jr-Lung

    2006-09-01

    This study reports an integrated microfluidic chip for polymerase chain reaction (PCR) applications utilizing digital microfluidic chip (DMC) technology. Several crucial procedures including sample transportation, mixing, and DNA amplification were performed on the integrated chip using electro-wetting-on-dielectric (EWOD) effect. An innovative concept of hydrophobic/hydrophilic structure has been successfully demonstrated to integrate the DMC chip with the on-chip PCR device. Sample droplets were generated, transported and mixed by the EWOD-actuation. Then the mixture droplets were transported to a PCR chamber by utilizing the hydrophilic/hydrophobic interface to generate required surface tension gradient. A micro temperature sensor and two micro heaters inside the PCR chamber along with a controller were used to form a micro temperature control module, which could perform precise PCR thermal cycling for DNA amplification. In order to demonstrate the performance of the integrated DMC/PCR chips, a detection gene for Dengue II virus was successfully amplified and detected. The new integrated DMC/PCR chips only required an operation voltage of 12V(RMS) at a frequency of 3 KHz for digital microfluidic actuation and 9V(DC) for thermal cycling. When compared to its large-scale counterparts for DNA amplification, the developed system consumed less sample and reagent and could reduce the detection time. The developed chips successfully demonstrated the feasibility of Lab-On-a-Chip (LOC) by utilizing EWOD-based digital microfluidics.

  1. Marzipan: polymerase chain reaction-driven methods for authenticity control.

    Science.gov (United States)

    Brüning, Philipp; Haase, Ilka; Matissek, Reinhard; Fischer, Markus

    2011-11-23

    According to German food guidelines, almonds are the only oilseed ingredient allowed for the production of marzipan. Persipan is a marzipan surrogate in which the almonds are replaced by apricot or peach kernels. Cross-contamination of marzipan products with persipan may occur if both products are produced using the same production line. Adulterations or dilutions, respectively, of marzipan with other plant-derived products, for example, lupine or pea, have also been found. Almond and apricot plants are closely related. Consequently, classical analytical methods for the identification/differentiation often fail or are not sensitive enough to quantify apricot concentrations below 1%. Polymerase chain reaction (PCR)-based methods have been shown to enable the differentiation of closely related plant species in the past. These methods are characterized by high specificity and low detection limits. Isolation methods were developed and evaluated especially with respect to the matrix marzipan in terms of yield, purity, integrity, and amplificability of the isolated DNA. For the reliable detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea, qualitative standard and duplex PCR methods were developed and established. The applicability of these methods was tested by cross-reaction studies and analysis of spiked raw pastes. Contaminations at the level of 0.1% could be detected.

  2. Role of multiplex polymerase chain reaction in diagnosing tubercular meningitis

    Directory of Open Access Journals (Sweden)

    Anupam Berwal

    2017-01-01

    Full Text Available Tuberculous meningitis (TBM is one of the most serious manifestations of extrapulmonary tuberculosis. Timely and accurate diagnosis provides a favorable prognosis in patients with TBM. The study evaluated the use of multiplex polymerase chain reaction (PCR in the diagnosis of TBM. A study was conducted on 74 patients clinically suspected with TBM. The cerebrospinal fluid (CSF specimens were processed for smear microscopy, middle brook 7H9 culture, and multiplex PCR using primers directed against IS6110 gene and 38 kD protein for detection of Mycobacterium tuberculosis. The results were analyzed to assess the role of multiplex PCR in the diagnosis of TBM. A total of 26 (35.1% patients were diagnosed with TBM. Microscopy was negative in all while culture was positive in two cases only. Comparing with clinical diagnosis and CSF adenosine deaminase levels of ≥10 U/L, multiplex PCR showed sensitivity, specificity, positive predictive value, and negative predictive value of 71.4%, 89.6%, 83.3%, and 81.2%, respectively, in the diagnosis of TBM.

  3. Polymerase chain reaction preparation of template for massively parallel pyrosequencing.

    Science.gov (United States)

    Whetten, Ross W; Sofía, Valenzuela A; Frampton, John

    2009-04-01

    Massively parallel pyrosequencing of DNA fragments immobilized on beads has been applied to genome survey sequencing and transcriptome analysis of a variety of eukaryotic organisms, including laboratory model species, agricultural crops and livestock, and species of interest to population biologists and ecologists. Preparation of sufficient high-quality template for sequencing has been an obstacle to sequence analysis of nucleic acids from tissues or cell types available in limited quantities. We report that the use of a biotinylated primer for polymerase chain reaction amplification allows removal of excess primer and poly(A) tract fragments from the sequencing templates, providing much higher yields of useful sequence information from pyrosequencing of amplified templates. This advance allows deep sequencing analysis of nucleic acids isolated from very small tissue samples. Massively parallel pyrosequencing is particularly useful for preliminary investigations of species that have not yet been the subject of significant genomic research, as genomic survey sequences and catalogs of expressed genes provide a means of linking the biology of less intensively studied species to that of more intensively studied model organisms. We obtained over 220 Mb of transcript DNA sequences from Abies fraseri (Pursh) Poir., a conifer species native to the southern Appalachian Mountains of eastern North America. Comparison of the resulting assembled putative transcripts with similar data obtained by other sequencing methods from other conifers demonstrates the utility of the improved sequencing template preparation.

  4. Multifunctional polyurethane sponge for polymerase chain reaction enhancement.

    Science.gov (United States)

    Seok, Seunghwan; Shin, Sujeong; Lee, Tae Jae; Jeong, Jae-Min; Yang, MinHo; Kim, Do Hyun; Park, Jung Youn; Lee, Seok Jae; Choi, Bong Gill; Lee, Kyoung G

    2015-03-04

    Selective filtering of target biomaterials from impurities is an important task in DNA amplification through polymerase chain reaction (PCR) enhancement and gene identification to save endangered animals and marine species. Conventional gene extraction methods require complicated steps, skilled persons, and expensive chemicals and instruments to improve DNA amplification. Herein, we proposed an alternative method for overcoming such challenges by imparting secondary functionality using commercially available polyurethane (PU) sponges and cost-effective fabrication approaches through polydopamine and polysiloxane coatings. The porous, highly flexible, and chemically modified superhydrophilic and superhydrophobic PU sponges allow large surface areas and mechanically stable frames for effective extraction of genomic DNA through selective filtering of fish tissues and oils. Furthermore, these chemically modified PU sponges allow separation of genes and improvement of PCR for DNA amplification for the identification of fish species. The combination of a simple fabrication method and functionalized PU sponges could be a useful platform for PCR enhancement and gene-based identification of species for practical applications.

  5. Identifying of meat species using polymerase chain reaction (PCR)

    Energy Technology Data Exchange (ETDEWEB)

    Foong, Chow Ming; Sani, Norrakiah Abdullah [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor (Malaysia)

    2013-11-27

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one’s diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  6. Human papillomavirus DNA detection in sperm using polymerase chain reaction.

    Science.gov (United States)

    Olatunbosun, O; Deneer, H; Pierson, R

    2001-03-01

    To detect human papillomavirus (HPV) in semen and find if sperm washing removes HPV DNA. Amplification by nested polymerase chain reaction (PCR) was used to detect viral DNA sequences in semen samples from 85 volunteers. Forty-five men had historical or clinical evidence of genital HPV infection (study group) and 40 were healthy, clinically HPV-negative semen donors. We detected HPV DNA in the sperm cells of 24 of 45 subjects (53%) with past or current HPV infections in contrast to three of 40 healthy subjects (8%) (P HPV in 21 of 32 subjects (66%) with identifiable lesions and six of 53 (11%) without them (P sperm cells with HPV reduced cellular HPV DNA below detectable levels in only two cases. HPV is present in sperm cells from infected and apparently healthy subjects, and sperm washing does not eliminate the risk of HPV transmission to recipients. We suggest that HPV DNA testing should be done on the semen of prospective donors, and those with positive tests should be excluded from donation.

  7. Identifying of meat species using polymerase chain reaction (PCR)

    Science.gov (United States)

    Foong, Chow Ming; Sani, Norrakiah Abdullah

    2013-11-01

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one's diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  8. Brucella contamination in raw milk by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Mohammad Khalili

    2016-10-01

    Full Text Available Background: Human brucellosis is a significant public health problem in many middle east countries including Iran. Brucella organisms, which are small aerobic, facultative intracellular coccobacilli, localize in the reproductive organs of host animals, causing abortions and sterility. They are shed in large numbers in the animal’s urine, milk, placental fluid, and other fluids. Dairy product from raw milk are a potential threat to public health in endemic developing countries. The gold standard for the diagnosis of brucellosis is isolation of Brucella species. However, isolation Brucella species is time consuming and needed to level 3 biocontainment facilities and highly skilled technical personnel to handle samples and live bacteria for eventual identification. Handling Brucella species increase risk of laboratory infection. Polymerase chain reaction (PCR with high sensitivity and specifity overcomed to these disadvantages. The aim of this study was to detect Brucella species in milk from dairy cattle farms in Kerman province, Iran by PCR technique. Methods: Forty and eight bulk tank milk (BTM were collected from October 2015 to March 2016 from 48 dairy cattle farm including 4200 cows. DNA of milk samples extracted by lysis buffer and proteinase K method. All milk samples were examined by PCR to detect Brucella-specific DNA targeting IS 711. Positive samples must be showed 317 bp amplified, corresponding to the expected size of the IS 711 genome region in all Brucella species. Results: Using IS711 primer were detected in 4 samples (8.3% Brucella spp. from 48 BTM samples in this area. Conclusion: The results indicate that brucellosis by Brucella species is endemic in the Kerman province dairy farms. Consumption of raw milk dairy products by individual farmers operating under poor hygienic conditions represents an high risk to public health. The need for implementing control measures and raising public awareness on zoonotic transmission of

  9. Identification of duck plague virus by polymerase chain reaction

    Science.gov (United States)

    Hansen, W.R.; Brown, Sean E.; Nashold, S.W.; Knudson, D.L.

    1999-01-01

    A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3a?? ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primer sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague. /// Se desarroll?? una prueba de reacci??n en cadena por la polimerasa para detectar el virus de la peste del pato. Un fragmento EcoRI de 765 pares de bases clonado del genoma del virus vacunal de la peste del pato fue secuenciado para la obtenci??n de los iniciadores de la prueba de la reacci??n en cadena por la polimerasa. En investigaciones de alineaci??n en el banco de genes ('GenBank') se encontr?? que la secuencia del fragmento era similar a los extremos 3a?? de un marco de lectura abierto

  10. Serotype specific polymerase chain reaction identifies a higher prevalence of streptococcus mutans serotype k and e in a random group of children with dental caries from the Southern region of India

    Directory of Open Access Journals (Sweden)

    Arun Prasad Rao

    2014-01-01

    Full Text Available Background: The development of dental caries has been associated with the oral prevalence of Streptococcus mutans. Four serotypes of S. mutans have been reported, namely serotype c, e, f, and k that are classified based on the composition and linkages of cell wall polysaccharides, response to physiological reactions, sero-specificity and 16s rRNA homology. Although the oral prevalence of S. mutans serotype c in Indian subjects with or without caries is known, the prevalence of the other three serotypes, e, f, and k are not known. Hence in this study, we have investigated the occurrence of the e, f, and k serotypes in children with or without caries within the age group of 6-12 years. Materials and Methods: Genomic DNA isolated from whole saliva of caries active (CA and caries free (CF groups were first screened for the presence of S. mutans by strain specific polymerase chain reaction (PCR. Those samples that tested positive for the presence of S. mutans were further analyzed by serotype specific PCR to identify the prevalence of the serotypes. Results: Strain specific PCR indicated a higher prevalence of S. mutans in CA group (80% relative to CF group (43%. Further analysis of the S. mutans positive samples in both groups indicated a higher prevalence of serotype k and e, followed by serotype f in CA group. Conclusion: The present data clearly establishes a novel S. mutans serotype prevalence hierarchy in children from this region, compared with those that have been reported elsewhere. Besides, the data are also clinically significant as the occurrence of serotype k has been associated with infective endocarditis.

  11. Serotype specific polymerase chain reaction identifies a higher prevalence of streptococcus mutans serotype k and e in a random group of children with dental caries from the Southern region of India.

    Science.gov (United States)

    Rao, Arun Prasad; Austin, Ravi David

    2014-07-01

    The development of dental caries has been associated with the oral prevalence of Streptococcus mutans. Four serotypes of S. mutans have been reported, namely serotype c, e, f, and k that are classified based on the composition and linkages of cell wall polysaccharides, response to physiological reactions, sero-specificity and 16s rRNA homology. Although the oral prevalence of S. mutans serotype c in Indian subjects with or without caries is known, the prevalence of the other three serotypes, e, f, and k are not known. Hence in this study, we have investigated the occurrence of the e, f, and k serotypes in children with or without caries within the age group of 6-12 years. Genomic DNA isolated from whole saliva of caries active (CA) and caries free (CF) groups were first screened for the presence of S. mutans by strain specific polymerase chain reaction (PCR). Those samples that tested positive for the presence of S. mutans were further analyzed by serotype specific PCR to identify the prevalence of the serotypes. Strain specific PCR indicated a higher prevalence of S. mutans in CA group (80%) relative to CF group (43%). Further analysis of the S. mutans positive samples in both groups indicated a higher prevalence of serotype k and e, followed by serotype f in CA group. The present data clearly establishes a novel S. mutans serotype prevalence hierarchy in children from this region, compared with those that have been reported elsewhere. Besides, the data are also clinically significant as the occurrence of serotype k has been associated with infective endocarditis.

  12. Blunt-end vectors generated by polymerase chain reaction (PCR) for ...

    African Journals Online (AJOL)

    Blunt-end cloning is a convenient way to clone polymerase chain reaction (PCR) products generated by proof-reading DNA polymerase. However, it is a time consuming procedure to prepare the linearized blunt-end vector, which usually involves plasmid extraction and restriction enzyme digestion. Moreover, 5' ...

  13. Blunt-end vectors generated by polymerase chain reaction (PCR) for ...

    African Journals Online (AJOL)

    Administrator

    2011-09-19

    Sep 19, 2011 ... Blunt-end cloning is a convenient way to clone polymerase chain reaction (PCR) products generated by proof-reading DNA polymerase. However, it is a time consuming procedure to prepare the linearized blunt-end vector, which usually involves plasmid extraction and restriction enzyme digestion.

  14. Detection of Enterococcus faecalis in Necrotic Teeth Root Canals by Culture and Polymerase Chain Reaction Methods

    OpenAIRE

    Cogulu, Dilsah; Uzel, Atac; Oncag, Ozant; Aksoy, Semiha C.; Eronat, Cemal

    2007-01-01

    Objectives The aim of this study was to investigate the presence of Enterococcus faecalis in endodontic infections in both deciduous and permanent teeth by culture and polymerase chain reaction (PCR) methods. Methods A total of 145 children aged 5?13 years old were involved in this study. The presence of E. faecalis in necrotic deciduous and permanent teeth root canals was studied using culture and polymerase chain reaction methods. Results Among 145 molar teeth, 57% (n=83) presented necrotic...

  15. Molecular typing of Lactobacillus brevis isolates from Korean food using repetitive element-polymerase chain reaction.

    Science.gov (United States)

    Kaur, Jasmine; Sharma, Anshul; Lee, Sulhee; Park, Young-Seo

    2018-01-01

    Lactobacillus brevis is a part of a large family of lactic acid bacteria that are present in cheese, sauerkraut, sourdough, silage, cow manure, feces, and the intestinal tract of humans and rats. It finds its use in food fermentation, and so is considered a "generally regarded as safe" organism. L. brevis strains are extensively used as probiotics and hence, there is a need for identifying and characterizing these strains. For identification and discrimination of the bacterial species at the subspecific level, repetitive element-polymerase chain reaction method is a reliable genomic fingerprinting tool. The objective of the present study was to characterize 13 strains of L. brevis isolated from various fermented foods using repetitive element-polymerase chain reaction. Repetitive element-polymerase chain reaction was performed using three primer sets, REP, Enterobacterial Repetitive Intergenic Consensus (ERIC), and (GTG) 5 , which produced different fingerprinting patterns that enable us to distinguish between the closely related strains. Fingerprinting patterns generated band range in between 150 and 5000 bp with REP, 200-7500 bp with ERIC, and 250-2000 bp with (GTG) 5 primers, respectively. The Jaccard's dissimilarity matrices were used to obtain dendrograms by the unweighted neighbor-joining method using genetic dissimilarities based on repetitive element-polymerase chain reaction fingerprinting data. Repetitive element-polymerase chain reaction proved to be a rapid and easy method that can produce reliable results in L. brevis species.

  16. Prenatal detection of trisomy 21 and 18 from amniotic fluid by quantitative fluorescent polymerase chain reaction.

    Science.gov (United States)

    Tóth, T; Findlay, I; Papp, C; Tóth-Pál, E; Marton, T; Nagy, B; Quirke, P; Papp, Z

    1998-01-01

    Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis on amniotic fluid. This requires lengthy laboratory procedures and high costs, and is unsuitable for large scale screening of pregnant women. An alternative method, which is both rapid and inexpensive and suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction using polymorphic small tandem repeats (STRs). In this paper we present the preliminary results of a larger study comparing parallel prenatal diagnoses of trisomies 21 and 18 using cytogenetics with quantitative fluorescent polymerase chain reaction using STR markers. The results obtained by the two techniques were concordant in all cases. This is the first study reporting significant numbers of prenatal diagnoses using the quantitative fluorescent polymerase chain reaction. We believe that further studies on greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for diagnosis of trisomy from single fetal cells isolated from maternal blood. PMID:9507392

  17. PCR (Polymerase Chain Reaction: Teknik dan Aplikasinya di Bidang Kedokteran Gigi

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-10-01

    Full Text Available This paper reviewed the ability of Polymerase Chain Reaction (PCR in multiplying DNA in order to amplify an extremely small amount of sample using an in-vitro chemical reaction which is needed for various purposes such as diagnostic in dentistry. Polymerase chain reaction (PCR is a technique of amplifying DNA in vitro which has been widely recognized as a powerful molecular-biology method. DNA template, primers, dNTP and DNA polymerase are needed, and this mixture will be exposed to three different temperatures for several cycles to multiply the DNA. The cycle consists of denaturation of double stranded DNA, annealing, and an extension phase which then will multiply the DNA twice. After n times of cycles, the amount of DNA will be multiplied 2n times.

  18. Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR

    Directory of Open Access Journals (Sweden)

    Seiki Kuramitsu

    2013-03-01

    Full Text Available Polymerase chain reaction (PCR-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3' end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science.

  19. A Woman with a Lobar Infiltrate due to Psittacosis Detected by Polymerase Chain Reaction

    NARCIS (Netherlands)

    Heddema, E.R.; Kraan, M.; Buys-Bergen, W.E.C.M.; Smith, H.E.; Wertheim-van Dillen, P.M.E.

    2003-01-01

    This report presents a case of community-acquired pneumonia due to Chlamydia psittaci presenting with a lobar infiltrate and diagnosed by a newly developed ompA gene-based polymerase chain reaction (PCR). This gene encodes a specific C. psittaci major outer membrane protein. This kind of PCR could

  20. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data

    NARCIS (Netherlands)

    Ramakers, Christian; Ruijter, Jan M.; Deprez, Ronald H. Lekanne; Moorman, Antoon F. M.

    2003-01-01

    Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR

  1. Case Report:False-negative HIV-1 polymerase chain reaction in a ...

    African Journals Online (AJOL)

    Polymerase chain reaction (PCR) testing is the gold standard for determining the HIV status in children <18 months of age. However, when clinical manifestations are not ... Consequently, the child was commenced on antiretroviral therapy and made a remarkable recovery. These findings indicate that more reliable PCR ...

  2. Using the Polymerase Chain Reaction in an Undergraduate Laboratory to Produce "DNA Fingerprints."

    Science.gov (United States)

    Phelps, Tara L.; And Others

    1996-01-01

    Presents a laboratory exercise that demonstrates the sensitivity of the Polymerase Chain Reaction as well as its potential application to forensic analysis during a criminal investigation. Can also be used to introduce, review, and integrate population and molecular genetics topics such as genotypes, multiple alleles, allelic and genotypic…

  3. Polymerase chain reaction-mediated DNA fingerprinting for epidemiological studies on Campylobacter spp

    NARCIS (Netherlands)

    Giesendorf, B A; Goossens, H; Niesters, H G; Van Belkum, A; Koeken, A; Endtz, H P; Stegeman, H; Quint, W G

    The applicability of polymerase chain reaction (PCR)-mediated DNA typing, with primers complementary to dispersed repetitive DNA sequences and arbitrarily chosen DNA motifs, to study the epidemiology of campylobacter infection was evaluated. With a single PCR reaction and simple gel electrophoresis,

  4. Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction

    NARCIS (Netherlands)

    Giesendorf, B A; Quint, W G; Henkens, M H; Stegeman, H; Huf, F A; Niesters, H G

    1992-01-01

    The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was

  5. FUNGAL SPECIATION USING QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) IN PATIENTS WITH AND WITHOUT CHRONIC RHINOSINUSITIS

    Science.gov (United States)

    Objectives/Hypothesis: 1. to determine the mycology of the middle meatus using an endoscopically guided brush sampling technique and polymerase chain reaction laboratory processing of nasal mucous. 2. To compare the mycology of the middle meatus in patients with sinus disease to...

  6. Detection of Salmonella typhi by nested polymerase chain reaction in blood, urine, and stool samples

    NARCIS (Netherlands)

    Hatta, Mochammad; Smits, Henk L.

    2007-01-01

    A nested polymerase chain reaction (PCR) specific for Salmonella enterica serovar Typhi was used for the detection of the pathogen in blood, urine, and stool samples from 131 patients with clinical suspicion of typhoid fever. The sensitivity of blood culture, the PCRs with blood, urine, and feces,

  7. Detection of infections of the eye with Chlamydia trachomatis by the polymerase chain reaction

    NARCIS (Netherlands)

    J. Fan (Jun); W.Y. Zhang (Wen); Y.Y. Wu (Yu); X.Y. Jing (Xiou); E.C.J. Claas (Eric)

    1993-01-01

    textabstractThe aim of this study was to test the diagnostic feasibility of the polymerase chain reaction (PCR) for detection of infections with Chlamydia trachomatis in eye swabs from patients with conjunctivitis, and to establish the basic technique of the PCR for epidemiological survey. The

  8. Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory

    Science.gov (United States)

    Elkins, Kelly M.

    2011-01-01

    The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for…

  9. Polymerase chain Reaction in molecular biotechnology; appropriate technology for developing countries

    NARCIS (Netherlands)

    Felice, A. E.; Alshinawi, C.

    1996-01-01

    The product of the Polymerase Chain Reaction (PCR) may be generically suitable for four types of investigations: Discovery PCR, Analytical PCR, Modification by PCR, and Synthetic PCR. Despite the potential problem of contamination with extraneous DNA, PCR is relatively simple and inexpensive, and

  10. [Software and hardware design for the temperature control system of quantitative polymerase chain reaction].

    Science.gov (United States)

    Qiu, Xian-bo; Yuan, Jing-qi; Li, Qi

    2005-07-01

    A temperature control system for quantitive polymerase chain reaction (PCR) is presented in the paper with both software and hardware configuration. The performance of the control system has been improved by optimizing the software and hardware design according to the system's properties. The control system has been proven to have a good repeatability and reliability as well as high control precision.

  11. Detection of adenovirus hexon sequence in a cat by polymerase chain reaction(short communication)

    NARCIS (Netherlands)

    Horzinek, M.C.; Lakatos, B.; Farkas, J.; Egberink, H.F.; Vennema, H.; Benko, M.

    1999-01-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal

  12. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078

    NARCIS (Netherlands)

    Goorhuis, Abraham; Bakker, Dennis; Corver, Jeroen; Debast, Sylvia B.; Harmanus, Celine; Notermans, Daan W.; Bergwerff, Aldert A.; Dekker, Frido W.; Kuijper, Ed J.

    2008-01-01

    Since 2005, an increase in the prevalence of Clostridium difficile infection (CDI) due to polymerase chain reaction ribotype 078 has been noticed in The Netherlands. This strain has also been identified as the predominant strain in pigs and calves. CDI caused by type 078 was studied in relation to

  13. Aspergillus Polymerase Chain Reaction: Systematic Review of Evidence for Clinical Use in Comparison With Antigen Testing

    NARCIS (Netherlands)

    White, P.L.; Wingard, J.R.; Bretagne, S.; Loffler, J.; Patterson, T.F.; Slavin, M.A.; Barnes, R.A.; Pappas, P.G.; Donnelly, J.P.

    2015-01-01

    BACKGROUND: Aspergillus polymerase chain reaction (PCR) was excluded from the European Organisation for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal disease because of limited standardization and validation. The definitions are being revised.

  14. Quantum Criticality of Hot Random Spin Chains

    Science.gov (United States)

    Vasseur, R.; Potter, A. C.; Parameswaran, S. A.

    2015-05-01

    We study the infinite-temperature properties of an infinite sequence of random quantum spin chains using a real-space renormalization group approach, and demonstrate that they exhibit nonergodic behavior at strong disorder. The analysis is conveniently implemented in terms of SU (2 )k anyon chains that include the Ising and Potts chains as notable examples. Highly excited eigenstates of these systems exhibit properties usually associated with quantum critical ground states, leading us to dub them "quantum critical glasses." We argue that random-bond Heisenberg chains self-thermalize and that the excited-state entanglement crosses over from volume-law to logarithmic scaling at a length scale that diverges in the Heisenberg limit k →∞. The excited state fixed points are generically distinct from their ground state counterparts, and represent novel nonequilibrium critical phases of matter.

  15. Diversity of Enterococcus faecalis Genotypes from Multiple Oral Sites Associated with Endodontic Failure Using Repetitive Sequence-based Polymerase Chain Reaction and Arbitrarily Primed Polymerase Chain Reaction.

    Science.gov (United States)

    Delboni, Maraísa G; Gomes, Brenda P F A; Francisco, Priscila A; Teixeira, Fabrício B; Drake, David

    2017-03-01

    The aim of this study was to evaluate the diversity and similarity of Enterococcus faecalis genotype isolates from multiple oral sites using repetitive sequence-based polymerase chain reaction and arbitrarily primed polymerase chain reaction (AP-PCR). Forty-two endodontically treated teeth with apical periodontitis were selected. A total of 126 microbial samples were collected from 3 different sites (saliva, pulp chamber, and root canals, all n = 42) during the nonsurgical retreatment procedures. After growth on m-Enterococcus agar, the colonies were isolated, characterized as gram-positive catalase negative cocci, and identified using an API 20 Strep kit (bioMérieux, Marcy-l'Etoile, France). Seventy-four colonies from 10 patients were confirmed as E. faecalis by polymerase chain reaction (16S ribosomal RNA). Repetitive sequence-based polymerase chain reactions using ERIC and AP-PCR using RW3A primers were performed in all 74 colonies. Fingerprints were analyzed and separated into genotypic groups based on the Dice coefficient percentage of similarity (82% or greater) as determined by ERIC reproducibility assays involving E. faecalis controls. Seven different E. faecalis genotypes (GTs) (GT1 = 27%, GT2 = 17.6%, GT3 = 1.3%, GT4 = 18.9%, GT5 = 9.5%, GT6 = 14.9%, and GT7 = 10.8%) were observed in different subjects and oral sites associated with endodontic failure. Remarkably, in 4 of 5 patients, the same GTs present in the infected root canals were also isolated from either the pulp chamber or the saliva samples. In particular, GT6 was detected in all 3 oral sites of patient 37. E. faecalis GTs isolated from saliva, the pulp chamber, and the root canal were similar using the Rep-PCR and AP-PCR methods. These findings suggest that coronal microleakage is a conceivable cause of endodontic failure. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  16. Polymerase chain reaction and conventional DNA tests in detection of HPV DNA in cytologically normal and abnormal cervical scrapes

    DEFF Research Database (Denmark)

    Kalia, A.; Jalava, T.; Nieminen, P.

    1992-01-01

    Med.mikrobiologi, polymerase chain reaction, DNA tests, human papillomavirus (HPV), cervical smear, hybridisation, cytologi, affiProbe HPV test, ViraType test......Med.mikrobiologi, polymerase chain reaction, DNA tests, human papillomavirus (HPV), cervical smear, hybridisation, cytologi, affiProbe HPV test, ViraType test...

  17. Evaluation of urogenital Chlamydia trachomatis infections by cell culture and the polymerase chain reaction using a closed system

    DEFF Research Database (Denmark)

    Østergaard, Lars; Traulsen, J; Birkelund, Svend

    1993-01-01

    the two test systems were compared, the overall sensitivity of the polymerase chain reaction was 96% and the specificity 94% when compared to the cell culture technique. By use of a closed system for DNA extraction and sample transfer for the polymerase chain reaction, contamination of the samples......Two hundred and fifty-four specimens from males and females consulting a clinic for sexually transmitted diseases were analyzed for genital Chlamydia trachomatis infection. Each clinical sample was tested by the cell culture technique and the polymerase chain reaction using a closed system. When...... was minimized. The polymerase chain reaction detected a higher number of Chlamydia trachomatis infections among both symptomatic and asymptomatic females and males, and it also detected Chlamydia trachomatis at an earlier stage of infection when compared to cell culture. The polymerase chain reaction did...

  18. Evaluation of urogenital Chlamydia trachomatis infections by cell culture and the polymerase chain reaction using a closed system

    DEFF Research Database (Denmark)

    Østergaard, Lars; Traulsen, J; Birkelund, Svend

    1991-01-01

    the two test systems were compared, the overall sensitivity of the polymerase chain reaction was 96% and the specificity 94% when compared to the cell culture technique. By use of a closed system for DNA extraction and sample transfer for the polymerase chain reaction, contamination of the samples......Two hundred and fifty-four specimens from males and females consulting a clinic for sexually transmitted diseases were analyzed for genital Chlamydia trachomatis infection. Each clinical sample was tested by the cell culture technique and the polymerase chain reaction using a closed system. When...... was minimized. The polymerase chain reaction detected a higher number of Chlamydia trachomatis infections among both symptomatic and asymptomatic females and males, and it also detected Chlamydia trachomatis at an earlier stage of infection when compared to cell culture. The polymerase chain reaction did...

  19. Studying the effect of graphene-ZnO nanocomposites on polymerase chain reaction

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Vinay, E-mail: winn201@gmail.com; Rajaura, Rajveer; Sharma, Preetam Kumar; Srivastava, Rishabh Ronin [Centre for Converging Technologies, University of Rajasthan, Jaipur 302004 (India); Sharma, Shyam Sundar [Govt. women Engineering College, Ajmer (India); Agrawal, Kailash [Centre for Converging Technologies, University of Rajasthan, Jaipur 302004 (India); Department of Botany, University of Rajasthan, Jaipur 302004 (India)

    2016-05-06

    An emerging area of research is improving the efficiency of the polymerase chain reaction (PCR) by using nanoparticles. With graphene nano-flakes showing promising results, in this paper we report the effect of Graphene-ZnO nanocomposites on Polymerase Chain reaction (PCR) efficiency. G-ZnO nanocomposites were efficiently synthesized via in situ chemical method. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) image confirms the formation of nanocomposites. ZnO nanoparticles of size range ~20-30 nm are uniformly attached on the graphene sheets. No amplification during PCR indicates inhibitory activity of G-ZnO nanocomposites which points the fingers at ZnO moiety of the G-ZnO composite for no amplification during our PCR reaction. Further work should concentrate on finding out the main inhibitory mechanism involved in inhibition of PCR using G-ZnO composites.

  20. Optimised sample preparation of synovial fluid for detection of Chlamydia trachomatis DNA by polymerase chain reaction

    Science.gov (United States)

    Kuipers, J.; Nietfeld, L.; Dreses-Werringloe..., U.; Koehler, L.; Wollenhaupt, J.; Zeidler, H.; Hammer, M.

    1999-01-01

    OBJECTIVE—To optimise sample preparation of synovial fluid for Chlamydia trachomatis (CT) specific polymerase chain reaction (PCR).
METHODS—Serial dilutions of purified CT elementary bodies in synovial fluid were prepared. The synovial fluid pellet was processed by eight different methods of sample preparation. Then samples were analysed by CT specific PCR. The sensitivity of PCR was the basis of ranking of the eight different methods.
RESULTS—Highest sensitivity was achieved by methods including an additional step of DNA isolation. Additional extraction of protein and polysaccharides by cetyltrimethylammonium bromide (CTAB) increased sensitivity. Addition of hyaluronidase did not increase sensitivity of QIAEX-DNA extraction but was necessary, however, before phenol-chloroform-DNA extraction.
CONCLUSIONS—The method of synovial fluid sample preparation significantly influences the sensitivity of subsequent PCR. Additional DNA isolation and extraction of PCR inhibitors by CTAB led to higher sensitivity.

 Keywords: Chlamydia trachomatis; polymerase chain reaction; synovial fluid PMID:10343525

  1. Polymerase chain reaction assay for detection of Staphylococcus aureus in buffalo milk

    Directory of Open Access Journals (Sweden)

    V.K. Jain

    2010-02-01

    Full Text Available In India, Haryana has the world’s best dairy type buffalo, the Murrah capable of milk yields as high as 35 kg a day. Clinical and Sub clinical mastitis exerts a negative impact on milk quality, quantity and animal health and profits. In India, Staphylococci are the main causative agents responsible for mastitis of economic importance. Therefore, a suitable and specific test is required for the rapid diagnosis of Staphylococcus aureus. For definitive diagnosis of Staphylococcus aureus in mastitic milk, a polymerase chain reaction assay was developed using target sequence of 16S to 23S rRNA spacer region. This test can be performed within hours and avoids cumbersome and lengthy steps involved in microbiological culture of milk and biochemical tests. Polymerase chain reaction assay can be used as a screening test for a large herd to detect Staphylococcus aureus in milk.

  2. Polymerase chain reaction of enterococcus faecalis and candida albicans in apical periodontitis from Turkish patients

    OpenAIRE

    Dumani, Aysin; Yoldas, Oguz; Yilmaz, Sehnaz; Koksal, Fatih; Kayar, Begum; Akcimen, Beril; Seydaoglu, Gulsah

    2012-01-01

    Aim: The aim of this study was to determine the frequency of two important pathogenic microorganisms associated with endodontic infections, Enterococcus faecalis and Candida albicans, in root canal samples from patients with necrotic pulps or failed canal therapy by polymerase chain reaction method. Method: Microbial samples were obtained from 117 teeth with necrotic pulp tissues and 114 teeth with failed endodontic treatment. Results: E.faecalis were identified in 16% of the necrotic and 10%...

  3. Detection of Mycoplasma pneumoniae by polymerase chain reaction and nonradioactive hybridization in microtiter plates.

    OpenAIRE

    Lüneberg, E; Jensen, J.S.; Frosch, M.

    1993-01-01

    In order to improve the diagnosis of a Mycoplasma pneumoniae infection, we developed a polymerase chain reaction (PCR)-based assay. The gene encoding elongation factor Tu (tuf) was selected as the target sequence. Oligonucleotides derived from variable stretches of the tuf gene were able to prime the amplification of a 950-bp fragment exclusively when M. pneumoniae DNA was used as the template. The sensitivity of the assay was increased 10-fold when the amplification products were hybridized ...

  4. Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

    OpenAIRE

    Oyofo, B A; Thornton, S A; Burr, D H; Trust, T J; Pavlovskis, O R; Guerry, P

    1992-01-01

    Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, ...

  5. STUDY OF UROGENITAL TRACT MICROFLORA OF DNEPROPETROVSK FEMALES BY POLYMERASE CHAIN REACTION

    Directory of Open Access Journals (Sweden)

    Honcharova S.Y.

    2015-08-01

    Full Text Available We isolated and identified the pathogens from the urogenital tract in 100 women of 26-55 years in Diagnostic Center of Dnepropetrovsk Medical Academy by polymerase chain reaction. It was found that all investigated microflora was represented by HPV of high and low cancer risk - HSV type 1+2, Ureaplasma urealyticum, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma hominis, and Candida yeast species. The most abundant pathogens from the urogenital tract were HPV, Ureaplasma urealyticum, and Chlamydia trachomatis.

  6. Detection of measles virus by reverse-transcriptase polymerase chain reaction in a placenta.

    Science.gov (United States)

    Bar-On, Shikma; Ochshorn, Yifat; Halutz, Ora; Aboudy, Yair; Many, Ariel

    2010-08-01

    Measles virus (MV) during pregnancy is associated with maternal morbidity and mortality and can put the fetus and newborn at risk of a wide range of complications. Reverse-transcriptase polymerase chain reaction (RT-PCR) for detecting MV in the placenta has not been reported. A case of RT-PCR detection of MV in the placenta of a 38-year-old woman who presented with premature rupture of membranes at 16 weeks' gestation is presented.

  7. The impact of meningococcal polymerase chain reaction testing on laboratory confirmation of invasive meningococcal disease.

    LENUS (Irish Health Repository)

    Drew, Richard J

    2012-03-01

    Laboratory methods of diagnosis were examined for 266 children with invasive meningococcal disease. Seventy-five (36%) of 207 cases with bloodstream infection had both positive blood culture and blood meningococcal polymerase chain reaction (PCR), 130 (63%) negative blood culture and positive blood PCR, and 2 (1%) had positive blood culture and negative blood PCR. Sixty-three percent of cases were diagnosed by PCR alone.

  8. Allergic fungal rhinosinusitis: detection of fungal DNA in sinus aspirate using polymerase chain reaction.

    Science.gov (United States)

    El-Morsy, S M; Khafagy, Y W; El-Naggar, M M; Beih, A A

    2010-02-01

    This study investigated allergic fungal rhinosinusitis cases, and aimed to compare the detection of fungi in sinus aspirate by culture and by polymerase chain reaction assay, and to relate the presence of fungi in the nasal sinuses to the type of fungal allergen causing disease. Sixty-eight cases of allergic fungal rhinosinusitis underwent fungal culture and polymerase chain reaction assay for universal fungal, aspergillus and bipolaris DNA. Aspergillus-specific immunoglobulin E levels were measured in sinus aspirate, and total serum immunoglobulin E levels were calculated. A control group of 10 cases was included in the study. Of the 68 allergic fungal rhinosinusitis cases, only 42 (61.7 per cent) had positive fungal cultures; of the 10 controls, only three (30 per cent) had positive cultures. Species from the dematiaceous family were most commonly grown, being isolated in 30 cases (71.4 per cent). Bipolaris was the most commonly isolated species (18 cases) followed by curvularia (11 cases) and alternaria (one case). Polymerase chain reaction assay detected fungal DNA in all the allergic fungal rhinosinusitis cases and also in four controls (40 per cent). Ten patients (of 68; 14.7 per cent) were positive for Aspergillus fumigatus specific immunoglobulin E. The mean concentration of this immunoglobulin was 11.32 +/- 4.12 IU/ml in patients and 0 IU/ml in controls, a statistically significant difference. Detection of fungal DNA in nasal aspirate by polymerase chain reaction was superior to fungal cultures as a method of detecting fungal growth. In allergic fungal rhinosinusitis, fungal growth is not always accompanied by an allergic reaction.

  9. Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA.

    OpenAIRE

    Fahrimal, Y; Goff, W L; Jasmer, D P

    1992-01-01

    Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequenc...

  10. Shortening Isolation of Patients With Suspected Tuberculosis by Using Polymerase Chain Reaction Analysis

    DEFF Research Database (Denmark)

    Fløe, Andreas; Hilberg, Ole; Thomsen, Vibeke Østergaard

    2015-01-01

    Background. Isolation of patients suspected for pulmonary tuberculosis is guided by serial sputum smears. This can result in isolation for days for patients with noncontagious tuberculosis. To determine whether a single sample negative for Mycobacterium tuberculosis complex at polymerase chain re......-positive/PCR-negative cases underlines the importance of increasing the quantity and quality of samples. Moreover, it is important that samples analyzed with PCR are cultured, owing to higher-sensitivity drug susceptibility testing, differential diagnosis, and surveillance....

  11. High density labeling of polymerase chain reaction products with the fluorescent base analogue tCo.

    Science.gov (United States)

    Stengel, Gudrun; Urban, Milan; Purse, Byron W; Kuchta, Robert D

    2009-11-01

    Fluorescent DNA of high molecular weight is an important tool for studying the physical properties of DNA and DNA-protein interactions, and it plays a key role in modern biotechnology for DNA sequencing and detection. While several DNA polymerases can incorporate large numbers of dye-linked nucleotides into primed DNA templates, the amplification of the resulting densely labeled DNA strands by polymerase chain reaction (PCR) is problematic. Here, we report a method for high density labeling of DNA in PCR reactions employing the 5'-triphosphate of 1,3-diaza-2-oxo-phenoxazine (tCo) and Deep Vent DNA polymerase. tCo is a fluorescent cytosine analogue that absorbs and emits light at 365 and 460 nm, respectively. We obtained PCR products that were fluorescent enough to directly visualize them in a gel by excitation with long UV light, thus eliminating the need for staining with ethidium bromide. Reactions with Taq polymerase failed to produce PCR products in the presence of only small amounts of dtCoTP. A comparative kinetic study of Taq and Deep Vent polymerase revealed that Taq polymerase, although it inserts dtCoTP with high efficiency opposite G, is prone to forming mutagenic tCo-A base pairs and does not efficiently extend base pairs containing tCo. These kinetics features explain the poor outcome of the PCR reactions with Taq polymerase. Since tCo substitutes structurally for cytosine, the presented labeling method is believed to be less invasive than labeling with dye-linked nucleotides and, therefore, produces DNA that is ideally suited for biophysical studies.

  12. Detection of stress resistance genes in transgenic maize by multiplex and touchdown polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Bannikova M. A.

    2015-10-01

    Full Text Available Aim. To develop a methodology for detection of the genes of resistance to the stress factors in transgenic maize by multiplex (mPCR and touchdown polymerase chain reactions. Methods. isolation of total DNA by CTAB method, purification of DNA from RNA and proteins, electrophoresis of total DNA and amplification products in agarose gel, polymerase chain reaction. Results. The protocol of multiplex and touchdown polymerase chain reactions has been developed for simultaneous verification of the quality of total DNA extracted from the studied maize plant samples and detection of the following genes that determine resistance to the stress factors in the transgenic maize and maize transformation events: BT176, MON810, MON88017, DAS1507, DAS59122, MIR604, GA21, NK603 (mPCR, Bt11, MON863, MON89034, T25 (touchdown PCR. The multiplex PCR and touchdown PCR were developed using the reference samples. Conclusions. The proposed protocol of mPCR and touchdown PCR reactions can be used for mass analysis of maize samples to detect the genes of tolerance/resistance to herbicides and genes of resistance to insects reliably, authentically, quickly and cheaply.

  13. Sensitivitas dan Spesifisitas Nested Polymerase Chain Reaction untuk Mendeteksi DNA Coxiella burnetii (SENSITIVITY AND SPECIFICITY OF NESTED POLYMERASE CHAIN REACTION FOR DETECTION OF COXIELLA BURNETII DNA

    Directory of Open Access Journals (Sweden)

    Trioso Purnawarman

    2014-04-01

    Full Text Available Sensitivity and specificity of nested polymerase chain reaction (nested PCR to detect Coxiella burnetii(C. burnetii DNA were studied. The primer system which consists of external primers (OMP1 and OMP2and internal primers (OMP3 and OMP4, was designed from the nucleotide sequence of the com I geneencoding for 27 kDa outer membrane protein and used to specifically amplify a 501 bp and 438 bp fragment.This nested PCR assay was 50 fold more sensitive than that of using PCR external primer only. TheNested PCR has a detection limit as low as 300 pg/?l. Specificity studies showed that nested PCR onlydetected C. burnetii DNA and did not happened Brucella abortus, Escherichia coli, Pseudomonas aeruginosaand Campylobacter Jejuni DNA. Nested PCR has high senstively and specificaly diagnostic method of C.burnetii as agent of Q fever disease.

  14. Randomly amplified polymorphic DNA-polymerase chain reaction ...

    Indian Academy of Sciences (India)

    Unknown

    genetically link to a trait of interest could be used for individual and pedigree identification, pathogenic diag- nostics, and trait improvement in genetics and breeding programmes. Morphological criteria (Bernardi and Talley. 2000), biochemical data (Jensen 2000), isozyme electro- phoresis (Smith et al 1997; Begg et al 1998 ...

  15. Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting

    NARCIS (Netherlands)

    Giesendorf, B A; van Belkum, A; Koeken, A; Stegeman, H; Henkens, M H; van der Plas, J; Goossens, H; Niesters, H G; Quint, W G

    The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial

  16. Xpert MTB/RIF(R), a novel automated polymerase chain reaction-based tool for the diagnosis of tuberculosis

    National Research Council Canada - National Science Library

    Bowles, E.C; Freyee, B; Ingen, J. van; Mulder, B; Boeree, M.J; Soolingen, D. van

    2011-01-01

    There is an urgent need for new point of care tests for tuberculosis (TB). Xpert MTB/RIF(R) is a real-time polymerase chain reaction-based system that detects Mycobacterium tuberculosis DNA and rifampicin...

  17. A simple protocol for the automation of DNA cycle sequencing reactions and polymerase chain reactions.

    Science.gov (United States)

    Civitello, A B; Richards, S; Gibbs, R A

    1992-01-01

    Automated DNA sequencing methods using robotic workstations have been previously reported, however it is often an arduous task to import these technologies into a laboratory. We describe protocols making use of a Beckman Biomek 1000 robotic workstation to prepare polymerase chain reactions (PCRs) and "cycle sequencing" reactions to be performed in a Perkin Elmer Cetus System 9600 thermocycler. The combination of these two instruments allows a high throughput of PCR and DNA sequencing reactions. The programs described are freely available via anonymous file transfer protocol (FTP).

  18. Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

    Science.gov (United States)

    Eychner, Alison M; Lebo, Roberta J; Elkins, Kelly M

    2015-06-01

    We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Identification of Chicken Originated Campylobacter coli and Campylobacter jejuni by Polymerase Chain Reaction (PCR)

    OpenAIRE

    ERTAŞ, Hasan Basri; ÇETİNKAYA, Burhan; MUZ, Adile; ÖNGÖR, Hasan

    2014-01-01

    The purpose of this study was to isolate Campylobacter species from the intestines and livers of chicken and to identify Campylobacter coli and Campylobacter jejuni by both conventional methods and Polymerase Chain Reaction (PCR). Four specific primers derived from the ceuE gene present in the genomes of C. coli and C. jejuni were used for PCR identification. In the examination of 150 intestine and liver samples by culture and PCR, 25 (16.6%) and 32 (21.3%) were identified as C. coli and C...

  20. Rare case of post-cataract-surgery Prevotella endophthalmitis diagnosed by polymerase chain reaction DNA sequencing.

    Science.gov (United States)

    Amissah-Arthur, Kwesi N; Farooq, Tahir A; Dhillon, Navpreet; Cunliffe, Ian A; Bansal, Atul

    2013-03-01

    We report the presentation, diagnosis, and management of endophthalmitis caused by the opportunistic Prevotella species. The case was referred to us following uneventful phacoemulsification and intraocular lens implantation. Accurate identification of this rare cause of endophthalmitis was made using bacterial polymerase chain reaction DNA sequencing. Subsequent prompt modification of antibacterial therapy allowed resolution of signs and symptoms and significant visual recovery. To our knowledge, this is the first reported case of diagnosis and management of post-cataract-surgery endophthalmitis caused by the opportunistic Prevotella species. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  1. An immunohistochemical and polymerase chain reaction evaluation of feline plasmacytic pododermatitis.

    Science.gov (United States)

    Bettenay, S V; Lappin, M R; Mueller, R S

    2007-01-01

    Sections of 14 skin biopsies of cats with plasmacytic pododermatitis and a clinical follow-up of 12-36 months were stained with a polyclonal anti-Mycobacterium bovis (Bacille Calmette-Guerin = BCG) antibody cross-reactive to a broad spectrum of fungi and bacteria. All sections were negative for organisms within the actual footpad tissue with the anti-BCG antibody stains. Polymerase chain reaction (PCR) assays that amplify the DNA of Bartonella spp., Ehrlichia spp., Anaplasma phagocytophilum, Chlamydophila felis, Mycoplasma spp., Toxoplasma gondii, and feline herpesvirus 1 (FHV-1) were applied to tissue digests. DNA of those pathogens assessed was not amplified from tissue.

  2. On-Chip integration of sample pretreatment and Multiplex polymerase chain reaction (PCR) for DNA analysis

    DEFF Research Database (Denmark)

    Brivio, Monica; Snakenborg, Detlef; Søgaard, E.

    2008-01-01

    In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple...... and efficient method for switching the liquid flow between the PT and PCR chamber. Purification of human genomic DNA from EDTA-treated blood and multiplex PCR were successfully carried out on-chip using the developed lab-on-a-chip system....

  3. [Antigenemia and real time polymerase chain reaction for diagnosis of cytomegalovirus disease in HIV infected adults].

    Science.gov (United States)

    Luchsinger, Vivían; Vásquez, Patricia; Silva, Matías; Bruno, M José; Siches, Izkia; Villarroel, Javier; Garmendia, M Luisa; Larrañaga, Carmen

    2015-12-01

    Cytomegalovirus (CMV) infection is frequent in HIV adults. It is unknown usefulness of quantitative methods for diagnosing the CMV disease in Chilean patients. To determine the performance of antigenemia and real time polymerase chain reaction (rtPCR) in the diagnosis of CMV disease in Chilean HIV adults. Detection of CMV by viral isolation (AVR), antigenemia and quantitative rtPCR in HIV adults. The 102 adults with suspected CMV disease had lower LTCD4 count and higher HIV viral load than 77 patients without suspicion (p HIV adults. AVR is inappropriate as a gold standard for its low performance.

  4. Detection of Brucella species in organs of naturally infected cattle by polymerase chain reaction.

    Science.gov (United States)

    Gallien, P; Dorn, C; Alban, G; Staak, C; Protz, D

    1998-05-09

    A polymerase chain reaction (PCR) assay was used to detect Brucella species in the uterus, udder, spleen, lymph nodes, kidney and liver of three cows which had been naturally infected in an outbreak of brucellosis, and the results were compared with the results of bacteriological investigations. All 18 samples reacted positively in the PCR, but five samples had weak bands after the electrophoretic separation of PCR mixtures. No Brucella strains could be detected in these five samples by bacterial cultivation, but all the other samples gave positive results. A pre-enrichment procedure was necessary for the PCR. A PCR with DNA from eight Yersinia strains gave no amplification product.

  5. Development and validation of a Myxoma virus real-time polymerase chain reaction assay.

    Science.gov (United States)

    Albini, Sarah; Sigrist, Brigitte; Güttinger, Regula; Schelling, Claude; Hoop, Richard K; Vögtlin, Andrea

    2012-01-01

    To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.

  6. [Arbitrary primed polymerase chain reaction for the genotypic identification of Streptococcus sanguis group].

    Science.gov (United States)

    Shi, Yu-cai; Zhang, Wei-dong; Chen, Hui

    2003-02-01

    To find out an ideal method used in identification of Streptococcus sanguis group (SSG) strains by arbitrary primed polymerase chain reaction (AP-PCR). AP-PCR was used to distinguish SSG strains by designing 25 bp arbitrary primer 5' AAG AGA GGA GCT AGC TCT TCT TGG A 3'. There were great differences in the main band of DNA polymorphism among SSG species. The similar band can be obtained from the different DNA extractions in the same species. AP-PCR may be useful in the identification and classification of SSG species.

  7. Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method

    OpenAIRE

    Hartung, J S; J.F. Daniel; Pruvost, O.P.

    1993-01-01

    pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and o...

  8. Metode Direct Polymerase Chain Reaction untuk Melacak Campylobacter sp. pada Daging Ayam (DIRECT POLYMERASE CHAIN REACTION METHOD FOR DETECTION CAMPYLOBACTER SP. OF POULTRY MEAT

    Directory of Open Access Journals (Sweden)

    Andriani .

    2013-08-01

    Full Text Available Campylobacter sp. is the most commonly reported as agent of foodborne zoonosis causing acutegastroenteritis in humans. Poultry meat is considered as a major source of C. jejuni infection in human.The conventional methods for detecting foodborne bacteria is time-consuming which rely on the of thebacteria in culture media, followed by biochemical identification. In this study polymerase chain reaction(PCR technique was used for rapid identification of the pathogenic Campylobacter sp. The samples usedwere 298 chicken carcass with sold in supermarkets and traditional markets, and were carried out inaccordance the isolation protocol ISO/ DIS 10272-1994. Identification was performed using biochemicalAPI Campy. The direct PCR (DPCR assay with two sets of primers was employed for isolation andidentification of C. jejuni and C. coli. The result of the isolation and identification both by conventional orPCR methods showed that chicken carcasses both from supermarket and traditional market werecontaminated with C. jejuni and or C. coli. Prevalence of Campylobacter sp. contamination in chicken meatwas higher by DPCR (62.6% than by conventional (19.8%, indicating that DPCR technique was moresensitive than conventional method with detection limit for C. jejuni was103 cfu/ml.

  9. Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction with Parasitological Methods for Detection of Strongyloides stercoralis in Human Fecal Samples

    Science.gov (United States)

    Sharifdini, Meysam; Mirhendi, Hossein; Ashrafi, Keyhan; Hosseini, Mostafa; Mohebali, Mehdi; Khodadadi, Hossein; Kia, Eshrat Beigom

    2015-01-01

    This study was performed to evaluate nested polymerase chain reaction (PCR) and real-time PCR methods for detection of Strongyloides stercoralis in fecal samples compared with parasitological methods. A total of 466 stool samples were examined by conventional parasitological methods (formalin ether concentration [FEC] and agar plate culture [APC]). DNA was extracted using an in-house method, and mitochondrial cytochrome c oxidase subunit 1 and 18S ribosomal genes were amplified by nested PCR and real-time PCR, respectively. Among 466 samples, 12.7% and 18.2% were found infected with S. stercoralis by FEC and APC, respectively. DNA of S. stercoralis was detected in 18.9% and 25.1% of samples by real-time PCR and nested PCR, respectively. Considering parasitological methods as the diagnostic gold standard, the sensitivity and specificity of nested PCR were 100% and 91.6%, respectively, and that of real-time PCR were 84.7% and 95.8%, respectively. However, considering sequence analyzes of the selected nested PCR products, the specificity of nested PCR is increased. In general, molecular methods were superior to parasitological methods. They were more sensitive and more reliable in detection of S. stercoralis in comparison with parasitological methods. Between the two molecular methods, the sensitivity of nested PCR was higher than real-time PCR. PMID:26350449

  10. Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

    Science.gov (United States)

    Yamaguchi, Akemi; Matsuda, Kazuyuki; Sueki, Akane; Taira, Chiaki; Uehara, Masayuki; Saito, Yasunori; Honda, Takayuki

    2015-08-25

    Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Evaluation of immunomagnetic separation for the detection of Salmonella in surface waters by polymerase chain reaction.

    Science.gov (United States)

    Hsu, Chao-Yu; Hsu, Bing-Mu; Chang, Tien-Yu; Hsu, Tsui-Kang; Shen, Shu-Min; Chiu, Yi-Chou; Wang, Hung-Jen; Ji, Wen-Tsai; Fan, Cheng-Wei; Chen, Jyh-Larng

    2014-09-19

    Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments. Instead of the traditional, time-consuming biochemical detection, polymerase chain reaction (PCR) allows rapid identification of Salmonella directly concentrated from water samples. However, prevalence of Salmonella may be underestimated because of the vulnerability of PCR to various environmental chemicals like humic acid, compounded by the fact that various DNA polymerases have different susceptibility to humic acid. Because immunomagnetic separation (IMS) theoretically could isolate Salmonella from other microbes and facilitate removal of aquatic PCR inhibitors of different sizes, this study aims to compare the efficiency of conventional PCR combined with immunomagnetic separation (IMS) for Salmonella detection within a moderately polluted watershed. In our study, the positive rate was increased from 17.6% to 47% with nearly ten-fold improvement in the detection limit. These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters. Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection.

  12. Evaluation of Immunomagnetic Separation for the Detection of Salmonella in Surface Waters by Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Chao-Yu Hsu

    2014-09-01

    Full Text Available Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments. Instead of the traditional, time-consuming biochemical detection, polymerase chain reaction (PCR allows rapid identification of Salmonella directly concentrated from water samples. However, prevalence of Salmonella may be underestimated because of the vulnerability of PCR to various environmental chemicals like humic acid, compounded by the fact that various DNA polymerases have different susceptibility to humic acid. Because immunomagnetic separation (IMS theoretically could isolate Salmonella from other microbes and facilitate removal of aquatic PCR inhibitors of different sizes, this study aims to compare the efficiency of conventional PCR combined with immunomagnetic separation (IMS for Salmonella detection within a moderately polluted watershed. In our study, the positive rate was increased from 17.6% to 47% with nearly ten-fold improvement in the detection limit. These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters. Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection.

  13. Detection of Enterococcus faecalis in Necrotic Teeth Root Canals by Culture and Polymerase Chain Reaction Methods.

    Science.gov (United States)

    Cogulu, Dilsah; Uzel, Atac; Oncag, Ozant; Aksoy, Semiha C; Eronat, Cemal

    2007-10-01

    The aim of this study was to investigate the presence of Enterococcus faecalis in endodontic infections in both deciduous and permanent teeth by culture and polymerase chain reaction (PCR) methods. A total of 145 children aged 5-13 years old were involved in this study. The presence of E. faecalis in necrotic deciduous and permanent teeth root canals was studied using culture and polymerase chain reaction methods. Among 145 molar teeth, 57% (n=83) presented necrotic asymptomatic pulp tissues and were included in this study. Culture and PCR methods detected the test species in 18 and 22 of 83 teeth involved, respectively. E. faecalis was cultured from 8 (18%) of 45 necrotic deciduous teeth and from 10 (26%) of 38 necrotic permanent teeth. PCR detection identified the target species in 10 (22%) and 12 (32%) of necrotic deciduous and permanent teeth respectively. Statistically significant difference in the presence of E. faecalis in deciduous and permanent teeth was found by culture and PCR methods (P=0.03 and 0.02, respectively). The difference in the presence of E. faecalis between two different methods was not statistically significant (P>.05). The results of the present study confirm that both culture and PCR methods are sensitive to detect E. faecalis in root canals.

  14. Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

    Directory of Open Access Journals (Sweden)

    Wen-Pin Chou

    2017-02-01

    Full Text Available Polymerase chain reaction (PCR has been one of the principal techniques of molecular biology and diagnosis for decades. Conventional PCR platforms, which work by rapidly heating and cooling the whole vessel, need complicated hardware designs, and cause energy waste and high cost. On the other hand, partial heating on the various locations of vessels to induce convective solution flows by buoyancy have been used for DNA amplification in recent years. In this research, we develop a new convective PCR platform, capillary loop convective polymerase chain reaction (clcPCR, which can generate one direction flow and make the PCR reaction more stable. The U-shaped loop capillaries with 1.6 mm inner diameter are designed as PCR reagent containers. The clcPCR platform utilizes one isothermal heater for heating the bottom of the loop capillary and a CCD device for detecting real-time amplifying fluorescence signals. The stable flow was generated in the U-shaped container and the amplification process could be finished in 25 min. Our experiments with different initial concentrations of DNA templates demonstrate that clcPCR can be applied for precise quantification. Multiple sample testing and real-time quantification will be achieved in future studies.

  15. Multiplex polymerase chain reaction for detection of Campylobacter from stool specimen.

    Science.gov (United States)

    Sarkar, S R; Ray, N C; Hossain, M A; Paul, S K; Sarkar, S; Kobayashi, N

    2014-07-01

    This was a study to prospectively evaluate the sensitivity and specificity of multiplex polymerase chain reaction (PCR) to identify Campylobacter. Multiplex polymerase chain reaction (PCR) based on cadF, hipO & asp gene for Campylobacter genus, C. jejuni & C. coli were tested for detection of Campylobacter jejuni & C. coli in naturally infected faecal samples of human. All the samples were subjected to the cultural isolation of organism and biochemical characterization. The samples resulted in the amplification of a DNA fragment of size 400 bp, 500 bp &735 bp in PCR assay. Two hundred faecal samples comprising diarrheal stools, 23(11.5%) could be detected by isolation whereas 24(12.0%) were found positive by PCR. All culture positive cases were positive by PCR and among 01 culture negative case, were positive by PCR. PCR was found to be more sensitive for Campylobacter detection in faecal samples 12.0% as relative to culture isolation which could detect the organism in 11.5% samples. Sensitivity and specificity of PCR were 100% and 99.4% respectively taking Culture as gold standard. The results depicted the superior efficacy of PCR for rapid screening of samples owing to its high sensitivity, specificity and automation potential.

  16. Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues.

    Science.gov (United States)

    Meason-Smith, Courtney; Edwards, Erin E; Older, Caitlin E; Branco, Mackenzie; Bryan, Laura K; Lawhon, Sara D; Suchodolski, Jan S; Gomez, Gabriel; Mansell, Joanne; Hoffmann, Aline Rodrigues

    2017-07-01

    Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

  17. ISOLASI Campylobacter DARI KARKAS AYAM MENGGUNAKAN METODE KONVENSIONAL DAN POLYMERASE CHAIN REACTIONS [Isolation of Campylobacter from Poultry Carcasses using Conventional and Polymerase Chain Reaction Methods

    Directory of Open Access Journals (Sweden)

    Surachmi Setiyaningsih2

    2013-06-01

    Full Text Available Campylobacter jejuni and Campylobacter coli are two spesies of Campylobacter sp. frequently found as pathogenic bacteria causing human gastrointestinal infections. Contaminated chicken carcasses have been reported as the source of human campylobacteriosis. In this study, Campylobacter were isolated from chicken carcasses sold in traditional markets and supermarkets. In traditional markets, chicken carcasses are sold without proper packaging or in an open space and stored at room temperature (25-30°C for prolonged period allowing pathogenic bacteria to grow. While at supermarkets, chicken carcasses are openly displayed or enclosed in plastic wrappings and stored in a refrigerator (4-8°C. A total of 298 samples of chicken carcasses from traditional markets and supermarkets in the area of DKI Jakarta, West Java (Bogor and Sukabumi and Central Java (Kudus and Demak were collected. Isolation and identification using conventional and Polymerase Chain Reactions (PCR methods were done to determine the prevalence of C. jejuni and C. coli contamination in poultry. The results showed that chicken carcasses sold in the sampling area, both traditional markets and supermarkets, were contaminated with C. jejuni and C. coli. The contamination rate of Campylobacter sp. in chicken carcasses sold in supermarkets, were 14.1% by conventional methods and 29.5% by PCR. This was higher than those in traditional markets, i.e. 5.7 and 12.1%, respectively. It is also confirmed that the prevalence for contamination of C. jejuni was higher than C. coli in 298 samples, i.e. 16.1% and 3.7% by conventional method and 23.5% and 18.1% by PCR method respectively.

  18. Performance characteristics of nested polymerase chain reaction vs real-time polymerase chain reaction methods for detecting Mycobacterium tuberculosis complex in paraffin-embedded human tissues.

    Science.gov (United States)

    Seo, An Na; Park, Hyo Jin; Lee, Hye Seung; Park, Jung Ok; Chang, Ho Eun; Nam, Kyung Han; Choe, Gheeyoung; Park, Kyoung Un

    2014-09-01

    Nucleic acid amplification tests on formalin-fixed, paraffin-embedded (FFPE) tissue specimens enable Mycobacterium tuberculosis complex (MTB) detection and rapid tuberculosis diagnosis in the absence of microbiologic culture tests. We aimed to evaluate the efficacy of different polymerase chain reaction (PCR) methods for detecting Mycobacterium species in FFPE tissues. We examined 110 FFPE specimens (56 nonmycobacterial cases, 32 MTB, and 22 nontuberculous mycobacteria [NTM] determined by acid-fast bacilli [AFB] culture) to assess five PCR methods: nested PCR (N-PCR) (Seeplex MTB Nested ACE Detection; Seegene, Seoul, South Korea), an in-house real-time PCR (RT-PCR) method, and three commercial RT-PCR methods (AccuPower MTB RT-PCR [Bioneer, Seoul, Korea], artus M tuberculosis TM PCR [Qiagen, Hilden, Germany], and AdvanSure tuberculosis/NTM RT-PCR [LG Life Sciences, Seoul, Korea]). The results of N-PCR, in-house RT-PCR, and AdvanSure RT-PCR correlated well with AFB culture results (concordance rates, 94.3%, 87.5%, and 89.5%, respectively). The sensitivity of N-PCR (87.5%) was higher than that of the RT-PCR methods, although these differences were not statistically significant between N-PCR and the in-house and AdvanSure RT-PCR methods (68.8% and 80.0%, respectively). All the PCR methods had high specificities, ranging from 98.2% to 100%. Only two NTM cases were detected by AdvanSure RT-PCR, implying a very low sensitivity. Well-designed RT-PCR and N-PCR can effectively identify MTB in FFPE specimens. Copyright© by the American Society for Clinical Pathology.

  19. α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

    Directory of Open Access Journals (Sweden)

    Olivier Martínez

    Full Text Available (S(C5', R(P α,β-D- Constrained Nucleic Acids (CNA are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

  20. Diagnosis of Leishmania infantum infection by Polymerase Chain Reaction in wild mammals

    Directory of Open Access Journals (Sweden)

    Mayara C. Lombardi

    2014-12-01

    Full Text Available Visceral leishmaniasis is a chronic infectious disease caused by Leishmania infantum (synonym: Leishmania chagasi and transmitted by the sandfly Lutzomyia longipalpis in Brazil. It is an endemic zoonosis in several regions of the country, including Belo Horizonte (State of Minas Gerais. In urban areas, the domestic dog is susceptible and considered the most important animal reservoir. However, L. infantum has been previously diagnosed in other species, including captive primates and canids. This study aimed to evaluate the presence of the agent DNA in captive animals as well as some free ranging animals from the Zoo-Botanical Foundation of Belo Horizonte by Polymerase Chain Reaction. Eighty one blood samples from primates, carnivores, ruminants, edentates, marsupial, and a monogastric herbivore were analyzed. Three primates Alouatta guariba (brown howler monkey, and two canids Speothos venaticus (bush dog were positive, demonstrating the importance of leishmaniasis control in endemic areas for preservation of wildlife species in captivity.

  1. Detection of Entamoeba histolytica using polymerase chain reaction in pus samples from amebic liver abscess.

    Science.gov (United States)

    Khan, Ujjawini; Mirdha, B R; Samantaray, J C; Sharma, M P

    2006-01-01

    Direct demonstration of Entamoeba histolytica by conventional microscopy and in vitro culture in pus obtained from amebic liver abscess (ALA) is often unsuccessful. We evaluated polymerase chain reaction (PCR) for detection of E. histolytica DNA in such pus. Species-specific primers were used for the amplification of E. histolytica DNA from liver pus obtained from 30 patients with ALA. Patients with pyogenic liver abscess and sterile (autoclaved) pus spiked with Entamoeba dispar and bacteria (Escherichia coli, Klebsiella spp. and Bacteroides spp.) were used as negative controls. PCR was positive in 83% of pus specimens from patients with ALA, and was negative in all 25 pus specimens obtained from pyogenic abscess and autoclaved pus spiked with known bacteria. Sensitivity and specificity of PCR were 83% and 100%, respectively. The overall positivity of PCR was higher compared to serological tests. PCR may be a more reliable and better alternative diagnostic modality for ALA.

  2. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.

    Science.gov (United States)

    Ririe, K M; Rasmussen, R P; Wittwer, C T

    1997-02-15

    A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.

  3. Identification of Campylobacter species isolates with phenotypic methods and polymerase chain reaction.

    Science.gov (United States)

    Miljković-Selimović, Biljana; Babić, Tatjana; Kocić, Branislava; Matkić, Aleksandra; Ristić, Ljiljana

    2014-01-01

    Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the most common bacterial causes of enterocolitis in humans. However, identification of the species level is not always possible using standard biochemical tests. Therefore, the goal of this study was to identify these microorganisms by both phenotyping and polymerase chain reaction (PCR) technique. A total of 153 species of thermophilic campylobacters were examined with standard biochemical tests and PCR technique to prove hipO genes of C. jejuni and asp genes of C. coli. Standard biochemical tests enabled the speciation of 121 strains of C. jejuni, while application of PCR detected 126 C. jejuni strains. PCR technique allowed not only identification of hippurate-positive C. jejuni, but also hipurat-negative strains of C. jejuni which otherwise would be detected as C. coli if only biochemical tests were applied.

  4. Probes and polymerase chain reaction for detection of food-borne bacterial pathogens.

    Science.gov (United States)

    Olsen, J E; Aabo, S; Hill, W; Notermans, S; Wernars, K; Granum, P E; Popovic, T; Rasmussen, H N; Olsvik, O

    1995-11-01

    DNA-hybridization and the polymerase chain reaction (PCR) are techniques commonly used to detect pathogenic bacteria. In this paper, the use of these techniques for detection of Salmonella, E. coli, V. cholerae, non-O1 Vibrio, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and C. botulinum is reviewed with emphasis on application in food microbiology. In food control, DNA-techniques have most often been used in a 'culture confirmation' fashion, i.e. bacteria are enriched and sometimes even purified by traditional culture procedures and thereafter identified by the use of DNA-based methods. The most desirable approach is, however, to detect organisms directly in the food, but major problems remain to be solved before this can be routinely performed.

  5. Evaluation of a real-time polymerase chain reaction assay for the detection of Dientamoeba fragilis.

    Science.gov (United States)

    Calderaro, Adriana; Gorrini, Chiara; Montecchini, Sara; Peruzzi, Simona; Piccolo, Giovanna; Rossi, Sabina; Gargiulo, Franco; Manca, Nino; Dettori, Giuseppe; Chezzi, Carlo

    2010-07-01

    The diagnostic value of a real-time polymerase chain reaction (PCR) assay targeting the 5.8S rDNA of Dientamoeba fragilis was investigated as compared with conventional parasitologic methods including cultivation testing 959 fecal samples from 491 patients attending a tertiary-care hospital and suspected of having an intestinal parasitosis. The real-time PCR assay revealed 117 additional D. fragilis-positive samples as compared with conventional methods, showing 100% sensitivity and specificity in our experience. On the whole, D. fragilis infection was detected in 186 samples from 105 patients (21.4%, third in frequency among the diagnosed intestinal parasitoses). The evaluated real-time PCR assay represents an effective tool to obtain both an accurate diagnosis and a reliable epidemiologic picture of dientamoebiasis.

  6. Detection of gonorrhea among HIV infected patients by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Pragya Poudel

    2015-07-01

    Full Text Available Objective: To determine the burden of gonorrhea among HIV patients using PCR technique. Methods: A cross sectional study was carried out in a HIV clinic of Sparsha Nepal from January to March, 2011. Standard microbiological procedures were followed during collection and processing of urine samples. DNA amplification, isolation and detection were carried out. Gonorrhoea diagnosis was done by multiplex polymerase chain reaction. Results: Among the 119 HIV positive patients, 12 (10.08% were positive for N. gonorrheae. The cases were more among the males than in females. The age of patients ranged from 20- 45 years and the highest prevalence was among the age group 20-35 years. The distribution of gonorrhea infected patients according to marital status revealed that 8 cases were among the married patients. Conclusions: This study revealed that the incidence of gonorrhoea was higher in males and in married people. So, the control measures should be targeted to these groups of people.

  7. Identification of campylobacter species isolates with phenotypic methods and polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Miljković-Selimović Biljana

    2014-01-01

    Full Text Available Introduction. Campylobacter jejuni (C. jejuni and Campylobacter coli (C. coli are the most common bacterial causes of enterocolitis in humans. However, identification of the species level is not always possible using standard biochemical tests. Objective. Therefore, the goal of this study was to identify these microorganisms by both phenotyping and polymerase chain reaction (PCR technique. Methods. A total of 153 species of thermophilic campylobacters were examined with standard biochemical tests and PCR technique to prove hipO genes of C. jejuni and asp genes of C. coli. Results Standard biochemical tests enabled the speciation of 121 strains of C. jejuni, while application of PCR detected 126 C. jejuni strains. Conclusion. PCR technique allowed not only identification of hippurate-positive C. jejuni, but also hipuratnegative strains of C. jejuni which otherwise would be detected as C. coli if only biochemical tests were applied.

  8. Vibrio cholerae Detection in Water and Wastewater by Polymerase Chain Reaction Assay

    Directory of Open Access Journals (Sweden)

    Behnaz Barzamini

    2014-11-01

    Full Text Available Background: Vibrio cholerae is a significant human pathogen worldwide and annually causes some cases of deaths. Contaminated water plays an important role in transmission of this pathogen, which indicates the importance of early diagnosis. Objectives: The current study aimed to perform Polymerase Chain Reaction (PCR on water and wastewater samples to determine the detection limit for Vibrio cholerae. Materials and Methods: PCR was performed on the DNA extracted from Vibrio cholerae of the contaminated water and wastewater using ctxA gene specific primers. The accuracy of PCR method to detect these bacteria was also assessed. Results: The result of PCR performed on the extracted DNA showed a specific 241 base pair band. The limit of bacterial detection for water and wastewater were 40 cfu/mL and 81 cfu/mL, respectively. Conclusions: In the current study, PCR performance using the ctxA gene specific primers to detect Vibrio cholerae was found highly accurate and specific.

  9. The polymerase chain reaction and its application to clinical plastic surgery.

    LENUS (Irish Health Repository)

    Rea, S

    2012-02-03

    Molecular biology has become an essential component in many fields of modern medical research, including plastic surgery. Research into the molecular mechanisms underlying many disease processes offer increased understanding of the pathogenesis of disease and provide exciting therapeutic possibilities. Yet for many clinicians, the presentation of much research into molecular biological processes is couched in confusing terminology and based on scientific techniques, the basis of which are frequently difficult for the clinician to understand. The purpose of this review is to present an introduction to some of the molecular biological techniques currently in use, namely the polymerase chain reaction (PCR) and explore its applications to different aspects of plastic surgery. This review explores the role PCR now plays in all aspects of modern plastic surgery practise, with particular emphasis on normal and abnormal wound healing, the diagnosis of craniofacial anomalies, the diagnosis and treatment of cancer including melanoma and squamous cell carcinoma of the head and neck, and burns.

  10. Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs.

    Science.gov (United States)

    Wang, Mugui; Wei, Chuchu; Ye, Xiufen; Liu, Jianping; Zhang, Cuicui; Chen, Hao; Zhang, Xiaobo; Tu, Jumin

    2015-04-15

    We developed an effective protocol for generating high-purity heteroduplexes via annealing single-stranded DNAs (ssDNAs) derived from plasmid DNA by asymmetric polymerase chain reaction (A-PCR). With the addition of dimethyl sulfoxide, a one-step A-PCR procedure can generate ssDNAs stably at a range of reaction temperatures. Several annealing buffers can anneal two ssDNAs into heteroduplexes effectively. We further developed a simple strategy to create d(GATC) hemimethylated heteroduplexes by annealing fully methylated homoduplexes in the presence of excessive unmethylated ssDNAs. The constructed heteroduplexes have been well tested as substrates for mismatch repair in Escherichia coli and, thus, can be used in various biotechnology applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Midtrimester fetal herpes simplex-2 diagnosis by serology, culture and quantitative polymerase chain reaction.

    Science.gov (United States)

    Curtin, William M; Menegus, Marilyn A; Patru, Maria-Magdalena; Peterson, C Jeanne; Metlay, Leon A; Mooney, Robert A; Stanwood, Nancy L; Scheible, Amy L; Dorgan, Angela

    2013-01-01

    The acquisition of herpes simplex virus (HSV) in utero comprises a minority of neonatal herpes infections. Prenatal diagnosis is rare. We describe a midtrimester diagnosis of fetal HSV-2 infection. Ultrasound at 20 weeks for elevated maternal serum α-fetoprotein (MSAFP) showed lagging fetal growth, echogenic bowel, echogenic myocardium, and liver with a mottled pattern of echogenicity. Amniocentesis demonstrated normal karyotype, elevated AFP and positive acetylcholinesterase. Culture isolated HSV-2 with an aberrant growth pattern. Maternal serology was positive for HSV-2. Quantitative DNA polymerase chain reaction (PCR) showed 59 million copies/ml. Fetal autopsy demonstrated widespread tissue necrosis but only sparse HSV-2 inclusions. Fetal HSV-2 infection can be suspected when an elevated MSAFP accompanies ultrasound findings suggesting perinatal infection. Maternal HSV serology, amniotic fluid culture and quantitative PCR are recommended for diagnostic certainty and counseling. Copyright © 2012 S. Karger AG, Basel.

  12. Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

    Energy Technology Data Exchange (ETDEWEB)

    Frank F. Roberto

    2008-08-01

    Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

  13. Validation study of HPV DNA detection from stained FNA smears by polymerase chain reaction

    DEFF Research Database (Denmark)

    Channir, Hani Ibrahim; Larsen, Christian Grønhøj; Ahlborn, Lise Barlebo

    2016-01-01

    BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) often presents with cystic cervical metastasis and a small primary tumor localized in the palatine tonsils or base of the tongue, which is diagnostically challenging. Testing for HPV DNA in fine......-needle aspiration (FNA) smears from metastases may facilitate a targeted diagnostic workup for identifying the primary tumor. This study was designed to assess the ability to detect HPV DNA in FNA smears with polymerase chain reaction (PCR). METHODS: May-Grünvald-Giemsa (MGG)-stained FNA smears from metastases...... and corresponding surgical specimens were collected from 71 patients with known HPV-positive OPSCC, 12 patients with oral squamous cell carcinoma (OSCC), 20 patients with branchial cleft cysts, and 20 patients with Warthin tumors. Thirty-eight patients with OPSCC and 7 patients with OSCC had FNA smears available...

  14. DNA extraction, Polymerase Chain Reaction, and Sequencing : Workshop in Clinical Genetics

    Directory of Open Access Journals (Sweden)

    Sumadi Lukman Anwar

    2017-02-01

    Full Text Available Abstract DNA extraction, Polymerase Chain Reaction (PCR, and Sequencing are basic methods in molecular biology and genetics. Those there are routinely performed as basic methods in genetic research and currently also for diagnostic lab especially for pathology and human genetics. With the advance in the genetics and clinical service for cancer management, mutation analysis is very important not only for diagnosis but also for prediction of therapeutic response. Detection of KRAS, BRAF, EGFR, and c-KIT mutations is presently performed in almost every molecular pathology lab as part of daily clinical service in cancer management. In this workshop we will discuss tips and tricks for those three basic lab methods. How to improve amount and purity of DNA extraction from blood and tissues, how to avoid DNA degradation during the procedure and storage, how to perform PCR, factors and substance that inhibit polymerases during PCR, how to design effective primer pairs, and how basic theory for sequencing, and interpretation of sequencing will be discussed. Although it has been widely discussed, this workshop is especially important for clinicians who previous do not have hands-on laboratory experience. In addition, number of labs with ability to perform and serve basic genetic and molecular analysis are still limited in Indonesia. With this workshop, we expect to improve knowledge and skill in DNA extraction, PCR, and Sequencing. Keywords : DNA, PCR, sequencing

  15. Neuro-genetic optimization of temperature control for a continuous flow polymerase chain reaction microdevice.

    Science.gov (United States)

    Lee, Hing Wah; Arunasalam, Parthiban; Laratta, William P; Seetharamu, Kankanhalli N; Azid, Ishak A

    2007-08-01

    In this study, a hybridized neuro-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA), is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated reaction zones of 94 degrees C, 65 degrees C, and 72 degrees C for the denaturing, annealing, and extension processes, respectively, to complete a cycle of polymerase chain reaction. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of +/-1 degree C or better, irrespective of changing ambient conditions. Results obtained from the FEA simulation shows good comparison with published experimental work for the temperature control in each reaction zone of the microfluidic channels. The simulation data are then used to train the ANN to predict the temperature distribution of the microfluidic channel for various heater input power and fluid flow rate. Once trained, the ANN analysis is able to predict the temperature distribution in the microchannel in less than 20 min, whereas the FEA simulation takes approximately 7 h to do so. The final optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. Finally, the GA optimized results are used to build a new FEA model for numerical simulation analysis. The simulation results for the neuro-genetic optimized CPCR model and the initial CPCR model are then compared. The neuro-genetic optimized model shows a significant improvement from the initial model, establishing the optimization method's superiority.

  16. Copy number ratios determined by two digital polymerase chain reaction systems in genetically modified grains

    Science.gov (United States)

    Pérez Urquiza, M.; Acatzi Silva, A. I.

    2014-02-01

    Three certified reference materials produced from powdered seeds to measure the copy number ratio sequences of p35S/hmgA in maize containing MON 810 event, p35S/Le1 in soybeans containing GTS 40-3-2 event and DREB1A/acc1 in wheat were produced according to the ISO Guides 34 and 35. In this paper, we report digital polymerase chain reaction (dPCR) protocols, performance parameters and results of copy number ratio content of genetically modified organisms (GMOs) in these materials using two new dPCR systems to detect and quantify molecular deoxyribonucleic acid: the BioMark® (Fluidigm) and the OpenArray® (Life Technologies) systems. These technologies were implemented at the National Institute of Metrology in Mexico (CENAM) and in the Reference Center for GMO Detection from the Ministry of Agriculture (CNRDOGM), respectively. The main advantage of this technique against the more-used quantitative polymerase chain reaction (qPCR) is that it generates an absolute number of target molecules in the sample, without reference to standards or an endogenous control, which is very useful when not much information is available for new developments or there are no standard reference materials in the market as in the wheat case presented, or when it was not possible to test the purity of seeds as in the maize case presented here. Both systems reported enhanced productivity, increased reliability and reduced instrument footprint. In this paper, the performance parameters and uncertainty of measurement obtained with both systems are presented and compared.

  17. Comparison of histopathology and real-time polymerase chain reaction (RT-PCR) for detection of Mycobacterium tuberculosis in fistula-in-ano.

    Science.gov (United States)

    Garg, Pankaj

    2017-07-01

    Histopathology is commonly used to diagnose tuberculosis in fistula-in-ano. The aim was to compare the sensitivity of polymerase chain reaction and histopathology in detecting tuberculosis in fistula-in-ano. The histopathology and polymerase chain-reaction of tissue (fistula tract) was done in all the consecutive operated cases. When pus sample was also available, polymerase chain reaction-pus was also done RESULTS: Three hundred forty seven samples (179 patients) were tested over 2 years (median 6.5 months). The mean age was 38.8 ± 10.7 years, and male/female was 170/9. Histopathology and polymerase chain reaction of tissue (fistula tract) was done in 152 and 165 patients, respectively. Polymerase chain reaction (pus) could be done in 30 patients. Overall, tuberculosis was detected in 20/179 (11.2%) patients. Of these, tuberculosis was detected by histopathology (tissue) in 1/152 (0.7%) and by polymerase chain reaction (tissue) in 14/165 (8.5%) patients. In pus, polymerase chain reaction detected tuberculosis in 6/30 (20%) patients. Both polymerase chain reaction of tissue and pus were positive in one patient. Polymerase chain reaction (tissue) and polymerase chain reaction (pus) were significantly more sensitive than histopathology (tissue) for detecting tuberculosis [histopathology 1/152 vs. polymerase chain reaction (tissue) 14/165, p = 0.0009] [histopathology 1/152 vs. polymerase chain reaction (pus) 6/30, p chain reaction was significantly more sensitive than histopathology in detecting tuberculosis in fistula-in-ano. Histopathology might be missing out tuberculosis in many patients leading to recurrence of the fistula.

  18. The use of polymerase chain reaction for early diagnosis of tuberculosis in Mycobacterium tuberculosis culture

    Directory of Open Access Journals (Sweden)

    M. Chagas

    2010-06-01

    Full Text Available Early diagnosis plays a vital role in controlling tuberculosis. The conventional methodology is slow, with results taking several weeks, in addition to having low sensitivity, especially in clinical paucibacillary samples. The objective of this study was to evaluate the use of polymerase chain reaction (PCR on solid medium culture for a rapid diagnosis of tuberculosis, mainly in cases of negative sputum smears. Forty sputum samples were collected from inpatients with tuberculosis treated for less than 2 days. Bacilloscopy, PCR for sputum, culture on Löwestein-Jensen (LJ solid medium, and daily PCR from culture were performed on each sample. DNA extracted from the BCG vaccine, which contains attenuated bacillus Calmette-Guérin, was used as the positive control. Smear microscopy showed 68.6% sensitivity, 80% specificity, 96% positive predictive value, and 26.7% negative predictive value, with culture on LJ medium as the gold standard. Culture at day 28 showed 74.3% sensitivity and 100% specificity. PCR of DNA extracted from sputum amplified a 1027-bp fragment of the 16s RNA gene, showing 22.9% sensitivity and 60% specificity. PCR performed with DNA extracted from daily culture showed that, from the 17th to the 40th day, the sensitivity (85.7% and specificity (60% were constant. We conclude that a 17-day culture is a good choice for rapid diagnosis and to interfere with the transmission chain of tuberculosis.

  19. Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus.

    Science.gov (United States)

    Nagaraj, Sowmya; Ramlal, Shylaja; Kingston, Joseph; Batra, Harsh Vardhan

    2016-05-13

    Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents entail a cold-chain facility to maintain their stability during storage and transportation. The main aim of the present study was to simplify the utility of a pre-optimized multiplex PCR format that was developed to detect toxigenic strains of Staphylococcus aureus by providing stable, pre-mixed, and ready-to-use master mix in a lyophilized formulation. Master mix containing all reagents except the template was lyophilized in the presence of an excipient lyoprotectant to achieve long-term stability without altering the sensitivity, specificity and PCR performance. Bromophenol blue was also included in the master mix to reduce the risk of external contamination during gel loading. The stability of lyophilized master mix was analyzed at different temperatures. The PCR performance was also examined after exposure of master mix to notable temperature fluctuations during transportation. The shelf-life of lyophilized master mix was estimated to be 1.5 months at ambient temperature and 6 months at 4°C. Stability was unaffected by temperature fluctuations during transportation even in cold-chain-free conditions, thus reducing the cost required for cold storage. The sensitive, cost-effective, ready-to-use, and ambient temperature stable formulation could be implemented as a detection tool in food analysis and diagnostic laboratories and hospitals and for on-field application outside the laboratories, as well as for detection of toxigenic strains of S. aureus. Copyright © 2016. Published by Elsevier B.V.

  20. A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

    Directory of Open Access Journals (Sweden)

    Figueiredo Luiz Tadeu M

    1997-01-01

    Full Text Available We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

  1. Impact of Fungicide Residues on Polymerase Chain Reaction and on Yeast Metabolism

    Directory of Open Access Journals (Sweden)

    Gildo Almeida da Silva

    Full Text Available ABSTRACT The indiscriminate use of pesticides on grape crops is harmful for consumers´ healthin “in natura” consumption and in the ingestion of wine and grape juice. During winemaking, a rapid and efficient fermentation stage is critical to avoid proliferation of contaminating microorganisms and to guarantee the product´s quality. Polymerase chain reaction (PCR has the advantage of detecting these contaminants in the early stages of fermentation. However,this enzymatic reaction may also be susceptible to specific problems, reducing its efficiency. Agricultural practices, such as fungicide treatments, may be a source of PCR inhibiting factors and may also interfere in the normal course of fermentation.The action of the pesticides captan and folpet on PCR and on yeast metabolism was evaluated, once these phthalimide compounds are widely employed in Brazilian vineyards. DNA amplification was only observed at 75 and 37.5 µg/mL of captan concentrations, whereas with folpet, amplification was observed only in the two lowest concentrations tested (42.2 and 21.1µg/mL.Besides the strong inhibition on Taq polymerase activity, phthalimides also inhibited yeast metabolism at all concentrations analyzed.Grape must containing captan and folpet residues could not be transformed into wine due to stuck fermentation caused by the inhibition of yeast metabolism. Non-compliance with the waiting period for phthalimide fungicides may result in financial liabilities to the viticulture sector.The use of yeasts with high fungicide sensitivity should be selected for must fermentation as a strategy for sustainable wine production and to assure that products comply with health and food safety standards.

  2. Experimental and simulation studies of a real-time polymerase chain reaction in the presence of a fullerene derivative

    Energy Technology Data Exchange (ETDEWEB)

    Shang Junjun; Knap, Halina T [Department of Entomology, Soils, and Plant Sciences, Clemson University, Clemson, SC 29634-0315 (United States); Ratnikova, Tatsiana A; Ke, Pu Chun [Department of Physics and Astronomy, COMSET, Clemson University, SC 29634-0978 (United States); Anttalainen, Sini; Salonen, Emppu, E-mail: pcke11@clemson.ed, E-mail: hskrpsk@clemson.ed [Department of Applied Physics, Helsinki University of Technology, FIN-02015 TKK (Finland)

    2009-10-14

    The real-time polymerase chain reaction of the plant gene heat shock transcription factor was fully inhibited in the presence of a fullerene derivative C{sub 60}(OH){sub 20} at a concentration of 4 x 10{sup -4} mM. This inhibition was attributed to the interaction between the nanoparticle and Taq DNA polymerase in the reaction. Atomistic molecular dynamics simulations showed a clear tendency for hydrogen bonding between C{sub 60}(OH){sub 20} and both the dNTPs and ssDNA components of the polymerase chain reaction. These studies facilitate our understanding of the fate of nanoparticles in biomolecular systems, a topic of tremendous importance for addressing the biological and environmental implications of nanomaterials.

  3. Development, validation, and standardization of polymerase chain reaction-based detection of E-coli O157

    DEFF Research Database (Denmark)

    Abdulmawjood, A.; Bulte, M.; Roth, S.

    2004-01-01

    A diagnostic polymerase chain reaction assay was developed for the detection of E. coli O157 as the first part of a multicenter validation and standardization project. The assay is based on amplification of sequences of the rfbE O157 gene and includes an internal amplification control. The select...

  4. Whole Blood Polymerase Chain Reaction in a Neonate with Disseminated Herpes Simplex Virus Infection and Liver Failure

    Directory of Open Access Journals (Sweden)

    Jennifer A. Scoble

    2013-10-01

    Full Text Available A late preterm neonate born by cesarean section with intact membranes presented at 9 days of life with shock and liver failure. Surface cultures were negative but whole blood polymerase chain reaction was positive for herpes simplex virus type 2, underscoring the value of this test in early diagnosis of perinatally acquired disseminated herpes simplex virus infection without skin lesions.

  5. Evaluation of Amplification Targets for the Specific Detection of Bordetella pertussis Using Real-Time Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Mohammad Rubayet Hasan

    2014-01-01

    Full Text Available BACKGROUND: Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.

  6. Pooling of porcine fecal samples for quantification of Lawsonia intracellularis by real-time polymerase chain reaction

    DEFF Research Database (Denmark)

    Pedersen, Ken Steen; Johansen, Markku; Jorsal, Sven Erik Lind

    2014-01-01

    obtained by averaging test results from individual fecal samples in relation to a quantitative polymerase chain reaction (qPCR) test for Lawsonia intracellularis. Ten diarrheic and 10 normal fecal samples were submitted from each of 43 Danish swine herds (n = 860 fecal samples). Pools (n = 43), each...

  7. Quantitation of RHD by real-time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism

    DEFF Research Database (Denmark)

    Krog, Grethe Risum; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

    2007-01-01

    Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage...

  8. Diagnosis of ventricular drainage-related bacterial meningitis by broad-range real-time polymerase chain reaction

    DEFF Research Database (Denmark)

    Deutch, Susanna; Dahlberg, Daniel; Hedegaard, Jesper

    2007-01-01

    OBJECTIVE: To compare a broad-range real-time polymerase chain reaction (PCR) diagnostic strategy with culture to evaluate additional effects on the etiological diagnosis and the quantification of the bacterial load during the course of ventricular drainage-related bacterial meningitis (VR...

  9. Testing vaccines in human experimental malaria: statistical analysis of parasitemia measured by a quantitative real-time polymerase chain reaction.

    NARCIS (Netherlands)

    Hermsen, C.C.; Vlas, S.J. de; Gemert, G.J.A. van; Telgt, D.S.C.; Verhage, D.F.; Sauerwein, R.W.

    2004-01-01

    Clinical trials are an essential step in evaluation of safety and efficacy of malaria vaccines, and human experimental malaria infections have been used for evaluation of protective immunity of Plasmodium falciparum malaria. In this study, a quantitative real-time polymerase chain reaction was used

  10. Molecular detection of the carriage rate of four intestinal protozoa with real-time polymerase chain reaction

    DEFF Research Database (Denmark)

    Efunshile, Michael A; Ngwu, Bethrand A F; Kurtzhals, Jørgen A L

    2015-01-01

    -Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia...

  11. Streptococcus mutans and Streptococcus sobrinus detection by Polymerase Chain Reaction and their relation to dental caries in 12 and 15 year-old schoolchildren in Valencia (Spain)

    OpenAIRE

    Sánchez Acedo, Mateo; Montiel Company, José María; Dasí Fernández, Francisco; Almerich Silla, José Manuel

    2013-01-01

    A cross-sectional study was carried out to determine the prevalence of Streptococcus mutans and Streptococcus sobrinus and the association of the two in a random sample (n=614) of the child population of the region of Valencia (Spain). Saliva samples were analyzed by the quantitative polymerase chain reaction (PCR) method to study the relation of these bacteria to caries prevalence and the DMFT index. The prevalence of S. mutans was 35.4% at age 12 and 22.9% at age 15, that of S. sobrinus 18....

  12. A novel method for detecting polymerase chain reaction product utilizing the 5 prime yields 3 prime exonuclease activity of Thermus aquaticus DNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Holland, P.M.; Watson, R.; Abramson, R.D.; Gelfand, D.H. (Cetus Corp., Emeryville, CA (United States))

    1991-03-11

    The polymerase chain reaction (PCR) method of DNA amplification is a powerful and sensitive technique which has been greatly simplified by the use of the thermostable enzyme Thermus aquaticus (Taq) DNA polymerase. In addition to its polymerase activity, Taq DNA polymerase also has a 5{prime}{yields}3{prime} exonuclease activity. Utilizing this activity, the authors have developed a PCR detection method which generates signal simultaneously with target sequence amplification. No additional probing, blotting or hybridization assays of amplified product are necessary. A 5{prime}{yields}3{prime} exonuclease cleaves 5{prime} terminal nucleotides of nicked double stranded DNA. The authors have generated a substrate suitable for exonuclease activity in a PCR assay by the addition of a labeled oligonucleotide probe designed to hybridize within the target sequence. During amplification, the 5{prime}{yields}3{prime} exonuclease activity of Taq DNA polymerase degrades the probe into smaller fragments which can be differentiated from undegraded probe. The presence of probe does not influence PCR product formation. Hydrolysis occurs only when probe is bound specifically to template. The presence of high complexity DNA in the PCR mixture does not compromise the specificity of probe interaction. In a PCR assay, the amount of detectable label may be modified by altering cycle number, target copy number or probe concentration. The size of labeled fragments may also be modified by varying base composition of the probe. This detection method is advantageous over present day procedures in that it is neither labor intensive nor requires significant skills. The minimal sample handling could reduce the risk of sample contamination, thereby increasing accuracy.

  13. Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis

    Directory of Open Access Journals (Sweden)

    Maria Cristina Nishiwaki-Dantas

    2011-01-01

    Full Text Available OBJECTIVES: To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibodyassay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detectingChlamydia trachomatis in these conditions. METHODS: Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: avernal keratoconjunctivitis group (group A and a control group (group B. The polymerase chain reaction and adirect fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves,and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curveswere plotted using a categorical variable with only two possible outcomes (positive and negative. RESULTS: Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. Allpatients in group A with positive polymerase chain reactions also presented with positive direct fluorescentantibody assays. CONCLUSION: The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection wasconfirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and bypositive polymerase chain reactions in 20% of these patients. The direct fluorescent antibody assay detectedChlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although thediagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients.Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggestconsidering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergicdisease.

  14. Accuracy of the serological ELISA test compared with the polymerase chain reaction for the diagnosis of cytomegalovirus infection in pregnancy

    Directory of Open Access Journals (Sweden)

    Silvana Varella Parmigiani

    Full Text Available CONTEXT: The most frequently used methods for detecting antibodies are the indirect immunofluorescence test and the enzymatic immunoassay (ELISA. The polymerase chain reaction is a molecular biology technique in which the production of large amounts of specific DNA fragments is induced from very low concentrations of complex substrates aloowing the detection of very low amounts of viral particles. OBJECTIVE: To assess the accuracy of serological/ELISA tests in comparison with the polymerase chain reaction in maternal blood to diagnose cytomegalovirus infection. DESIGN: A descriptive study was performed. SETTING: High-risk outpatient clinic of Campinas University (Unicamp. PARTICIPANTS: We selected 243 pregnant women. All of them had been indicated for blood sampling because of suspicions of cytomegalovirus infection and also because of other infections. MAIN MEASUREMENTS: The group was tested for cytomegalovirus. Serological tests were run and compared to the polymerase chain reaction, which was considered to be the gold standard. Status analyses were done using Fisher's exact test, via the SAS software. RESULTS: The previous cytomegalovirus infection rate was 94.6%. The main reasons for inclusion in the study were fetal nervous system malformation (25.5%, maternal toxoplasmosis (25.5% and Rh isoimmunization (14.8%. Only two women were included because of positive serological immunoglobulin M test for cytomegalovirus. The sensitivity and specificity of the serological tests were 94% and 6% for immunoglobulin G. CONCLUSION: Serological tests had lower sensitivity in comparison with the polymerase chain reaction test when diagnosing cytomegalovirus infection. The consequences of positive polymerase chain reaction and negative immunoglobulin M in women remain unknown.

  15. Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis.

    Science.gov (United States)

    Nishiwaki-Dantas, Maria Cristina; de Abreu, Mariza Toledo; de Melo, Cynthia Mendonça; Romero, Ivana Lopes; Neto, Rubens Belfort Matos; Dantas, Paulo Elias Correa

    2011-01-01

    To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibody assay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detecting Chlamydia trachomatis in these conditions. Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: a vernal keratoconjunctivitis group (group A) and a control group (group B). The polymerase chain reaction and a direct fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves, and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curves were plotted using a categorical variable with only two possible outcomes (positive and negative). Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. All patients in group A with positive polymerase chain reactions also presented with positive direct fluorescent antibody assays. The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection was confirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and by positive polymerase chain reactions in 20% of these patients. The direct fluorescent antibody assay detected Chlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although the diagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients. Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggest considering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergic disease.

  16. Detection of clarithromycin-resistant Helicobacter pylori by polymerase chain reaction using residual samples from rapid urease test

    Directory of Open Access Journals (Sweden)

    Jae-Sik Jeon

    2017-01-01

    Full Text Available Background: Approximately 50% of the world population is infected with Helicobacter pylori, which corresponds to a high infection rate. Furthermore, the incidence of antibiotic-resistant H. pylori has increased with the recent rise in use of antibiotics for H. pylori elimination, suggesting growing treatment failures. Aim: The study was aimed to assess the use of residual samples from rapid urease test (RUT for biomolecular testing as an effective and accurate method to detect antibiotic-resistant H. pylori. Settings and Design: This study was a retrospective study performed using data obtained from medical records of previously isolated H. pylori strains. Materials and Methods: RUT was conducted for 5440 biopsy samples from individuals who underwent health examination in South Korea. Subsequently, 469 RUT residual samples were randomly selected and subjected to polymerase chain reaction (PCR to detect antibiotic-resistant H. pylori. Statistical Analysis Used: The Chi-square test was used to analyse categorical data. P < 0.05 was considered statistically significant. Results: The results showed a concordance between the results of PCR and conventional RUT in 450 of 469 samples, suggesting that the H. pylori PCR test is a time- and cost-effective detection method. Conclusions: This study demonstrated that PCR test can aid physicians to prescribe the appropriate antibiotics at the time of diagnosis, thus preventing the reduction in H. pylori eradication due to antibiotic resistance, averting progression to serious diseases and increasing the treatment success rate.

  17. Nested polymerase chain reaction technique for the detection of Gpc3 and Afp mRNA in liver cancer micrometastases.

    Science.gov (United States)

    Luo, J; Yang, K; Wen, Y G

    2017-02-08

    The incidence of liver cancer has gradually risen to a high level in China, and tumor metastasis occurs via multiple pathways. Alpha fetal protein (AFP) is the main biomarker of liver cancer micrometastases. A recent study showed that glypican-3 (GPC3), which is abundant in hepatoma cells, has promising specificity and could be used to determine the presence of malignant cells. The nested polymerase chain reaction (PCR) technique is superior in experimental sensitivity. Using rat models of liver cancer in the current study, we utilized nested PCR to detect Gpc3 and Afp mRNA to determine their relationship with liver cancer micrometastases. The aim was to provide an experimental basis for clinical examination. We randomly assigned male Sprague-Dawley rats to sham and experimental groups. The experimental group constituted a liver cancer model induced by diethylnitrosamine, whereas the sham group was administered with an equivalent volume of normal saline. Gpc3 and Afp mRNA was detected using nested PCR. Analysis was performed to determine statistical significance. Compared with the sham group, the rates of occurrence of Gpc3 and Afp mRNA were significantly higher in the experimental group (P Afp mRNA increased significantly in the four subgroups of the experimental group (P Afp mRNA in liver cancer micrometastases.

  18. Automated Microfluidic Platform for Serial Polymerase Chain Reaction and High-Resolution Melting Analysis.

    Science.gov (United States)

    Cao, Weidong; Bean, Brian; Corey, Scott; Coursey, Johnathan S; Hasson, Kenton C; Inoue, Hiroshi; Isano, Taisuke; Kanderian, Sami; Lane, Ben; Liang, Hongye; Murphy, Brian; Owen, Greg; Shinoda, Nobuhiko; Zeng, Shulin; Knight, Ivor T

    2016-06-01

    We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing. © 2015 Society for Laboratory Automation and Screening.

  19. Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

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    Márcia Andreia Barge Loução Terra

    2004-03-01

    Full Text Available Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations.Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG, or infected with tachyzoites (RH strain, the acute infection group (AIG. Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.

  20. Detection of Brucella sp. and Leptospira sp. in dogs using conventional polymerase chain reaction

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    Khamesipour Faham

    2014-12-01

    Full Text Available The study was conducted to detect Brucella sp. and Leptospira sp. in blood samples of dogs in Isfahan and Shahrekord province in Iran. A total of 94 blood samples were collected from dogs of different breed, age, sex, and dogs’ type (stray or nonstray. The samples were examined using conventional polymerase chain reaction (PCR. Fourteen (14.89% dogs were positive for Brucella sp. and 18 (19.15%. dogs for Leptospira sp. There were no significant differences between the prevalence of the pathogens, provinces, sex, and age groups (P > 0.05. However, there was a statistically significant difference in prevalence of Brucella sp. and Leptospira sp. between stray and non-stray dogs (P < 0.0001; χ2 = 30.3767. The study also demonstrated that PCR was successfully used for the first time in Iran for the detection of Brucella sp. and Leptospira sp. in blood samples of dogs. Therefore, we recommend the PCR as a supplementary method with other commonly recognised methods (e.g. serological methods for the diagnosis of subclinical infections with the microorganisms. Strict measures for the control of stray dogs are also highly recommended.

  1. Detection and differentiation of Aino and Akabane Simbu serogroup bunyaviruses by nested polymerase chain reaction.

    Science.gov (United States)

    Akashi, H; Onuma, S; Nagano, H; Ohta, M; Fukutomi, T

    1999-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR were developed to detect and differentiate Aino (AINO) and Akabane (AKA) virus S RNA. Two pairs of AINO- and AKA-specific primers for nested PCR were synthesized and examined for their capacity to amplify PCR products using 7 Simbu serogroup viruses isolated in Japan and Australia. RT- and nested PCR using AKA-specific primers amplified cDNA from Tinaroo virus RNA as well as homologous RNA. Nested PCR products were differentiated by Hph I and Bst EII digestion. Peaton (PEA) virus S cDNA was also amplified using AINO- specific primers. The nested PCR products of PEA virus were not digested by 3 restriction enzymes (Ava II, Eco RI and Hae II), whereras those of AINO virus were digested as expected. Using this technique, AINO and AKA viruses were detected at concentrations as low as 10(-3) plaque-forming units (PFU) and 10(-5) PFU, respectively, in a supernatant of virus-infected cells. It was possible to detect AINO and AKA genome from various tissues of experimentally infected mice, and also the AKA nested PCR products from serum samples from sentinel cattle naturally infected with AKA virus. The present nested PCR appears a simple, rapid and valuable method for diagnosing AINO and AKA infection.

  2. Simultaneous detection of bovine arboviruses using single-tube multiplex reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Ohashi, Seiichi; Yoshida, Kazuo; Yanase, Tohru; Kato, Tomoko; Tsuda, Tomoyuki

    2004-09-01

    Single-tube multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed to detect and identify arboviruses in infected cell-culture fluids and field specimens. The technique was equally sensitive for detecting five different viruses in cell cultures, namely the Chuzan, Ibaraki, and Bluetongue viruses belonging to Orbivirus, and the Akabane virus and Peaton virus belonging to Orthobunyavirus, and was less sensitive than former viruses for detecting Aino virus belonging to Orthobunyavirus. The mRT-PCR reliably detected 0.6-10(3.1) median tissue culture infective doses. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. The technique was as sensitive as virus isolation in detecting single infected plasma in five plasmas from sentinel cattle and in detecting two infectious homogenates in eight homogenates of Culicoides biting midges. The mRT-PCR may be a sensitive and rapid assay for surveillance of bovine arboviruses in field specimens.

  3. Polymerase chain reaction detection of circulating tumour cells. EORTC Melanoma Cooperative Group, Immunotherapy Subgroup.

    Science.gov (United States)

    Keilholz, U; Willhauck, M; Scheibenbogen, C; de Vries, T J; Burchill, S

    1997-08-01

    Reverse transcriptase polymerase chain reaction (RT-PCR)-based assays to detect occult neoplastic cells offer the highest sensitivity for the study of tumour dissemination and minimal residual disease. The detection of small numbers of tumour cells in a clinical sample may result in a redefinition of what constitutes residual disease and relapse, affecting future patient management. However, there remains disparity in the published data on the clinical value of RT-PCR for the detection of circulating tumour cells. This most likely reflects differences in the methods for sample preparation, RNA extraction, and cDNA synthesis among laboratories. Consequently the need for implementation of standard quality control measures is pressing in order to facilitate meaningful assessment of the methodology and it's clinical value. A 2-day workshop organized by the immunotherapy subgroup of the EORTC Melanoma Cooperative Group was held on this topic at the Ludwig Institute in Epalinges-sur-Lausanne, Switzerland in January 1996, with Stefan Carrel as the local host. Many pertinent issues were discussed in great detail, covering every step from sample handling to quality control. This workshop resulted in a concerted action leading to the preparation of laboratory guidelines, which are summarized in this review.

  4. Characterization of fecal microbiota across seven Chinese ethnic groups by quantitative polymerase chain reaction.

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    Lai-yu Kwok

    Full Text Available The human gut microbiota consists of complex microbial communities, which possibly play crucial roles in physiological functioning and health maintenance. China has evolved into a multicultural society consisting of the major ethnic group, Han, and 55 official ethnic minority groups. Nowadays, these minority groups inhabit in different Chinese provinces and some of them still keep their unique culture and lifestyle. Currently, only limited data are available on the gut microbiota of these Chinese ethnic groups. In this study, 10 major fecal bacterial groups of 314 healthy individuals from 7 Chinese ethnic origins were enumerated by quantitative polymerase chain reaction. Our data confirmed that the selected bacterial groups were common to all 7 surveyed ethnicities, but the amount of the individual bacterial groups varied to different degree. By principal component and canonical variate analyses of the 314 individuals or the 91 Han subjects, no distinct group clustering pattern was observed. Nevertheless, weak differences were noted between the Han and Zhuang from other ethnic minority groups, and between the Heilongjiang Hans from those of the other provinces. Thus, our results suggest that the ethnic origin may contribute to shaping the human gut microbiota.

  5. A new reverse transcription-polymerase chain reaction method for accurate quantification

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    Shiao Yih-Horng

    2003-12-01

    Full Text Available Abstract Background Reverse transcription-polymerase chain reaction (RT-PCR is a very sensitive technique to measure and to compare mRNA levels among samples. However, it is extremely difficult to maintain linearity across the entire procedure, especially at the step of PCR amplification. Specific genes have been used as baseline controls to be co-amplified with target genes to normalize the amplification efficiency, but development or selection of reliable controls itself has created a new challenge. Results Here, we describe a new quantitative RT-PCR to compare two mRNA samples directly without the requirement of synthetic control DNAs for reference. First, chimeric RT primers carrying gene-specific and universal PCR priming sequences with or without a linker for size distinction were utilized to generate cDNAs. The size-different cDNAs were then combined in a single reaction for PCR amplification using the same primer set. The two amplified products were resolved and detected with gel electrophoresis and fluorescence imaging. Relative abundance of the two products was obtained after a baseline correction. Conclusion This methodology is simple and accurate as indicated by equal amplification efficiency throughout PCR cycling. It is also easily implemented for many existing protocols. In addition, parameters affecting RT linearity are characterized in this report.

  6. Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction

    Science.gov (United States)

    OH, In Young; KIM, Kyung Tae; SUNG, Ho Joong

    2017-01-01

    Background: Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR). Methods: The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI) genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing. Results: Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously. Conclusion: This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection. PMID:28979354

  7. Toxin genotyping of Clostridium perfringens strains using a polymerase chain reaction protocol

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    Elisabetta Di Giannatale

    2010-03-01

    Full Text Available A polymerase chain reaction protocol consisting of a multiplex to identify the cpa, cpb1, cpetx, cpi genes and a duplex to identify the cpe and cpb2 genes encoding for a, b1, e, i, enterotoxin and b2 toxins, respectively, was applied to DNA extracted from two collections of Clostridium perfringens strains. The first collection involved 19 isolates from rabbits. The second collection of 41 isolates came from routine necropsies. The cpa gene alone, or in association with the cpb2 gene, was detected in all DNA samples examined. The cpa gene, together with cpb2 gene, were detected in seven of the rabbit C. perfringens strains (36.8% and in nine isolates from necropsies (21.9%. The cpa gene was found in 63.2% of rabbit strains and 76.9% of strains from other animal species. In rabbits, the pathological lesions associated with C. perfringens detection were predominantly forms of non-inflammatory enteropathies. In other species, C. perfringens was mainly associated with congestive-haemorrhagic enteropathy, but also with fatal traumatic lesions, degenerative diseases and organs with post-mortem autolysis. No clear correlation was observed between detection of b2 toxin gene and species-specific pathological features.

  8. Identification of aflatoxigenic fungi using polymerase chain reaction-based assay

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    Šošo Vladislava M.

    2014-01-01

    Full Text Available As the aflatoxins represent a health-risk for humans because of their proven carcinogenicity, food-borne fungi that produce them as secondary metabolites, mainly Aspergillus flavus and Aspergillus parasiticus, have to be isolated and identified. The best argument for identifying problem fungi is that it indicates control points within the food system as part of a hazard analysis critical control point (HACCP approach. This assumes there is a close link between fungus and toxin. Conventional methods for isolation and identification of fungi are time consuming and require admirably dedicated taxonomists. Hence, it is imperative to develop methodologies that are relatively rapid, highly specific and as an alternative to the existing methods. The polymerase chain reaction (PCR facilitates the in vitro amplification of the target sequence. The main advantages of PCR is that organisms need not be cultured, at least not for a long time, prior to their detection, target DNA can be detected even in a complex mixture, no radioactive probes are required, it is rapid, sensitive and highly versatile. The gene afl-2 has been isolated and shown to regulate aflatoxin biosynthesis in A. flavus. Also, the PCR reaction was targeted against aflatoxin synthesis regulatory gene (aflR1 since these genes are nearly identical in A. flavus and A. parasiticus in order to indicate the possibility of detection of both the species with the same PCR system (primers/reaction. [Projekat Ministarstva nauke Republike Srbije, br. III46009

  9. Diagnosis of whooping cough in Switzerland: differentiating Bordetella pertussis from Bordetella holmesii by polymerase chain reaction.

    Science.gov (United States)

    Pittet, Laure F; Emonet, Stéphane; François, Patrice; Bonetti, Eve-Julie; Schrenzel, Jacques; Hug, Melanie; Altwegg, Martin; Siegrist, Claire-Anne; Posfay-Barbe, Klara M

    2014-01-01

    Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR). In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old), formerly diagnosed as Bordetella pertussis (IS481+). No B. holmesii (IS481+, IS1001-, hIS1001+) was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested.

  10. Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens.

    Science.gov (United States)

    Furini, Adriana Antônia da Cruz; Pedro, Heloisa da Silveira Paro; Rodrigues, Jean Francisco; Montenegro, Lilian Maria Lapa; Machado, Ricardo Luiz Dantas; Franco, Célia; Schindler, Haiana Charifker; Batista, Ida Maria Foschiani Dias; Rossit, Andrea Regina Baptista

    2013-01-01

    To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis.

  11. Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics.

    Science.gov (United States)

    Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

    2014-02-20

    Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

  12. Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction.

    Science.gov (United States)

    Noda, Naohiro; Tani, Hidenori; Morita, Nao; Kurata, Shinya; Nakamura, Kazunori; Kanagawa, Takahiro; Tsuneda, Satoshi; Sekiguchi, Yuji

    2008-02-11

    Estimation of single-nucleotide polymorphism (SNP) allele frequency in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present a simple, accurate, and cost-effective method for estimating SNP allele frequency, called alternately binding probe (ABProbe) competitive polymerase chain reaction (ABC-PCR) that entails no expensive devices for real-time fluorescence measurement and complex post-PCR steps. We prepared DNA pools of PCR products derived from homozygous samples of three different SNPs (ALDH2, GNB3, and HTR2A) in different portions, and the allele frequencies of these samples were estimated by ABC-PCR. Two alleles were coamplified by PCR with a fluorescent probe that binds to either alleles, and then fluorescence intensity was measured using a simple fluorometer. The ratio of the two alleles was calculated from the fluorescence intensity of the probe at the end-point. The estimated allele frequencies strongly correlated to the expected ratios for all three SNPs with high accuracy. When the allele frequencies were more than 5%, the relative standard deviations (R.S.D.s) of ABC-PCR were less than 20%. Moreover, we also confirmed that this method was applicable to SNP genotyping.

  13. Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

    Science.gov (United States)

    Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

    2014-02-01

    Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

  14. Trends and advances in food analysis by real-time polymerase chain reaction.

    Science.gov (United States)

    Salihah, Nur Thaqifah; Hossain, Mohammad Mosharraf; Lubis, Hamadah; Ahmed, Minhaz Uddin

    2016-05-01

    Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling.

  15. Fabrication of Polymerase Chain Reaction Plastic Lab-on-a-Chip Device for Rapid Molecular Diagnoses.

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    Trinh, Kieu The Loan; Zhang, Hainan; Kang, Dong-Jin; Kahng, Sung-Hyun; Tall, Ben D; Lee, Nae Yoon

    2016-05-01

    We aim to fabricate a thermoplastic poly(methylmethacrylate) (PMMA) Lab-on-a-Chip device to perform continuous- flow polymerase chain reactions (PCRs) for rapid molecular detection of foodborne pathogen bacteria. A miniaturized plastic device was fabricated by utilizing PMMA substrates mediated by poly(dimethylsiloxane) interfacial coating, enabling bonding under mild conditions, and thus avoiding the deformation or collapse of microchannels. Surface characterizations were carried out and bond strength was measured. The feasibility of the Lab-on-a-Chip device for performing on-chip PCR utilizing a lab-made, portable dual heater was evaluated. The results were compared with those obtained using a commercially available thermal cycler. A PMMA Lab-on-a-Chip device was designed and fabricated for conducting PCR using foodborne pathogens as sample targets. A robust bond was established between the PMMA substrates, which is essential for performing miniaturized PCR on plastic. The feasibility of on-chip PCR was evaluated using Escherichia coli O157:H7 and Cronobacter condimenti, two worldwide foodborne pathogens, and the target amplicons were successfully amplified within 25 minutes. In this study, we present a novel design of a low-cost and high-throughput thermoplastic PMMA Lab-on-a-Chip device for conducting microscale PCR, and we enable rapid molecular diagnoses of two important foodborne pathogens in minute resolution using this device. In this regard, the introduced highly portable system design has the potential to enable PCR investigations of many diseases quickly and accurately.

  16. An improved electrochemiluminescence polymerase chain reaction method for highly sensitive detection of plant viruses

    Energy Technology Data Exchange (ETDEWEB)

    Tang Yabing [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China); Xing Da [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China)]. E-mail: xingda@scnu.edu.cn; Zhu Debin [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China); Liu Jinfeng [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China)

    2007-01-23

    Recently, we have reported an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection of genetically modified organisms. The ECL-PCR method was further improved in the current study by introducing a multi-purpose nucleic acid sequence that was specific to the tris(bipyridine) ruthenium (TBR) labeled probe, into the 5' terminal of the primers. The method was applied to detect plant viruses. Conserved sequence of the plant viruses was amplified by PCR. The product was hybridized with a biotin labeled probe and a TBR labeled probe. The hybridization product was separated by streptavidin-coated magnetic beads, and detected by measuring the ECL signals of the TBR labeled. Under the optimized conditions, the experiment results show that the detection limit is 50 fmol of PCR products, and the signal-to-noise ratio is in excess of 14.6. The method was used to detect banana streak virus, banana bunchy top virus, and papaya leaf curl virus. The experiment results show that this method could reliably identity viruses infected plant samples. The improved ECL-PCR approach has higher sensitivity and lower cost than previous approach. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.

  17. Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum

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    Ching Giap Tan

    2014-02-01

    Full Text Available The present study was based on the reverse transcription polymerase chain reaction (RT-PCR of the 16S ribosomal nucleic acid (rRNA of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20–25 h at 37 °C, 22–25 h at 16 °C, and 23–27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h. The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.

  18. Development and performance of a microwell-plate-based polymerase chain reaction assay for Mycoplasma genitalium.

    Science.gov (United States)

    Dutro, Susan M; Hebb, Jennifer K; Garin, Cresley A; Hughes, James P; Kenny, George E; Totten, Patricia A

    2003-10-01

    Mycoplasma genitalium is associated with, and could be the cause of, idiopathic cases of urethritis, endometritis, and cervicitis. Further epidemiologic studies on this organism are needed, but currently used polymerase chain reaction (PCR) assays are labor-intensive and culture is insensitive. The goal was to develop and evaluate a microwell-plate-based PCR assay for M. genitalium. We adapted an M. genitalium PCR assay targeting the MgPa gene to a 96-microwell plate format with colorimetric detection of PCR products and incorporation of an internal inhibition control to determine the limit of detection of this assay (termed MgPa-IMW) for M. genitalium DNA and evaluate its performance on cervical and male urine specimens. The MgPa-IMW PCR assay detected 1 and 17 genome copies of M. genitalium (with 27% and 95% confidence) and was able to detect specimens inhibited for amplification. This assay was 100% concordant (50 positive and 50 negative) with the Southern-blot-based PCR assay with cervical specimens. Similarly, this test was 89% concordant with the Southern-blot-based assay for 64 male urine specimens (25 positive, 32 negative, 7 discordant), 97% concordant after correcting for specimens no longer positive by the Southern blot-based assay after freezer storage. The MgPa-IMW assay is sensitive and specific for the detection of M. genitalium in patient specimens and should facilitate large-scale screening for this organism.

  19. A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance

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    Wang Xiaowei

    2008-12-01

    Full Text Available Abstract Background Quantitative polymerase chain reaction (QPCR is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile. Results We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method. Conclusion We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays.

  20. Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

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    Richard Atherton

    2013-06-01

    Full Text Available The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61% were classified as assemblage B (26 as BIII and 16 as BIV, 22 (32% as assemblage A (3 as AI and 19 as AII and five (7% as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

  1. Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis.

    Science.gov (United States)

    Clavijo, Maria J; Oliveira, Simone; Zimmerman, Jeffrey; Rendahl, Aaron; Rovira, Albert

    2014-11-01

    Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy. © 2014 The Author(s).

  2. Detecting Malaria Hotspots: A Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction.

    Science.gov (United States)

    Mogeni, Polycarp; Williams, Thomas N; Omedo, Irene; Kimani, Domtila; Ngoi, Joyce M; Mwacharo, Jedida; Morter, Richard; Nyundo, Christopher; Wambua, Juliana; Nyangweso, George; Kapulu, Melissa; Fegan, Gregory; Bejon, Philip

    2017-11-27

    Malaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used. We conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic. Eight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT. Malaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls.

  3. Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

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    Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung [College of Pharmacy, Seoul National University, Seoul 151-742 (Korea, Republic of); Han, Myun Soo; Kim, Jong Jin [Forensic DNA Center, National Forensic Service, Seoul 158-707 (Korea, Republic of); Kim, Wook [Department of Biological Sciences, Dankook University, Cheonan 330-714 (Korea, Republic of); Lee, Jong Eun [DNA Link, VICHEN CITY Building 3F, Seoul 121-850 (Korea, Republic of); Song, Joon Myong, E-mail: jmsong@snu.ac.kr [College of Pharmacy, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. Black-Right-Pointing-Pointer This probe provided PCR-free determination of human genomic DNA. Black-Right-Pointing-Pointer Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. Black-Right-Pointing-Pointer Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

  4. Survey of pathogenic Leptospira in rats by polymerase chain reaction in Sisaket Province.

    Science.gov (United States)

    Jittimanee, Jutharat; Wongbutdee, Jaruwan

    2014-04-01

    Leptospirosis, a zoonotic disease caused by Leptospira, has been a health problem in Thailand for several years. It can be transmitted to humans via rats' urine, which may contaminate the environment. The prevalence rate of Leptospira infection in rats may result in the spread of leptospirosis in humans. The present study aimed to determine the prevalence rate of Leptospira infection in a total of 87 rats from areas where patients with leptospirosis had been reported compared to areas with no reports of leptospirosis in Sisaket Province. DNA samples were isolated from rats' kidneys. The polymerase chain reaction (PCR) technique was used for the detection of 16s rRNA and LipL32 genes specific to genus and pathogenic Leptospira, respectively. In areas where patients with leptospirosis had been reported, 8.7% (4/46) of rats were infected with pathogenic Leptospira; no infected rats were found in non-endemic areas. This indicated the prevalence rate of Leptospira infection in rats between endemic and non-endemic areas of human leptospirosis. The prevalence rate of Leptospira infection in rats may result in the spread of leptospirosis to humans. These results may be of benefit in the prevention and/or control of the spread of leptospirosis in humans due to Leptospira-infected rats.

  5. Polymerase chain reaction based epidemiological investigation of canine parvoviral disease in dogs at Bareilly region

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    Jobin Thomas

    2014-11-01

    Full Text Available Aim: The aim of this study was to screen the suspected samples by polymerase chain reaction (PCR and epidemiological analysis of positive cases of canine parvovirus type2. Materials and Methods: Fecal samples were collected from dogs suspected for canine parvovirus type 2 (CPV-2 and viral DNA was extracted. Primers were designed, and PCR was done with all extracted DNA samples. Age, sex and breed wise distribution of positive cases were analyzed. Results: Out of a total 44 collected fecal samples, 23 were found to be positive for CPV-2 by developed PCR. The disease was found to be more common in Labrador male pups of 3-6 months of age. The percentage of positive cases in vaccinated dogs was found to be around 17.4%. Conclusion: Almost half (52.3% of total collected samples were found to be positive by PCR. However, number of field samples are needed to further validate this test and additionally sequence analysis needs to be done to ensure the prevalent field strain of CPV-2.

  6. Accuracy of mucocutaneous leishmaniasis diagnosis using polymerase chain reaction: systematic literature review and meta-analysis

    Science.gov (United States)

    Gomes, Ciro Martins; Mazin, Suleimy Cristina; dos Santos, Elisa Raphael; Cesetti, Mariana Vicente; Bächtold, Guilherme Albergaria Brízida; Cordeiro, João Henrique de Freitas; Theodoro, Fabrício Claudino Estrela Terra; Damasco, Fabiana dos Santos; Carranza, Sebastián Andrés Vernal; Santos, Adriana de Oliveira; Roselino, Ana Maria; Sampaio, Raimunda Nonata Ribeiro

    2015-01-01

    The diagnosis of mucocutaneous leishmaniasis (MCL) is hampered by the absence of a gold standard. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. This study aimed to assess the ability of polymerase chain reaction (PCR) to identify MCL and to compare these results with clinical research recently published by the authors. A systematic literature review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA Statement was performed using comprehensive search criteria and communication with the authors. A meta-analysis considering the estimates of the univariate and bivariate models was performed. Specificity near 100% was common among the papers. The primary reason for accuracy differences was sensitivity. The meta-analysis, which was only possible for PCR samples of lesion fragments, revealed a sensitivity of 71% [95% confidence interval (CI) = 0.59; 0.81] and a specificity of 93% (95% CI = 0.83; 0.98) in the bivariate model. The search for measures that could increase the sensitivity of PCR should be encouraged. The quality of the collected material and the optimisation of the amplification of genetic material should be prioritised. PMID:25946238

  7. Application of the arbitrarily primed polymerase chain reaction for the detection of DNA damage

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    Atienzar, F.; Evenden, A.; Jha, A.; Depledge, M. [University of Plymouth (United Kingdom). Environmental Research Centre; Child, P. [ADAS Boxworth (United Kingdom); Savva, D.; Walker, C. [University of Reading (United Kingdom). School of Animal and Microbial Sciences

    1998-07-01

    The technique of arbitrarily primed polymerase chain reaction (AP-PCR) shows potential as a selective and sensitive assay for the detection of xenobiotic-induced DNA damage. Problems, however, may occur in AP-PCR, diminishing its discriminative abilities. These problems include the presence of spurious amplification products in non-template-containing negative control reactions, and a lack of reproducibility amongst amplification patterns. Experiments designed to remove contaminated nucleic acids by ultraviolet (UV) treatment indicated that spurious bands are the result of aberrant primer-induced polymerisation, an event shown to be influenced by the concentration of deoxynucleotide triphosphates (dNTP) present in the reaction mixtures. Optimisation of dNTP concentration from 0.22 to 0.33 MM resulted in clear negative controls and highly reproducible amplification patterns with all DNA templates. As an example of the application of the method, in the present study, the macroalga Palmaria palmata (Rhodophyta) was exposed to UV A and B radiations. The study shows that the AP-PCR method can detect DNA damage and may be useful in detecting such damage following exposure of cells to xenobiotics. (author)

  8. Real-time polymerase chain reaction for detection of Strongyloides stercoralis in stool.

    Science.gov (United States)

    Sultana, Yasmin; Jeoffreys, Neisha; Watts, Matthew R; Gilbert, Gwendolyn L; Lee, Rogan

    2013-06-01

    The use of real-time polymerase chain reaction (PCR) for detection of Strongyloides stercoralis in stool has recently been described. We compared five DNA extraction methods by using normal human stool spiked with Strongyloides ratti and tested by using a real-time PCR. The PowerSoil kit was found to be the best technique in terms of sensitivity and ease of use. The PCR detected DNA extracted from one spiked S. ratti larva diluted 10⁻². The PowerSoil kit was then used to extract DNA from 160 human survey samples. All culture positive specimens with a high and moderate larval load were identified by real-time PCR, but only 15% of specimens with low larval load were positive. Specificity was greater than 99%. The combination of the PowerSoil kit and real-time PCR reliably detected high to moderate larval numbers of S. stercoralis in stools but was less sensitive when the larval load was low.

  9. Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

    Directory of Open Access Journals (Sweden)

    Ana Lisa do Vale Gomes

    2006-10-01

    Full Text Available This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA. The efficiency was 0.99 and the correlation coefficient (R² was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC. The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

  10. Introduction of a dermatophyte polymerase chain reaction assay to the diagnostic mycology service in Scotland.

    Science.gov (United States)

    Alexander, C L; Shankland, G S; Carman, W; Williams, C

    2011-05-01

    Dermatophytes are the major cause of superficial mycoses in samples submitted to Clinical Mycology, Glasgow. The most prevalent species is Trichophyton rubrum as identified classically by microscopy and culture. Recent advances in polymerase chain reaction (PCR) technology were examined for the feasibility of introducing a T. rubrum real-time PCR assay into a routine diagnostic service. To improve the diagnostic mycology service by the introduction of a real-time PCR test for T. rubrum. The DNA from 4972 nail and skin samples was obtained using the Qiagen QIAsymphony automated extractor. This DNA was subjected to real-time PCR using T. rubrum-specific primers and a probe. During phase 1 of the study, 862 samples were analysed; 446 of 470 specimens that grew T. rubrum were detected by PCR. Out of 4110 samples analysed during phase 2, 753 T. rubrum infections were diagnosed and reported within 72 h. A total of 3357 samples were negative for a fungal infection by PCR and microscopy; these were also reported within 72 h. A vast reduction in the turnaround times can be achieved using this technique as opposed to classical methods. Samples which are PCR negative but microscopy positive are still subjected to culture. Screening samples for their suitability for PCR prior to processing eliminates the application of PCR for T. rubrum on inappropriate samples such those from the scalp or pityriasis versicolor. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

  11. Detection of beer spoilage bacteria Megasphaera and Pectinatus by polymerase chain reaction and colorimetric microplate hybridization.

    Science.gov (United States)

    Satokari, R; Juvonen, R; Mallison, K; von Wright, A; Haikara, A

    1998-12-08

    Anaerobic bacteria of the genera Megasphaera and Pectinatus cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. A polymerase chain reaction (PCR) method and a colorimetric microplate hybridization assay were developed for the rapid and specific detection of Megasphaera cerevisiae and Pectinatus spp. A biotinylated primer pair was designed for the amplification of a 403 base pair (bp) fragment of the M. cerevisiae 16S rRNA gene and a primer pair from literature was used for the amplification of an 816 bp fragment of Pectinatus 16S rRNA gene. Amplified PCR products were analyzed by the colorimetric microplate hybridization method in which a biotinylated PCR product was captured by streptavidin and hybridized with a digoxigenin-labelled oligonucleotide probe. In the final step an enzyme-linked antibody and a colorimetric reaction were utilized. A simple and rapid sample treatment was set up for the PCR detection of contaminants in beer. Detection of M. cerevisiae (> or = 5 x 10(3) colony forming units [cfu]/100 ml) and Pectinatus frisingensis (> or = 5 x 10(5) cfu/100 ml) in beer was successful, but the sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

  12. Use of the polymerase chain reaction for detection of Fusarium graminearum in bulgur wheat

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    Carla Bertechini Faria

    2012-03-01

    Full Text Available The detection of mycotoxigenic fungi in foodstuff is important because their presence may indicate the possible associated mycotoxin contamination. Fusarium graminearum is a wheat pathogen and a producer of micotoxins. The polymerase chain reaction (PCR has been employed for the specific identification of F. graminearum. However, this methodology has not been commonly used for detection of F. graminearum in food. Thus, the objective of the present study was to develop a molecular methodology to detect F. graminearum in commercial samples of bulgur wheat. Two methods were tested. In the first method, a sample of this cereal was contaminated with F. graminearum mycelia. The genomic DNA was extracted from this mixture and used in a F. graminearum specific PCR reaction. The F. graminearum species was detected only in samples that were heavily contaminated. In the second method, samples of bulgur wheat were inoculated on a solid medium, and isolates having F. graminearum culture characteristics were obtained. The DNA extracted from these isolates was tested in F. graminearum specific PCR reactions. An isolate obtained had its trichothecene genotype identified by PCR. The established methodology could be used in surveys of food contamination with F. graminearum.

  13. Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels.

    Science.gov (United States)

    Stavis, Samuel M; Corgié, Stéphane C; Cipriany, Benjamin R; Craighead, Harold G; Walker, Larry P

    2007-09-20

    Laser induced fluorescence in submicrometer fluidic channels was used to characterize the synthesis of polymerase chain reaction (PCR) products from a model bacterial system in order to explore the advantages and limitations of on chip real time single molecule PCR analysis. Single oligonucleotide universal bacterial primers and PCR amplicons from the 16S rDNA of Thermobifida fusca (325 bp) were directly detected at all phases of the reaction with low sample consumption and without post-amplification purification or size screening. Primers were fluorescently labeled with single Alexa Fluor 488 or Alexa Fluor 594 fluorophores, resulting in double labeled, two color amplicons. PCR products were driven electrokinetically through a fused silica channel with a 250 nm by 500 nm rectangular cross section. Lasers with 488 nm and 568 nm wavelengths were focused and overlapped on the channel for fluorescence excitation. All molecules entering the channel were rapidly and uniformly analyzed. Photon burst analysis was used to detect and identify individual primers and amplicons, and fluorescence correlation and cross-correlation spectroscopy were used to account for analyte flow speed. Conventional gel and capillary electrophoresis were also used to characterize the PCR amplification, and the results of differences in detection sensitivity and analyte discrimination were examined. Limits were imposed by the purity and labeling efficiency of the PCR reagents, which must be improved in parallel with increases in detection sensitivity.

  14. Detection of helicobacter pylori in benign laryngeal lesions by polymerase chain reaction: a cross sectional study

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    Izadi Farzad

    2012-04-01

    Full Text Available Abstract Background Although Helicobacter Pylori (HP was detected in some cases of chronic laryngitis, the results were not confirmed by polymerase chain reaction (PCR. By this time, it has not been found in laryngeal lesions by in house PCR, the most sensitive method for detecting the genome tracks. Regarding the previous results and also few numbers of studies about the presence of HP in benign laryngeal lesions, specifically by PCR, we aimed to investigate the presence of HP in benign laryngeal lesions by in-house PCR. Methods The samples were taken from 55 patients with benign laryngeal lesions and frozen in −20°C. One milliliter (ml of lysis buffer was added to 100 mg (mg of each sample and the tube was placed in 56°C overnight. Then DNA extraction was carried out. Results To find HP DNA, in-house PCR was performed that revealed 5 positive results among 55 patients with benign laryngeal lesions. Of them, 3 were polyp, 1 was nodule and 1 was papilloma. Conclusion Although the number of positive results was not a lot in this study, it was in contrast with previous studies which could not find any HP tracks in benign laryngeal lesions by other methods. More studies about the prevalence of HP in benign laryngeal lesions improve judging about the effect of this infection on benign laryngeal lesions.

  15. Analysis ulcerative colitis for presence Epstein-Barr virus DNA sequences by polymerase chain reaction technique

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    Sahar Mehrabani khasraghi

    2016-02-01

    Full Text Available Introduction: Ulcerative colitis (UC is one type of inflammatory bowel disease (IBD. The purpose of this study is to explore the prevalence of Epstein–Barr virus (EBV in UC patients in comparison with healthy subjects using the polymerase chain reaction (PCR method. Methods: In this case-control study, five biopsies of patients with UC and 30 healthy people as controls were selected. Sampling was performed by endoscopic biopsy operation. After DNA extraction, PCR was used to determine EBV genome by specific primers. Statistical analysis was performed using the chi-square test. Results: The results of PCR indicated that EBV genome was detected in 60.0% of samples in the case group, and 36.7% of samples in the control group were positive for EBV. Thus, no significant association was observed between the prevalence of EBV and incidence of UC in comparison with the control group (P = 0.36. Conclusion: The findings presented herein demonstrate no direct molecular evidence to support an association of EBV with UC. These results, do not exclude the possibility oncogenic role of EBV to infect the different colon cell.

  16. Rapid detection of Listeria monocytogenes in food by polymerase chain reaction.

    Science.gov (United States)

    Ennaji, H; Timinouni, M; Ennaji, M M; Ait m'hand, R; Hassar, M; Cohen, N

    2009-02-25

    The standard conventional methods for the detection of Listeria monocytogenes in foods require high time 7 to 10 days to give ready results. To dissolve this problem we have evaluate a short method using Polymerase Chain Reaction (PCR) to analyze food samples. In parallel with this study, a comparison was made between PCR amplification from templates directly prepared from food and the official standard ISO procedure 11290-1. In this study we have used a Half Frazer broth as an enrichment medium; there were positive results of PCR detection of L. monocytogenes in different food sample analyzed (milk, cheese and meat) with approximately 1.5 10(1) Colony Forming Units /25 g in less than 36 h. This PCR procedure has proved to be rapid and sensitive method suitable for the routine analysis; firstly, because this assay required just a short pre-enrichment step before PCR. Secondly, this procedure is very simple and time-saving; it could take less than one working day to obtain results if initial microbiological load was very important.

  17. Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens

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    Adriana Antônia da Cruz Furini

    2013-12-01

    Full Text Available OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens.METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results.RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard, we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively.CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis.

  18. Prescription Surveillance and Polymerase Chain Reaction Testing to Identify Pathogens during Outbreaks of Infection

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    Hiroaki Sugiura

    2013-01-01

    Full Text Available Syndromic surveillance, including prescription surveillance, offers a rapid method for the early detection of agents of bioterrorism and emerging infectious diseases. However, it has the disadvantage of not considering definitive diagnoses. Here, we attempted to definitively diagnose pathogens using polymerase chain reaction (PCR immediately after the prescription surveillance system detected an outbreak. Specimens were collected from 50 patients with respiratory infections. PCR was used to identify the pathogens, which included 14 types of common respiratory viruses and Mycoplasma pneumoniae. Infectious agents including M. pneumoniae, respiratory syncytial virus (RSV, rhinovirus, enterovirus, and parainfluenza virus were detected in 54% of patients. For the rapid RSV diagnosis kit, sensitivity was 80% and specificity was 85%. For the rapid adenovirus diagnosis kit, no positive results were obtained; therefore, sensitivity could not be calculated and specificity was 100%. Many patients were found to be treated for upper respiratory tract infections without the diagnosis of a specific pathogen. In Japan, an outbreak of M. pneumoniae infection began in 2011, and our results suggested that this outbreak may have included false-positive cases. By combining syndromic surveillance and PCR, we were able to rapidly and accurately identify causative pathogens during a recent respiratory infection outbreak.

  19. Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

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    Daniela Camargos Costa

    2014-02-01

    Full Text Available The polymerase chain reaction (PCR-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34 of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

  20. Multiplex polymerase chain reaction assay developed to diagnose adult bacterial meningitis in Taiwan.

    Science.gov (United States)

    Lee, Chi-Tsung; Hsiao, Kuang-Ming; Chen, Jin-Cherng; Su, Cheng-Chuan

    2015-11-01

    Acute bacterial meningitis causes high morbidity and mortality; the associated clinical symptoms often are insensitive or non-specific; and the pathogenic bacteria are geographically diverse. Clinical diagnosis requires a rapid and accurate methodology. This study aimed to develop a new multiplex polymerase chain reaction (mPCR) assay to detect simultaneously six major bacteria that cause adult bacterial meningitis in Taiwan: Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, and Acinetobacter baumannii. Species-specific primers for the six bacteria were developed using reference strains. The specificities of the mPCRs for these bacteria were validated, and the sensitivities were evaluated via serial dilutions. The mPCR assay specifically detected all of the six pathogens, particularly with sensitivities of 12 colony forming units (CFU)/mL, 90 CFU/mL, and 390 CFU/mL for E. coli, S. pneumoniae, and K. pneumoniae, respectively. This mPCR assay is a rapid and specific tool to detect the six major bacterial pathogens that cause acute adult meningitis in Taiwan, particularly sensitive for detecting E. coli, S. pneumoniae, and K. pneumoniae. The assay may facilitate early diagnosis and guidance for antimicrobial therapy for adult patients with this deadly disease in Taiwan. © 2015 APMIS. Published by John Wiley & Sons Ltd.

  1. Diagnosis of neonatal sepsis by broad-range 16S real-time polymerase chain reaction.

    Science.gov (United States)

    Ohlin, Andreas; Bäckman, Anders; Ewald, Uwe; Schollin, Jens; Björkqvist, Maria

    2012-01-01

    The standard diagnostic test (blood culture) for suspected neonatal sepsis has limitations in sensitivity and specificity, and 16S polymerase chain reaction (PCR) has been suggested as a new diagnostic tool for neonatal sepsis. To develop and evaluate a new real-time PCR method for detection of bacterial DNA in blood samples collected from infants with suspected neonatal sepsis. Immediately after blood culture, a study sample of 0.5-1.0 ml whole blood was collected and used for a novel 16S real-time PCR assay. All positive samples were sequenced. Detailed case studies were performed in all cases with conflicting results, to verify if PCR could detect pathogens in culture negative sepsis. 368 samples from 317 infants were included. When compared with blood culture, the assay yielded a sensitivity of 79%, a specificity of 90%, a positive predictive value of 59%, and a negative predictive value of 96%. Seven of the 31 samples with a positive PCR result and a negative blood culture had definite or suspected bacterial sepsis. In five samples, PCR (but not blood culture) could detect a pathogen that was present in a blood culture collected more than 24 h prior to the PCR sample. This study presents an evaluation of a new real-time PCR technique that can detect culture-positive sepsis, and suggests that PCR has the potential to detect bacteria in culture-negative samples even after the initiation of intravenous antibiotics. Copyright © 2011 S. Karger AG, Basel.

  2. A Novel, Essential Control for Clonality Analysis with Human Androgen Receptor Gene Polymerase Chain Reaction

    Science.gov (United States)

    van Dijk, Jeroen P.; Heuver, Leonie H.; van der Reijden, Bert A.; Raymakers, Reinier A.; de Witte, Theo; Jansen, Joop H.

    2002-01-01

    The most widely used technique for determining clonality based on X-chromosome inactivation is the human androgen receptor gene polymerase chain reaction (PCR). The reliability of this assay depends critically on the digestion of DNA before PCR with the methylation-sensitive restriction enzyme HpaII. We have developed a novel method for quantitatively monitoring the HpaII digestion in individual samples. Using real-time quantitative PCR we measured the efficiency of HpaII digestion by measuring the amplification of a gene that escapes X-chromosome inactivation (XE169) before and after digestion. This method was tested in blood samples from 30 individuals: 2 healthy donors and 28 patients with myelodysplastic syndrome. We found a lack of XE169 DNA reduction after digestion in the granulocytes of two myelodysplastic syndrome patients leading to a false polyclonal X-chromosome inactivation pattern. In all other samples a significant reduction of XE169 DNA was observed after HpaII digestion. The median reduction was 220-fold, ranging from a 9.0-fold to a 57,000-fold reduction. Also paraffin-embedded malignant tissue was investigated from two samples of patients with mantle cell lymphoma and two samples of patients with colon carcinoma. In three of these cases inefficient HpaII digestion led to inaccurate X-chromosome inactivation pattern ratios. We conclude that monitoring the efficiency of the HpaII digestion in a human androgen receptor gene PCR setting is both necessary and feasible. PMID:12213708

  3. Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction.

    Science.gov (United States)

    Malaguti, Natália; Bahls, Larissa Danielle; Uchimura, Nelson Shozo; Gimenes, Fabrícia; Consolaro, Marcia Edilaine Lopes

    2015-01-01

    Bacterial vaginosis (BV) is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR) assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs) related to symptomatic BV: Gardnerella vaginalis, Mobiluncus curtisii, Mobiluncus mulieris, Bacteroides fragilis, Mycoplasma hominis, Atopobium vaginae, Ureaplasma urealyticum, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs) 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR) were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%), and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

  4. Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction

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    Natália Malaguti

    2015-01-01

    Full Text Available Bacterial vaginosis (BV is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs related to symptomatic BV: Gardnerella vaginalis, Mobiluncus curtisii, Mobiluncus mulieris, Bacteroides fragilis, Mycoplasma hominis, Atopobium vaginae, Ureaplasma urealyticum, Megasphaera type I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs 1, 2, and 3, Sneathia sanguinegens, and Mycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%, and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

  5. Sensitivitas Metode Serologi dan Polymerase Chain Reaction untuk Mendeteksi Bean Common Mosaic Potyvirus pada Kacang Panjang

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    Sherli Anggraini

    2014-08-01

    Full Text Available Mosaic disease in yard long bean is caused by Bean common mosaic potyvirus (BCMV and has been reported to affect yield. Common method to detect infection of BCMV involves serological assay and polymerase chain reaction (PCR. The aims of this research is to assess the sensitivity of three methods, i.e. Indirect Enzym-Linked Immunosorbent Assay (I-ELISA, Dot Immunobinding Assay (DIBA, and reverse transcription (RT-PCR as detection method for BCMV infection in yard long bean. Sensitivity level of the methods was evaluated by diluting plant extract and antisera for I-ELISA and DIBA, and cDNA as template in RT-PCR. Virus isolate from Cirebon was maintained in yard long bean in screenhouse and used for the assessment. Absorbance value of ELISA showed that dilution end point for I-ELISA was reached at 10-3 and 10-2 of plant extract and antisera dilution, respectively. Positive infection was still detected using DIBA when the plant extract was diluted up to 10-5 based on development of color intensity on nitrocellulose membrane. Specific viral DNA fragment was still amplified when cDNA was diluted up to 10-4, indicated higher sensitivity level of RT-PCR method.

  6. Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction

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    In Young OH

    2017-09-01

    Full Text Available Background: Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR.Methods: The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing.Results: Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously.Conclusion: This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection.

  7. Immunohistochemistry and Polymerase Chain Reaction for Detection Human Papilloma Virus in Warts: A Comparative Study

    Science.gov (United States)

    Lee, Hong Sun; Lee, Ji Hyun; Choo, Ji Yoon; Byun, Hee Jin; Jun, Jin Hyun

    2016-01-01

    Background Immunohistochemistry and polymerase chain reaction (PCR) are the most widely used methods for the detection of viruses. PCR is known to be a more sensitive and specific method than the immunohistochemical method at this time, but PCR has the disadvantages of high cost and skilled work to use widely. With the progress of technology, the immunohistochemical methods used in these days has come to be highly sensitive and actively used in the diagnostic fields. Objective To evaluate and compare the usefulness of immunohistochemistry and PCR for detection human papilloma virus (HPV) in wart lesions. Methods Nine biopsy samples of verruca vulgaris and 10 of condyloma accuminatum were examined. Immunohistochemical staining using monoclonal antibody to HPV L1 capsid protein and PCR were done for the samples. DNA sequencing of the PCR products and HPV genotyping were also done. Results HPV detection rate was 78.9% (88.9% in verruca vulgaris, 70.0% in condyloma accuminatum) on immunohistochemistry and 100.0% for PCR. HPV-6 genotype showed a lower positivity rate on immunohistochemistry (50.0%) as compared to that of the other HPV genotypes. Conclusion Immunohistochemistry for HPV L1 capsid protein showed comparable sensitivity for detection HPV. Considering the high cost and great effort needed for the PCR methods, we can use immunohistochemistry for HPV L1 capsid protein with the advantage of lower cost and simple methods for HPV detection. PMID:27489431

  8. Automation of polymerase chain reaction tests. Reduction of human errors leading to contamination.

    Science.gov (United States)

    Wilke, W W; Jones, R N; Sutton, L D

    1995-04-01

    We compared the performance over 21 months of manually performed polymerase chain reaction (PCR)-based DNA analysis experiments with 25 months of automated PCR performed by a Zymark robotic system. Automation of the PCR technique resulted in a sixfold reduction in the number of experiments reporting carryover contamination and decreased the overall rate of contamination among total reactions 68-fold. Whereas contamination occurrences among manual experiments were evenly dispersed over the study interval and correlated with the lack of experience of laboratory personnel, the contamination that occurred with the robotic system was confined to the first 10 months of operation. In manual experiments, many of the 81 no-target false positives were sufficiently strong to result in the invalidation of 151 samples and positive controls. The seven no-target control false positives in the automated system were weak bands that were easily subtracted as background. Because none of the negative samples had DNA bands, no sample on the automated system has ever been invalidated as a result of contamination. Automation of PCR tests appears to offer great promise in reducing contamination to acceptable levels (e.g., < or = 0.1%).

  9. Polymerase chain reaction for detection of Chlamydia trachomatis in conjunctival swabs

    Science.gov (United States)

    Elnifro, E.; Storey, C.; Morris, D.; Tullo, A.

    1997-01-01

    AIMS/BACKGROUND—Ocular Chlamydia trachomatis infection in the west occurs as ophthalmia neonatorum, acquired from the mother, or adult paratrachoma which is also associated with current genital tract infection. Accurate rapid laboratory diagnosis facilitates management, but the relative merits of antigen detection or DNA amplification tests are unresolved.
METHODS—A polymerase chain reaction (PCR) test was developed which amplified part of the plasmid shared by all the serovars of C trachomatis. Conjunctival swabs were tested using an in house immune dot-blot test (IDBT) for chlamydial lipopolysaccharide antigen, a commercial direct fluorescent antibody (DFA) test for chlamydial elementary bodies, and the PCR (DNA extracted using guanidinium lysis buffer).
RESULTS—The PCR achieved a detection limit of 100 plasmid copies (10 elementary bodies). In a combined retrospective and prospective clinical evaluation, the PCR and IDBT gave identical results with 21 positive and 57 negative eye swabs. However, interpretation of the DFA test required meticulous examination of the stained smear, sometimes by two microscopists.
CONCLUSIONS—The PCR is likely to play an increasing role in the diagnosis of ocular C trachomatis infection because of its excellent sensitivity and specificity.

 PMID:9274416

  10. Accuracy of mucocutaneous leishmaniasis diagnosis using polymerase chain reaction: systematic literature review and meta-analysis

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    Ciro Martins Gomes

    2015-04-01

    Full Text Available The diagnosis of mucocutaneous leishmaniasis (MCL is hampered by the absence of a gold standard. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. This study aimed to assess the ability of polymerase chain reaction (PCR to identify MCL and to compare these results with clinical research recently published by the authors. A systematic literature review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA Statement was performed using comprehensive search criteria and communication with the authors. A meta-analysis considering the estimates of the univariate and bivariate models was performed. Specificity near 100% was common among the papers. The primary reason for accuracy differences was sensitivity. The meta-analysis, which was only possible for PCR samples of lesion fragments, revealed a sensitivity of 71% [95% confidence interval (CI = 0.59; 0.81] and a specificity of 93% (95% CI = 0.83; 0.98 in the bivariate model. The search for measures that could increase the sensitivity of PCR should be encouraged. The quality of the collected material and the optimisation of the amplification of genetic material should be prioritised.

  11. Accuracy of mucocutaneous leishmaniasis diagnosis using polymerase chain reaction: systematic literature review and meta-analysis.

    Science.gov (United States)

    Gomes, Ciro Martins; Mazin, Suleimy Cristina; Santos, Elisa Raphael dos; Cesetti, Mariana Vicente; Bächtold, Guilherme Albergaria Brízida; Cordeiro, João Henrique de Freitas; Theodoro, Fabrício Claudino Estrela Terra; Damasco, Fabiana dos Santos; Carranza, Sebastián Andrés Vernal; Santos, Adriana de Oliveira; Roselino, Ana Maria; Sampaio, Raimunda Nonata Ribeiro

    2015-04-01

    The diagnosis of mucocutaneous leishmaniasis (MCL) is hampered by the absence of a gold standard. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. This study aimed to assess the ability of polymerase chain reaction (PCR) to identify MCL and to compare these results with clinical research recently published by the authors. A systematic literature review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA Statement was performed using comprehensive search criteria and communication with the authors. A meta-analysis considering the estimates of the univariate and bivariate models was performed. Specificity near 100% was common among the papers. The primary reason for accuracy differences was sensitivity. The meta-analysis, which was only possible for PCR samples of lesion fragments, revealed a sensitivity of 71% [95% confidence interval (CI) = 0.59; 0.81] and a specificity of 93% (95% CI = 0.83; 0.98) in the bivariate model. The search for measures that could increase the sensitivity of PCR should be encouraged. The quality of the collected material and the optimisation of the amplification of genetic material should be prioritised.

  12. Pouched Rats’ Detection of Tuberculosis in Human Sputum: Comparison to Culturing and Polymerase Chain Reaction

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    Amanda Mahoney

    2012-01-01

    Full Text Available Setting. Tanzania. Objective. To compare microscopy as conducted in direct observation of treatment, short course centers to pouched rats as detectors of Mycobacterium tuberculosis. Design. Ten pouched rats were trained to detect tuberculosis in sputum using operant conditioning techniques. The rats evaluated 910 samples previously evaluated by smear microscopy. All samples were also evaluated through culturing and multiplex polymerase chain reaction was performed on culture growths to classify the bacteria. Results. The patientwise sensitivity of microscopy was 58.0%, and the patient-wise specificity was 97.3%. Used as a group of 10 with a cutoff (defined as the number of rat indications to classify a sample as positive for Mycobacterium tuberculosis of 1, the rats increased new case detection by 46.8% relative to microscopy alone. The average samplewise sensitivity of the individual rats was 68.4% (range 61.1–73.8%, and the mean specificity was 87.3% (range 84.7–90.3%. Conclusion. These results suggest that pouched rats are a valuable adjunct to, and may be a viable substitute for, sputum smear microscopy as a tuberculosis diagnostic in resource-poor countries.

  13. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction.

    Science.gov (United States)

    Loeffelholz, M J; Lewinski, C A; Silver, S R; Purohit, A P; Herman, S A; Buonagurio, D A; Dragon, E A

    1992-11-01

    A rapid and sensitive polymerase chain reaction (PCR)-based assay for detection of Chlamydia trachomatis in cervical specimens is described. This assay consists of (i) sample preparation which avoids the use of heat, centrifugation, or organic extractions; (ii) rapid, two-temperature PCR amplification of C. trachomatis cryptic plasmid sequences; and (iii) capture and colorimetric detection of amplified DNA in microwell plates. PCR was compared with culture by using 503 cervical specimens. After resolution of discrepant specimens with a confirmatory PCR assay directed against the chlamydial major outer membrane protein gene, PCR had a sensitivity of 97% and a specificity of 99.7% while culture had a sensitivity of 85.7% and a specificity of 100%. In a separate study, PCR was compared with a direct specimen enzyme immunoassay (Chlamydiazyme; Abbott Diagnostics) by using 375 cervical specimens. After resolution of discrepant specimens, PCR had a sensitivity and specificity of 100%, while the enzyme immunoassay had a sensitivity of 58.8% and a specificity of 100%.

  14. Isolation and polymerase chain reaction-based identification of Riemerella anatipestifer from ducks in Kerala, India

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    Manju Soman

    2014-10-01

    Full Text Available Aim: The aim was to isolate and characterize Riemerella anatipestifer organisms from disease outbreaks in ducks in Kerala. Materials and Methods: Ducklings, suspected of Riemerella infection, were sacrificed and subjected to post-mortem examination. Heart blood smears and impression smears from liver and spleen were examined for the presence of pathogenic organisms. Heart blood, lung, liver, and spleen collected aseptically from the birds were subjected to isolation trials in brain heart infusion agar and 10% bovine blood agar. The isolates were characterized based on morphology, cultural characteristics and biochemical tests, and their identity were confirmed by polymerase chain reaction (PCR and the PCR amplified DNA was sequenced. The antibiotic sensitivity testing of the isolates were carried out using six antibiotics viz ciprofloxacin, chloramphenicol, enrofloxacin, amoxycillin, cotrimoxazole, and gentamicin. Results: Colonies suggestive of Riemerella organisms could be isolated on blood agar. Biochemical characterization and PCR confirmed the identity of isolates as R. anatipestifer. The nucleotide sequence of the PCR product showed 99% homology to the R. anatipestifer sequences in the NCBI. The antibiogram revealed that the organisms were sensitive to ciprofloxacin, enrofloxacin, and gentamicin. Conclusion: The present study suggests that the PCR assay can facilitate fast and proper identification of R. anatipestifer infection in ducks. The assay can also differentiate between R. anatipestifer and Pasteurella multocida and can replace the traditional methods of differentiation which are cumbersome and time-consuming.

  15. Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA.

    Science.gov (United States)

    Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

    2012-04-06

    Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism.

    Science.gov (United States)

    Taylor, D B; Szalanski, A L; Peterson, R D

    1996-01-01

    Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty-seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (F.). Five restriction fragment length patterns were polymorphic in C.hominivorax while all fragment patterns were fixed in C.macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than $2.50.

  17. Diagnosis of whooping cough in Switzerland: differentiating Bordetella pertussis from Bordetella holmesii by polymerase chain reaction.

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    Laure F Pittet

    Full Text Available Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR. In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old, formerly diagnosed as Bordetella pertussis (IS481+. No B. holmesii (IS481+, IS1001-, hIS1001+ was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested.

  18. [Use of polymerase chain reaction for the diagnosis of central nervous system infections].

    Science.gov (United States)

    Zambrano, Yelitza; Chiarello, Anna; Soca, Alain; Villalobos, Iris; Marrero, Miguel; Soler, Maritza; Laferte, José; Alvarez, Maritza

    2006-12-01

    In the present work, the polymerase chain reaction (PCR) assay and his variants RT-PCR and Multiplex PCR were applied for the detection of specific sequences of Enterovirus, Human Herpes viruses (Herpes simple virus, Human Herpes virus type 6, Cytomegalovirus, Epstein Barr virus, and Varicella Zoster), Human Immunodeficiency virus, Toxoplasma gondii, Mycobacterium tuberculosis and Mycoplasma pneumoniae in patients' cohorts grouped by medical suspicion of meningoencephalitis. Of 326 samples of processed cerebrospinal fluid (CSF), 93 samples (28.5%) were positive for the different infectious agents. In the group of patients with clinical diagnosis of viral meningoencephalitis (n=212), there was obtained a whole of 73 positive samples (34.4%), of which 37 patients were positive to Enterovirus (50.7%), 19 were positive to VHS (26%) and 10 patients (13.7%) were positive to CMV. Other viral agents as VZV, EBV and HVH6 were detected in minor frequency. The 114 remaining samples were analyzed applying specific PCR to each pathogen for strict medical indication, being able to detect the presence of Human Immunodeficiency Virus (40%), Mycoplasma pneumoniae (40%), Toxoplasma gondii (14%) and Mycobacterium tuberculosis (12%) in CSF samples. The results obtained in this study demonstrate the convenience of the application of the molecular assays in the laboratory diagnosis of the meningoencefalitis of different etiology. Besides this, it is also a very valuable tool for the clinical management of the patients and for the execution of the epidemiological studies.

  19. Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

    Science.gov (United States)

    Méndez, María C; Domingo, Cristina; Tenorio, Antonio; Pardo, Lissethe C; Rey, Gloria J; Méndez, Jairo A

    2013-09-01

    Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

  20. [Detection of enteric pathogenic bacteria from surface waters by quantitative polymerase chain reaction (QPCR)].

    Science.gov (United States)

    Liu, Yong-Jun; Zhang, Chong-Miao; Wang, Xiao-Chang; Lü, Ying-Jun; Zuo, Li-Li

    2008-05-01

    A rapid quantitative polymerase chain reaction (QPCR) analysis method with universal primers was developed to detect cell densities of the enteric pathogenic bacteria from 5 surface water of Xi'an City for 4 months continuously. And the detection results by QPCR method were compared with counts of coliforms colony-forming units (CFU) determined by membrane filter (MF) analysis. The results showed that QPCR method had an estimated 94% confidence, and detection limit was 2.7 Escherichia coli cells per sample in undiluted DNA extracts. For five surface waters (N = 60), the geometric mean of pathogenic bacteria concentration determined by QPCR was 2.2-5 times of corresponding coliform CFU determined by MF analysis. Using QPCR analysis, these geometric means of pathogenic bacteria concentration ranged from 25 CCE/100 mL to 67 000 CCE/100 mL. Using MF culture analysis, coliforms ranged from 3 CFU/100 mL to 45 000 CFU/100 mL. Regression analysis showed that there was a significant positive correlation between pathogenic bacteria determined by QPCR method and coliforms determined by MF method, the correlation coefficient (r) was 0.983.

  1. Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction.

    Science.gov (United States)

    Stensvold, Rune; Brillowska-Dabrowska, Anna; Nielsen, Henrik Vedel; Arendrup, Maiken Cavling

    2006-10-01

    Blastocystis hominis is a common enteric parasite of worldwide distribution. Its pathogenetic potential has not yet been established, although numerous case reports suggest that B. hominis may cause the development of various gastrointestinal symptoms and disorders. The detection of the parasite in stool specimens is conventionally done by microscopy of direct smears, fecal concentrates, or permanently stained smears; however, morphology-based diagnosis is problematic. The aim of this study was to develop and evaluate a polymerase chain reaction (PCR) technique for the direct detection of B. hominis in human stool samples. Primers were based on small subunit ribosomal DNA and able to detect > or =32 parasites/200 mg stool artificially spiked with cultured B. hominis. In the evaluation of 43 clinical specimens, the PCR was tested against the formol ethyl acetate concentration technique (FECT) and a culture technique, proving 100% test specificity and a significantly higher sensitivity than the FECT. The PCR method is recommended for screening clinical specimens for B. hominis infection and for use in prevalence studies.

  2. Risk assessment of transfusion-associated babesiosis in Tyrol: appraisal by seroepidemiology and polymerase chain reaction.

    Science.gov (United States)

    Sonnleitner, Sissy Therese; Fritz, Josef; Bednarska, Malgorzata; Baumgartner, Raphaela; Simeoni, Josef; Zelger, Roland; Schennach, Harald; Lass-Flörl, Cornelia; Edelhofer, Renate; Pfister, Kurt; Milhakov, Andrea; Walder, Gernot

    2014-07-01

    After malaria, babesiosis is the second most common transfusion-transmitted parasitic disease in the United States. In Europe, one reported transfusion case, concerning Babesia microti, occurred in Germany. Due to the fact that Babesia spp. are present in Tyrolean ticks, the aim of this study is to assess the occurrence of immunoglobulin (Ig)G antibodies against the Babesia divergens complex, including B. divergens and Babesia venatorum (EU1), as well as B. microti by screening a representative collective of 988 blood donors from North and East Tyrol (Austria) with indirect immunofluorescence antibody test. Additionally, we investigated 206 local ixodid ticks for the presence of babesial DNA by polymerase chain reaction. Seroprevalence data resulted in rates of 2.1% for IgG antibodies against the B. divergens complex and 0.6% against B. microti in Tyrolean blood donors. All sera could be confirmed by independent retesting. Our data indicate that cross-reactivity is high between B. divergens and B. venatorum and lower than 19.8% between B. divergens and B. microti. This study shows that Babesia spp. are present in the Tyrols, which blood donors come into serologic contact with, and that we have to consider how to sustain blood product safety concerning this new challenge. Additionally, it is the first description of B. venatorum in the Tyrols, found in one Ixodes ricinus at the Italian border. © 2014 AABB.

  3. Bovine thrombin enhances the efficiency and specificity of polymerase chain reaction.

    Science.gov (United States)

    Zhang, Ying; Li, Xiaoning; Zou, Ruxing; Xue, Ying; Lou, Xinhui; He, Miao

    2014-12-01

    The polymerase chain reaction (PCR) has become one of the central techniques in molecular biology since its invention. However, PCR can be fraught with difficulties in various situations, and it is desirable to find novel PCR enhancers suitable for universal applications. Here we show that bovine thrombin (BT), a well-known coagulation protein, is exceptionally effective at preventing the formation of primer dimers and enhancing the formation of the desired PCR products. The PCR enhancement effects of BT were demonstrated by testing various types of samples, including low-copy synthetic single-stranded DNAs (ssDNAs), synthetic ssDNA pools, human genomic DNA, and hepatitis B virus genomic DNA. In addition, BT was also able to effectively relieve PCR inhibition by nanomaterial inhibitors such as gold nanoparticles (AuNPs) and graphene oxide (GO). Compared with BSA, one of the most popular PCR enhancers, BT was more effective and required concentrations 18-178 times less than that of BSA to achieve a similar level of PCR enhancement.

  4. Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

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    Šramková Zuzana

    2016-06-01

    Full Text Available Staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (SE-s and staphylococcal enterotoxin-like proteins (SEl-s. Therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. The present work is focused on Staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23S rRNA gene for identification of S. aureus at the species level, genes for classical SE-s (SEA, SEC, SED, new SE-s (SEH, SEI, SEl-s (SEK, SEL and tsst-1 gene (toxic shock syndrome toxin. Primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. According to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and DNA extraction.

  5. Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction.

    Science.gov (United States)

    Oh, In Young; Kim, Kyung Tae; Sung, Ho Joong

    2017-01-01

    Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR). The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI) genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing. Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously. This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection.

  6. Detection of Theileria lestoquardi (hirci) in ticks, sheep, and goats using the polymerase chain reaction.

    Science.gov (United States)

    Kirvar, E; Ilhan, T; Katzer, F; Wilkie, G; Hooshmand-Rad, P; Brown, D

    1998-06-29

    Theileria lestoquardi (= T. hirci) is a protozoan parasite of sheep and goats that is morphologically and biologically similar to T. annulata, the causative agent of bovine tropical theileriosis. Both parasites are transmitted by ixodid ticks of the genus Hyalomma. However, because of their morphological similarity, they cannot be distinguished in the salivary glands of infected ticks by traditional staining methods such as Feulgen or Methyl green-pyronin. Thus a need has arisen for sensitive and specific diagnostic tests that will distinguish between the two species in the vector tick, allowing the epidemiology of both diseases to be clearly defined. A contribution to this has been the development of a polymerase chain reaction using specific primers which amplify, only in T. lestoquardi-infected ticks, a 785 bp fragment of the gene that codes for a 30 kD merozoite surface protein. The sensitivity of this test and its application to the detection of T. lestoquardi in infected H. anatolicum anatolicum ticks, in the blood of three species of domestic ruminants and in cell cultures established in mononuclear cells of sheep and goats is also discussed.

  7. Cell-based polymerase chain reaction for canine transmissible venereal tumor (CTVT) diagnosis.

    Science.gov (United States)

    Setthawongsin, Chanokchon; Techangamsuwan, Somporn; Tangkawattana, Sirikachorn; Rungsipipat, Anudep

    2016-08-01

    Canine transmissible venereal tumor (CTVT) is the only naturally contagious tumor that is transmitted during coitus or social behaviors. Based on the tumor's location, the diagnosis of genital TVT (GTVT) is comparably easier than those in the extragenital area (ETVT) that are more easily incorrectly diagnosed. Fortunately, CTVT cells contain a specific long interspersed nuclear elements (LINE), inserted upstream of the myc gene, allowing a diagnostic polymerase chain reaction (PCR) based detection assay. The objectives of this study were aimed to improve the diagnostic accuracy by applying the diagnostic LINE1-c-myc PCR assay and fine needle aspiration (FNA) collection in direct comparison with standard cytological and histopathological analyses. Seventy-four dogs, comprised of 41 and 31 dogs with tumor masses at their external genitalia and extragenital areas (e.g. skin and nasal cavity), respectively, were included in this study. The signalment of these 65 dogs and clinical history of 20 client-owned dogs were collected. Samples were taken by biopsy for both histopathological examination and FNA for cytological examination and diagnostic PCR. The PCR products from 10 apparently CTVT samples were purified and sequenced. Sixty-one CTVT cases were diagnosed by cytological and histological analyses, but 65 were positive by the PCR assay. Overall, the PCR assay improved the accuracy of diagnostic CTVT results, especially for the more difficult ETVT tumors. Moreover, this PCR-based approach can facilitate the decision as to discontinue chemotherapy by discrimination between residual tumor cell masses and fibrotic tissue.

  8. Clinical Significance of Fecal Lactoferrin and Multiplex Polymerase Chain Reaction in Patients with Acute Diarrhea.

    Science.gov (United States)

    Lee, Hae Mi; Lee, Seungok; Lee, Bo-In; Jekarl, Dong Wook; Song, Joo-Yong; Choi, Hye-Jung; Kang, Bong Koo; Im, Eun Joo; Kim, Joon Sung; Kim, Jong In; Kim, Byung-Wook; Choi, Hwang

    2015-09-23

    The diagnostic yield of fecal leukocyte and stool cultures is unsatisfactory in patients with acute diarrhea. This study was performed to evaluate the clinical significance of the fecal lactoferrin test and fecal multiplex polymerase chain reaction (PCR) in patients with acute diarrhea. Clinical parameters and laboratory findings, including fecal leukocytes, fecal lactoferrin, stool cultures and stool multiplex PCR for bacteria and viruses, were evaluated prospectively for patients who were hospitalized due to acute diarrhea. A total of 54 patients were included (male, 23; median age, 42.5 years). Fecal leukocytes and fecal lactoferrin were positive in 33 (61.1%) and 14 (25.4%) patients, respectively. Among the 31 patients who were available for fecal pathogen evaluation, fecal multiplex PCR detected bacterial pathogens in 21 patients, whereas conventional stool cultures were positive in only one patient (67.7% vs 3.2%, p=0.000). Positive fecal lactoferrin was associated with presence of moderate to severe dehydration and detection of bacterial pathogens by multiplex PCR (21.4% vs 2.5%, p=0.049; 100% vs 56.5%, p=0.032, respectively). Fecal lactoferrin is a useful marker for more severe dehydration and bacterial etiology in patients with acute diarrhea. Fecal multiplex PCR can detect more causative organisms than conventional stool cultures in patients with acute diarrhea.

  9. The effect of DNA-dispersed single-walled carbon nanotubes on the polymerase chain reaction.

    Directory of Open Access Journals (Sweden)

    Ryan M Williams

    Full Text Available The unique properties of single-wall carbon nanotubes (SWCNT make them useful in many new technologies and applications. The interaction of DNA and SWCNT is of interest for many uses, including molecular sensors. This study determined polymerase chain reaction (PCR efficiency in amplifying a 76 base pair DNA sequence in the presence of SWCNT, of heterogeneous "Mix" and (6,5-enriched chiralities, associated with three DNA sequences. The dependence of PCR efficiency on the concentration of DNA:SWCNT preparations was measured, as well as their age and level of dispersion (less than one month or between four and ten months. Additionally, the ability to directly amplify the DNA sequence associated with the SWCNT scaffold was investigated. In PCRs with DNA:SWCNT preparations less than one month old, concentrations greater than or equal to 0.1 mg/mL inhibited the PCR reaction. In PCRs with older preparations, no inhibition was seen at 0.01 or 0.1 mg/mL, with amplification at 1 mg/mL in some samples. Additionally, our studies showed that the DNA directly associated with the SWCNT can be amplified using PCR. This work provides an inhibitory concentration of DNA-dispersed SWCNT in PCR reactions for different preparations as well as a basis for future DNA:SWCNT studies that require PCR amplification. This will be useful for future studies focused on the use of SWCNT in molecular sensing technologies.

  10. Low-Cost Temperature Logger for a Polymerase Chain Reaction Thermal Cycler

    Directory of Open Access Journals (Sweden)

    Chan-Young Park

    2016-10-01

    Full Text Available Polymerase chain reaction (PCR is a method of amplifying DNA which is normally carried out with a thermal cycler. To obtain more accurate and reliable PCR results, the temperature change within the chamber of the thermal cycler needs to be verified and calibrated regularly. Commercially available temperature loggers commonly used for temperature verification tests usually require a graphical user interface (GUI attached to the logger for convenience and straightforward understanding of the device. In this study, a host-local architecture for the temperature logger that significantly reduces the development time and cost is proposed. Employing standard computing devices as the host gives better development environment and user-friendly GUI. This paper presents the hardware and software design of the host-local temperature logger, and demonstrates the use of the local temperature logger connected to a personal computer with a Windows operating system. The probe design, thermistor resistance measurement, temperature filtering, and temperature calibration is described in detail. The thermistor self-heating problem was investigated in particular to determine the reference resistor that was serially connected to the thermistor. The temperature accuracy and temporal precision of the proposed system was 0.1 K.

  11. Detection of Chlamydia trachomatis in endocervical specimens by an enzyme-linked polymerase chain reaction assay

    Directory of Open Access Journals (Sweden)

    Hashemi F.B.

    2007-05-01

    Full Text Available Chlamydia trachomatis (CT is the most common cause of sexually transmitted infections (STI worldwide and its early detection and treatment can reduces the high morbidity associated with this infection. In this study a sensitive diagnostic polymerase chain reaction (PCR-based enzyme immunoassay (PCR-EIA method was developed which detects CT in women with cervicitis. Endocervical swabs collected from 123 women (20-55 years with cervicitis were tested by both conventional PCR, and PCR-EIA assays, using identical sets of primers to amplify a CT-specific plasmid. For the conventional PCR, amplicons were detected by agarose gel electrophoretic analysis and the PCR-EIA assay used biotin-labeled primers, strepavidin-coated plates, a digoxigenin-labeled probe, and a final enzyme-linked colorometric analysis (405 nm was used to measure the CT amplicon. The frequency of positive CT infection by conventional PCR and PCR-EIA assay was 7% and 17%, respectively. The highest frequencies of CT infection were among women of 31-40 years old group (25%. The PCR-EIA limit of detection, calculated by linear regression analysis, was10 pg of CT DNA (r2=0.9642. The degree of agreement (Kappa between the conventional PCR and PCR-EIA method was 0.556 (p<0.0001.

  12. Isolation of eicosapentaenoic acid-producing fungi from soil based on polymerase chain reaction amplification.

    Science.gov (United States)

    Hu, Jing-Rong; Zhou, Peng-Peng; Zhu, Yuan-Min; Ren, Liang; Yu, Long-Jiang

    2011-01-01

    A method was developed for rapid screening and isolation of eicosapentaenoic acid (EPA)-producing soil fungi through polymerase chain reaction (PCR) amplification. Genes coding for delta6 fatty acid desaturase and delta5 fatty acid desaturase were used as molecular markers for screening these EPA-producing fungi from soil. Three out of 65 soil fungi gave positive results through PCR amplification. Two out of these three strains were found to produce EPA when they had grown in 80 ml potato/dextrose liquid medium at (25 +/- 1) degrees C for 144 h. The EPA yields were 215.81 mg 1(-1) and 263.80 mg 1(-1), respectively. The other positive strain was detected to produce arachidonic acid (AA). This study indicates that molecular detection of genes encoding delta6 and delta5 desaturases is an efficient method for primary screening of EPA- or its related polyunsaturated fatty acids (PuFAs)-producing fungi, which can improve the screening efficiency prominently.

  13. Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection.

    Science.gov (United States)

    Zhang, Mao-Jun; Qiao, Bo; Xu, Xue-Bin; Zhang, Jian-Zhong

    2013-05-28

    To develop a real-time polymerase chain reaction (PCR) method to detect and quantify Campylobacter jejuni (C. jejuni) from stool specimens. Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C. jejuni. The specificity of the primers and probe were tested against a set of Campylobacter spp. and other enteric pathogens. The optimal PCR conditions were determined by testing a series of conditions with standard a C. jejuni template. The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen. Two hundred and forty-two specimens were analyzed for the presence of C. jejuni by direct bacterial culture and real-time PCR. The optimal PCR system was determined using reference DNA templates, 1 × uracil-DNA glycosylase, 3.5 mmol/L MgCl2, 1.25 U platinum Taq polymerase, 0.4 mmol/L PCR nucleotide mix, 0.48 μmol/L of each primer, 0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL. The PCR reaction was carried as follows: 95 °C for 4 min, followed by 45 cycles of 10 s at 95 °C and 30 s at 59 °C. The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10(3) CFU/g using DNA from stool specimens. Twenty (8.3%, 20/242) C. jejuni strains were isolated from bacterial culture, while 41 (16.9%, 41/242) samples were found to be positive by real-time PCR. DNA sequencing of the PCR product indicated the presence of C. jejuni in the specimen. One mixed infection of C. jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive. The sensitivity of detection of C. jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

  14. Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction

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    Xu Q

    2015-06-01

    Full Text Available Qing Xu,1,* Yazhen Zhu,2,* Yali Bai,1 Xiumin Wei,1 Xirun Zheng,2 Mao Mao,1 Guangjuan Zheng21Translational Bioscience and Diagnostics, WuXi AppTec, Shanghai, 2Department of Pathology, Guangdong Provincial Hospital of TCM, Guangzhou University of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou, People’s Republic of China*These authors contributed equally to this workBackground: Two types of epidermal growth factor receptor (EGFR mutations in exon 19 and exon 21 (ex19del and L858R are prevalent in lung cancer patients and sensitive to targeted EGFR inhibition. A resistance mutation in exon 20 (T790M has been found to accompany drug treatment when patients relapse. These three mutations are valuable companion diagnostic biomarkers for guiding personalized treatment. Quantitative polymerase chain reaction (qPCR-based methods have been widely used in the clinic by physicians to guide treatment decisions. The aim of this study was to evaluate the technical and clinical sensitivity and specificity of the droplet digital polymerase chain reaction (ddPCR method in detecting the three EGFR mutations in patients with lung cancer.Methods: Genomic DNA from H1975 and PC-9 cells, as well as 92 normal human blood specimens, was used to determine the technical sensitivity and specificity of the ddPCR assays. Genomic DNA of formalin-fixed, paraffin-embedded specimens from 78 Chinese patients with lung adenocarcinoma were assayed using both qPCR and ddPCR.Results: The three ddPCR assays had a limit of detection of 0.02% and a wide dynamic range from 1 to 20,000 copies measurement. The L858R and ex19del assays had a 0% background level in the technical and clinical settings. The T790M assay appeared to have a 0.03% technical background. The ddPCR assays were robust for correct determination of EGFR mutation status in patients, and the dynamic range appeared to be better than qPCR methods. The ddPCR assay for T790M could detect

  15. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering.

    Science.gov (United States)

    Hoseini, Sayed Shahabuddin; Sauer, Martin G

    2015-01-01

    Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning. Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells. Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.

  16. Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

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    M.S. Santos

    2010-08-01

    Full Text Available Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP. More specifically: a to evaluate 3 different amplification regions, b to investigate 3 different restriction enzymes, and c to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2 were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas - FMTAM were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

  17. Multiplexed real-time polymerase chain reaction on a digital microfluidic platform.

    Science.gov (United States)

    Hua, Zhishan; Rouse, Jeremy L; Eckhardt, Allen E; Srinivasan, Vijay; Pamula, Vamsee K; Schell, Wiley A; Benton, Jonathan L; Mitchell, Thomas G; Pollack, Michael G

    2010-03-15

    This paper details the development of a digital microfluidic platform for multiplexed real-time polymerase chain reactions (PCR). Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting. Rapid PCR thermocycling is performed in a closed-loop flow-through format where for each cycle the reaction droplets are cyclically transported between different temperature zones within an oil-filled cartridge. The cartridge is fabricated using low-cost printed-circuit-board technology and is intended to be a single-use disposable device. The PCR system exhibited remarkable amplification efficiency of 94.7%. To test its potential application in infectious diseases, this novel PCR system reliably detected diagnostic DNA levels of methicillin-resistant Staphylococcus aureus (MRSA), Mycoplasma pneumoniae , and Candida albicans . Amplification of genomic DNA samples was consistently repeatable across multiple PCR loops both within and between cartridges. In addition, simultaneous real-time PCR amplification of both multiple different samples and multiple different targets on a single cartridge was demonstrated. A novel method of PCR speed optimization using variable cycle times has also been proposed and proven feasible. The versatile system includes magnetic bead handling capability, which was applied to the analysis of simulated clinical samples that were prepared from whole blood using a magnetic bead capture protocol. Other salient features of this versatile digital microfluidic PCR system are also discussed, including the configurability and scalability of microfluidic operations, instrument portability, and substrate-level integration with other pre- and post-PCR processes.

  18. Prognostic value of circulating melanoma cells detected by reverse transcriptase-polymerase chain reaction.

    Science.gov (United States)

    Palmieri, Giuseppe; Ascierto, Paolo A; Perrone, Francesco; Satriano, Sabrina M R; Ottaiano, Alessandro; Daponte, Antonio; Napolitano, Maria; Caracò, Corrado; Mozzillo, Nicola; Melucci, Maria T; Cossu, Antonio; Tanda, Francesco; Gallo, Ciro; Satriano, Rocco A; Castello, Giuseppe

    2003-03-01

    Factors that are predictive of prognosis in patients who are diagnosed with malignant melanoma (MM) are widely awaited. Detection of circulating melanoma cells (CMCs) by reverse transcriptase-polymerase chain reaction (RT-PCR) has recently been postulated as a possible negative prognostic factor. Two main questions were addressed: first, whether the presence of CMCs, defined as the patient being positive for any of the three markers, had a prognostic role; and second, what the predictive value of each individual marker was. A consecutive series of 200 melanoma patients observed between January 1997 and December 1997, with stage of disease ranging from I to IV, was analyzed by semiquantitative RT-PCR. Tyrosinase, p97, and MelanA/MART1 were used as markers to CMCs on baseline peripheral blood samples. Progression-free survival (PFS) was used as a unique end point and was described by the product limit method. Multivariable analysis was applied to verify whether the auspicated prognostic value of these markers was independent of the stage of disease, and a subgroup analysis was performed that excluded patients with stage IV disease. Overall, 32% (64 of 200) of patients progressed, and a median PFS of 52 months in the whole series was observed. The presence of CMCs and the markers individually or combined was predictive of prognosis in the univariate analysis but did not provide additional prognostic information to the stage of disease in multivariable models. In the subgroup analysis of stage (ie, I-III subgroup), similar results were observed. Detection of CMCs in peripheral blood samples at the time of MM diagnosis by semiquantitative RT-PCR does not add any significant predictive value to the stage of disease. Thus, this approach should not be used in clinical practice, and further studies are required to determine its usefulness.

  19. Use of the polymerase chain reaction to detect Mycobacterium leprae in urine

    Directory of Open Access Journals (Sweden)

    K.R. Caleffi

    2012-02-01

    Full Text Available Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT and dapsone, rifampicin and clofazimine for borderline (BB and lepromatous (LL forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients’ urine samples was successfully amplified by PCR-Pra in 46.6% (34/73 of the cases. The positivity of PCR-Pra for patients with the TT form was 75% for both patients under treatment and non-treated patients (P = 0.1306. In patients with the LL form, PCR-Pra positivity was 52 and 30% for patients under treatment and non-treated patients, respectively (P = 0.2386. PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033. Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

  20. Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction.

    Science.gov (United States)

    Corbisier, Philippe; Bhat, Somanath; Partis, Lina; Xie, Vicki Rui Dan; Emslie, Kerry R

    2010-03-01

    Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.

  1. Detection of Listeria monocytogenes in salmon using the Probelia polymerase chain reaction system.

    Science.gov (United States)

    Wan, Jason; King, Kerryn; Forsyth, Santina; Coventry, M John

    2003-03-01

    A validation was conducted on the performance of a commercially available polymerase chain reaction (PCR) kit (Probelia) in comparison with International Organization for Standardization (ISO) method 11290-1 (adopted as an Australian New Zealand Standard Method, AS/NZS 1766.2.16.1:1998) for the detection of Listeria monocytogenes in salmon samples. The validation was conducted following the guidelines of an Australian New Zealand Standard (Guide to Determining the Equivalence of Food Microbiology Test Methods, Part 1, Qualitative Tests, AS/NZS 4659.1:1999), which adopts an approach similar to that recommended by the Association of Analytical Communities Microbiology Method Validation Program for Performance Tested and Peer Verified Methods. The validation study involved the use of five cultures of L. monocytogenes, each challenged at a single level of inoculation into five different types of salmon samples. A total of 60 salmon samples (30 unchallenged and 30 challenged) were tested using both the PCR method and the ISO method. Results from this study indicated that the Probelia PCR method is equivalent to the ISO method. In addition, the detection sensitivity of the Probelia PCR system was determined as approximately 0.5 CFU per PCR assay (equivalent to 20 CFU/ml broth culture) for a pure culture of L. monocytogenes. The Probelia PCR method offers the advantage of detecting L. monocytogenes to genetic specificity within 48 to 50 h, whereas the ISO method requires 5 days for negative results with additional days for confirmed positive results by the use of other biochemical and cultural tests.

  2. Fabrication of Polymerase Chain Reaction Plastic Lab-on-a-Chip Device for Rapid Molecular Diagnoses

    Science.gov (United States)

    2016-01-01

    Purpose: We aim to fabricate a thermoplastic poly(methylmethacrylate) (PMMA) Lab-on-a-Chip device to perform continuous- flow polymerase chain reactions (PCRs) for rapid molecular detection of foodborne pathogen bacteria. Methods: A miniaturized plastic device was fabricated by utilizing PMMA substrates mediated by poly(dimethylsiloxane) interfacial coating, enabling bonding under mild conditions, and thus avoiding the deformation or collapse of microchannels. Surface characterizations were carried out and bond strength was measured. The feasibility of the Lab-on-a-Chip device for performing on-chip PCR utilizing a lab-made, portable dual heater was evaluated. The results were compared with those obtained using a commercially available thermal cycler. Results: A PMMA Lab-on-a-Chip device was designed and fabricated for conducting PCR using foodborne pathogens as sample targets. A robust bond was established between the PMMA substrates, which is essential for performing miniaturized PCR on plastic. The feasibility of on-chip PCR was evaluated using Escherichia coli O157:H7 and Cronobacter condimenti, two worldwide foodborne pathogens, and the target amplicons were successfully amplified within 25 minutes. Conclusions: In this study, we present a novel design of a low-cost and high-throughput thermoplastic PMMA Lab-on-a-Chip device for conducting microscale PCR, and we enable rapid molecular diagnoses of two important foodborne pathogens in minute resolution using this device. In this regard, the introduced highly portable system design has the potential to enable PCR investigations of many diseases quickly and accurately. PMID:27230459

  3. Polymerase chain reaction-miniexon: a promising diagnostic method for mucocutaneous leishmaniasis.

    Science.gov (United States)

    Ovalle-Bracho, Clemencia; Díaz-Toro, Yira Rosalba; Muvdi-Arenas, Sandra

    2016-05-01

    Mucocutaneous leishmaniasis is a chronic disease caused mainly by Leishmania species that belong to Viannia subgenus. It affects upper respiratory airways and may lead to deformity, dysphagia, and even death in severe cases. Diagnosis is a challenge because clinical and histopathologic changes are easily confused with other diseases, and conventional methods for parasite identification and culture have a low sensitivity. Molecular methods have been used in the last two decades. In 2007, we published a validation study using internal transcript spacers and kinetoplast DNA as molecular targets with satisfactory results. In this research, we tested miniexon gene as the target. Mucosal tissue samples from 60 Colombian patients with clinical signs of mucocutaneous leishmaniasis were included. A composite reference standard defined 30 cases and 30 controls. Two blind observers performed patient classification and test application independently. Miniexon gene amplification generated: 226-230 bp fragment for subgenus Viannia; 308 bp fragment for L. amazonensis; 340 bp fragment for L. mexicana; and 418 bp fragment for L. infantum-chagasi. Polymerase chain reaction (PCR) sensitivity for fresh samples was 87.5% (95% confidence interval [CI] 72.2-100), specificity, 95% (95% CI 83.0-100), and positive likelihood ratio was 17.5 (95% CI 2.58-118.93), similar to results obtained with paraffin-embedded samples. Agreement between observers was 96% (kappa = 0.912; 95% CI 0.815-1.000) for both subgenus Viannia and Leishmania. We consider PCR-miniexon as a diagnostic method of first choice for mucocutaneous leishmaniasis due to its excellent diagnostic performance and its ability to discriminate between Leishmania and Viannia subgenera as well as between species belonging to Leishmania subgenus. © 2015 The International Society of Dermatology.

  4. Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

    Directory of Open Access Journals (Sweden)

    Clemencia Ovalle Bracho

    2007-08-01

    Full Text Available We validated the polymerase chain reaction (PCR with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 were non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS rDNA. PCR showed 68.6% (95% CI 59.2-72.6 sensitivity and 92% (95% CI 78.9-97.7 specificity; positive likelihood ratio: 8.6 (95% CI 2.8-31.3 and negative likelihood ratio: 0.3 (95% CI 0.3-0.5, when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40% (95% CI 31.5-42.3 sensitivity and 96% (95% CI 84.1-99.3 specificity; positive likelihood ratio: 10 (95% CI 2.0-58.8 and negative likelihood ratio: 0.6 (95% CI 0.6-0.8. The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species.

  5. Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA.

    Science.gov (United States)

    Fahrimal, Y; Goff, W L; Jasmer, D P

    1992-01-01

    Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequence is associated with a 7.4-kb DNA element in undigested B. bovis genomic DNA (as shown previously), and the amplified product was detected by Southern and dot blot hybridization. The assay was specific for B. bovis, since no amplification was detected with Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, or leukocyte DNA. The target sequence was amplified in DNA from B. bovis Mexico, Texas, and Australia S and L strains, demonstrating the applicability of the method to strains from different geographic regions. The sensitivity of the method ranged from 1 to 10 infected erythrocytes extracted from 0.5 ml of blood. This sensitivity was about 1,000 times greater than that from the use of unamplified parasite DNA. By the PCR method, six B. bovis carrier cattle were detected 86% of the time (range, 66 to 100%) when they were tested 11 times, while with microscopic examination of thick blood smears, the same carrier cattle were detected only 36% of the time (range, 17 to 66%). The method provides a useful diagnostic tool for detecting B. bovis carrier cattle, and the sensitivity is significantly improved over that of current methods. The results also suggest that characteristics of the apocytchrome b gene may make this a valuable target DNA for PCR-based detection of other hemoparasites. Images PMID:1624551

  6. A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

    Science.gov (United States)

    Rocke, T.E.; Smith, S.R.; Miyamoto, A.; Shadduck, D.J.

    2002-01-01

    A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

  7. A novel multiplex polymerase chain reaction assay for profile analyses of gene expression in peripheral blood

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    Jia Xingwang

    2012-07-01

    Full Text Available Abstract Background Studies have demonstrated that inflammation has a key role in the pathogenesis of atherosclerosis due to the abnormal gene expressions of multiple cytokines. We established an accurate and precise method to observe gene expression in whole blood that might provide specific diagnostic information for coronary artery disease (CAD and other related diseases. Methods The fifteen selected CAD-related genes (IL1B, IL6, IL8, IFNG, MCP-1, VWF, MTHFR, SELL, TNFalpha, ubiquitin, MCSF, ICAM1, ID2, HMOX1 and LDLR and two housekeeping genes (ACTB and GK as internal references have been measured simultaneously with a newly developed multiplex polymerase chain reaction (multi-PCR method. Moreover, the precision was evaluated, and a procedure for distinguishing patients from the normal population has been developed based upon analyses of peripheral blood. A total of 148 subjects were divided into group A (control group without plaques, group B (calcified plaques and group C (non-calcified plaques, and combination group according dual-source CT criteria. Gene expression in blood was analyzed by multi-PCR, and levels of glucose and lipids measured in 50 subjects to explore the relationship among them. Results The precision results of the multi-PCR system revealed within-run and between-run CV values of 3.695–12.537% and 4.405–13.405%, respectively. The profiles of cytokine gene expression in peripheral blood were set: a positive correlation between glucose and MCSF, HMOX1 or TNFalpha were found. We also found that triglyceride levels were negatively correlated with SELL gene expression in 50 subjects. Compared with controls, gene expression levels of IL1B, IL6, IL8 and MCP-1 increased significantly in group C. Conclusions A new multiple gene expression analysis system has been developed. The primary data suggested that gene expression was related to CAD. This system might be used for risk assessment of CVDs and other related diseases.

  8. A new era of uveitis: impact of polymerase chain reaction in intraocular inflammatory diseases.

    Science.gov (United States)

    Mochizuki, Manabu; Sugita, Sunao; Kamoi, Koju; Takase, Hiroshi

    2017-01-01

    Uveitis is a sight-threatening intraocular inflammatory disorder which may occur from both infectious and non-infectious or autoimmune causes. The frequency of infectious uveitis and autoimmune uveitis varies depending on countries and regions. According to a nationwide survey conducted by the Japanese Ocular Inflammation Society, infectious and non-infectious uveitis accounted for 16.4 and 50.1% of new patients, respectively while the remaining 33.5% of new uveitis cases were not classified or were idiopathic uveitis. Infectious uveitis is particularly important because it causes tissue damage to the eye and may result in blindness unless treated. However, it can be treated if the pathogenic microorganisms are identified promptly and accurately. Remarkable advancements in molecular and immunological technologies have been made in the last decade, and the diagnosis of infectious uveitis has been greatly improved by the application of molecular and immunological investigations, particularly polymerase chain reaction (PCR). PCR performed on a small amount of ocular samples provides a prompt, sensitive, and specific molecular diagnosis of pathogenic microorganisms in the eye. This technology has opened a new era in the diagnosis and treatment of uveitis, enabling physicians to establish new clinical entities of uveitis caused by infectious microorganisms, identify pathogens in the eyes of many patients with uveitis, and determine prompt diagnosis and appropriate therapy. Here we review the PCR process, new PCR tests specialized for ocular diseases, microorganisms detected by the PCR tests, diseases in the eye caused by these microorganisms, and the clinical characteristics, diagnosis, and therapy of uveitis.

  9. Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis.

    Science.gov (United States)

    Ote, Isabelle; Taminiau, Bernard; Duprez, Jean-Noël; Dizier, Isabelle; Mainil, Jacques G

    2011-12-15

    Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. A highly effective polymerase chain reaction enhancer based on dendrimer-entrapped gold nanoparticles.

    Science.gov (United States)

    Chen, Jingjing; Cao, Xueyan; Guo, Rui; Shen, Mingwu; Peng, Chen; Xiao, Tongyu; Shi, Xiangyang

    2012-01-07

    In molecular biology, polymerase chain reaction (PCR) has played an important role but suffers a general problem of low efficiency and specificity. Development of suitable PCR additives to improve the specificity and efficiency still remains a great challenge. Here we report the use of dendrimer-entrapped gold nanoparticles (Au DENPs) as a novel class of enhancers to improve the specificity and efficiency of PCR. We show that the Au DENPs prepared using amine-terminated generation 5 poly(amidoamine) dendrimers (G5.NH(2)) as templates are much more effective than the same dendrimers without AuNPs entrapped in improving the specificity and efficiency of an error-prone two-round PCR system. With the increase of the molar ratio between Au atom and G5.NH(2) dendrimer in the Au DENPs, the optimum concentration of Au DENPs used to improve the PCR specificity and efficiency is decreased and can be as low as 0.37 nM when the Au atom/G5.NH(2) dendrimer molar ratio reaches 100:1. Our PCR results along with the dynamic light scattering data suggest that unlike the flexible soft dendrimers without NPs entrapped that may display a non-spherical shape when interacting with the PCR components, the Au DENPs with increasing Au atom/dendrimer molar ratio are able to reserve the spherical shape of dendrimers, enabling much more efficient interaction with the PCR components. Therefore, as a NP-based PCR enhancer, both the surface charge and the shape of the particles should be responsible for effective interaction with the PCR components for improving the PCR specificity and efficiency. Furthermore, the used Au DENPs were proved to be stable after the PCR process, enabling them to be potentially used for enhancing different PCR systems. This journal is © The Royal Society of Chemistry 2012

  11. THE METHOD OF POLYMERASE CHAIN REACTION FOR SEX DETERMINATION IN HUCHEN (HUCHO HUCHO

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    Yu. Rud

    2013-12-01

    Full Text Available Purpose. To analyse the nucleotide sequences of salmonids Y chromosome and to determine the fragment for specific primers selection and also to develop the PCR based method for sex determination in huchen H. hucho. Methodology. Using the ClustalW algorithm in MEGA 5.2, the nucleotide sequences of salmonids Y chromosome were analysed. For developing of method for rapid diagnostic of huchen sex the polymerase chain reaction (PCR assay was used. Tne nucleotide sequences of amplified products were investigated by sequencing. Findings. Using PCR assay the method of sex determination in huchen H. hucho was developed. It was shown that specific PCR products in size of 450 nucleotides were visible in huchen males only. In addition we showed that selected primers can be used in sex determination of rainbow trout Oncorhynchus mykiss and this fact is proved the high rate of sdY locus similarity and its wide destribution in salmonids. Originality. The nucleotide sequences of salmonids Y chromosome were analysed and highly conservative region of sdY locus for specific primers selection, which covers sex-linked marker, was identified. Practical Value. Rapid sex determination in huchen by the developed method will allow to identify reversal males in process of gormonal sex reversion. At the stage of reversal males screening, this method will allow to identify the genotypic males (XY in experimental group and discard them because only phenotypic males with XX genotype (reversal males must be used in the crosses with native femelas for getting of 100 % all-females stock.

  12. Real-time polymerase chain reaction correlates well with clinical diagnosis of Clostridium difficile infection.

    Science.gov (United States)

    Berry, N; Sewell, B; Jafri, S; Puli, C; Vagia, S; Lewis, A M; Davies, D; Rees, E; Ch'ng, C L

    2014-06-01

    To determine the clinical utility of a rapid molecular assay for Clostridium difficile infection (CDI) in an acute hospital setting. From March to September 2011, stool specimens from inpatients in two acute hospitals with suspected CDI were tested prospectively by routine cell culture cytotoxin neutralization assay (CCNA), real-time polymerase chain reaction (PCR) using the GeneXpert (Cepheid Inc., Sunnyvale, CA, USA), and a dual testing algorithm [glutamate dehydrogenase (GDH)/toxin enzyme immuno-assay, Premier, Launch Diagnostics, Longfield, UK]. All patients with positive PCR, CCNA or discrepant results were reviewed by a multi-disciplinary team (treating clinician, gastroenterologist, microbiologist and infection control nurse). C. difficile detection rates were 11.7% (PCR), 6% (CCNA) and 13.8% (GDH). Out of 1034 stool specimens included in the study, 974 (94.1%) had concordant CCNA and PCR results. Eighty-nine percent (886/985) had concordant CCNA, PCR and GDH results, and 94.4% (930/985) had concordant GDH and PCR results. Using clinical diagnosis as the reference, PCR had sensitivity of 99.1%, specificity of 98.9%, positive predictive value (PPV) of 91.9% and negative predictive value (NPV) of 99.9%. CCNA on a single sample had sensitivity of 51%, specificity of 99.4%, PPV of 91.9% and NPV of 94.3%. GDH had sensitivity of 83.8%, specificity of 94.5%, PPV of 64.7% and NPV of 97.9%. Almost twice as many patients were positive by PCR compared with CCNA (121 vs 62); 54/59 of those with discrepant results were clinically confirmed as CDI. Rapid diagnosis of CDI using PCR was timely, accurate and correlated well with clinical diagnosis. Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  13. Evaluation of polymerase chain reaction in nerve biopsy specimens of patients with Hansen's disease.

    Science.gov (United States)

    Tiwari, Vandana; Malhotra, Kiranpreet; Khan, Kainat; Maurya, Pradeep K; Singh, Ajai Kumar; Thacker, Anup Kumar; Husain, Nuzhat; Kulshreshtha, Dinkar

    2017-09-15

    Pure neuritic variety of leprosy (PNL) presents as peripheral neuropathy with absent skin lesions and negative skin smears. Diagnosing PNL is an uphill task as most of these patients have nonspecific changes on nerve biopsy. In such circumstances, additional molecular diagnostic tools like polymerase chain reaction (PCR) has proven to be useful in diagnosing leprosy. The present study was planned to evaluate the role of PCR in nerve biopsy specimens of patients with PNL. Patients attending the neuromuscular clinic from January 2013 to June 2014 with mononeuropathy multiplex underwent detailed diagnostic evaluation to ascertain the cause of neuropathy. Patients where this evaluation failed to establish an etiology underwent a nerve biopsy. Nerve biopsy was done in 52 patients, of which 35 were diagnosed as pure neuritic leprosy. Definite leprosy with positive wade fite staining for lepra bacilli was seen in 13 patients and 22 biopsies revealed a probable leprosy without lepra bacilli being identified. PCR for M. leprae was positive in 22 patients (62%). 12 of the 13 cases with definite leprosy on histopathology were PCR positive while in the AFB negative group, PCR was positive in 10 cases. PCR had a sensitivity of 92.3%, specificity of 54.5%. The positive and negative predictive value of PCR was 54.5% and 92.3% respectively. PCR helps in diagnosing PNL in doubtful cases. A positive PCR increases the sensitivity of detection of M. leprae especially in cases of probable PNL group where AFB cannot be demonstrated on histopathology. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Prevalence of Helicobacter pylori detected by polymerase chain reaction in the oral cavity of periodontitis patients.

    Science.gov (United States)

    Gebara, E C E; Pannuti, C; Faria, C M; Chehter, L; Mayer, M P A; Lima, L A P A

    2004-08-01

    Helicobacter pylori is an important gastrointestinal pathogen associated with gastritis, peptic ulcers, and an increased risk of gastric carcinoma. The oral cavity has been indicated as a possible H. pylori reservoir, and may therefore be involved in the reinfection of the stomach which sometimes follows treatment of H. pylori infection. The objective of the present study was to evaluate the prevalence of H. pylori as detected by polymerase chain reaction (PCR) in the oral cavity of periodontitis patients testing positive for this bacterium in the stomach. Thirty adult patients with alterations of the superior digestive tract, testing urease positive after endoscopy and biopsy, were selected. A full-mouth periodontal examination was performed in every patient and the subjects were allocated to two groups: gingivitis (15 patients) and chronic periodontitis (15 patients). Plaque and saliva samples collected from each patient were stored in 0.5 ml of TE buffer. DNA was extracted from the samples by the boiling method and was evaluated for the presence of H. pylori using the PCR method. JW 22/23 primers were used. The DNA of ATCC H. pylori 43629 (positive control) and water (negative control) were used for controlling the reactions. Of the 30 evaluated patients, 13 (43.3%) harbored H. pylori in the mouth. The bacterium was not found on the dorsum of the tongue of any patient, but was found in saliva in three patients (10%), in the supragingival plaque in six patients (20%), and in the subgingival plaque in eight patients (26.6%). The presence of H. pylori was similar in the gingivitis and chronic periodontitis groups. In conclusion, a high percentage of patients harbored H. pylori in their mouth. The bacterium was detected in saliva, supragingival and subgingival plaque, suggesting that these sites may be considered reservoirs for H. pylori in urease-positive patients.

  15. Polymerase chain reaction targeting insertion sequence for the diagnosis of extrapulmonary tuberculosis

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    V Makeshkumar

    2014-01-01

    Full Text Available Background & objectives: Diagnosis of extrapulmonary tuberculosis (EPTB is difficult using conventional diagnostic methods. This study was conducted to evaluate the use of polymerase chain reaction (PCR in diagnosis of definitive and probable extrapulmonary tuberculosis patients, and to assess the performance of insertion sequence (IS 6110 based PCR assay as compared to conventional culture by Lowenstein-Jensen (LJ method for the diagnosis of EPTB. Methods: A total of 178 non repeated clinical specimens were collected from clinically suspected extrapulmonary tuberculosis patients. The specimens included 59 ascitic fluid, 54 pleural fluid, 25 cerebrospinal fluid (CSF, 12 fine needle aspiration (FNA, 8 urine, 7 pus, 6 synovial fluid, 2 skin tissue, one pericardial fluid, one liver abscess, one pancreatic cyst fluid, one omental biopsy and one semen sample. All these clinical samples were subjected to Ziehl-Neelsen staining (ZN for acid fast bacilli (AFB and culture on LJ medium. PCR was performed by targeting 123bp fragment of insertion sequence IS6110 of Mycobacterium tuberculosis (MTB. Results: Of the 178 specimens, 10 (5.61% were ZN smear positive for AFB, six (3.37% were L-J culture positive from 10 AFB smear positive cases and 48 (26.96% were PCR IS 6110 positive for M. tuberculosis. Interpretation & conclusions: PCR using IS6110 primer was able to pick up more EPTB patients compared to conventional L-J culture method for detection of M. tuberculosis. False positive PCR IS6110 in three CSF samples may be due to latent TB infection which was limitation in this study.

  16. Does Polymerase Chain Reaction of Tissue Specimens Aid in the Diagnosis of Tuberculosis?

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    Yoo Jin Lee

    2016-11-01

    Full Text Available Background Mycobacterial culture is the gold standard test for diagnosing tuberculosis (TB, but it is time-consuming. Polymerase chain reaction (PCR is a highly sensitive and specific method that can reduce the time required for diagnosis. The diagnostic efficacy of PCR differs, so this study determined the actual sensitivity of TB-PCR in tissue specimens. Methods We retrospectively reviewed 574 cases. The results of the nested PCR of the IS6110 gene, mycobacterial culture, TB-specific antigen-induced interferon-γ release assay (IGRA, acid-fast bacilli (AFB staining, and histological findings were evaluated. Results The positivity rates were 17.6% for PCR, 3.3% for the AFB stain, 22.2% for mycobacterial culture, and 55.4% for IGRA. PCR had a low sensitivity (51.1% and a high specificity (86.3% based on the culture results of other studies. The sensitivity was higher (65.5% in cases with necrotizing granuloma but showed the highest sensitivity (66.7% in those with necrosis only. The concordance rate between the methods indicated that PCR was the best method compared to mycobacterial culture, and the concordance rate increased for the methods using positive result for PCR or histologic features. Conclusions PCR of tissue specimens is a good alternative to detect tuberculosis, but it may not be as sensitive as previously suggested. Its reliability may also be influenced by some histological features. Our data showed a higher sensitivity when specimens contained necrosis, which indicated that only specimens with necrosis should be used for PCR to detect tuberculosis.

  17. Detection rates of trichomonas vaginalis, in different age groups, using real-time polymerase chain reaction.

    Science.gov (United States)

    Stemmer, Shlomo M; Adelson, Martin E; Trama, Jason P; Dorak, M Tevfik; Mordechai, Eli

    2012-10-01

    The study aimed to compare the overall detection rate of Trichomonas vaginalis to Chlamydia trachomatis and Neiserria gonorrhea and report detection rates by age groups. Real-time polymerase chain reaction was used to detect the presence of T. vaginalis, C. trachomatis, and N. gonorrhea in cervical samples obtained from patients during gynecological examinations. A total of 78,428, 119,451, and 117,494 samples from women age 12 to 75 years were retrospectively analyzed for the presence of T. vaginalis, C. trachomatis, and N. gonorrhea, respectively. T. vaginalis and C. trachomatis detection rates in Florida, New Jersey, and Texas were calculated in different age groups. The overall detection rate was 4.3% for T. vaginalis, 3.8% for C. trachomatis, and 0.6% for N. gonorrhea. The overall detection rate of T. vaginalis in Florida was 4.7% (n = 22,504), in New Jersey was 3.6% (n = 22,249), and in Texas was 4.5% (n = 33,675). Calculation of infection rates with T. vaginalis revealed differences between selected age groups with the highest detection rates in all 3 states found in age group 46 to 55 years (6.2%), which was higher than the overall detection rates in other age groups (p rate was found in age group 12 to 25 years (7.3%). The overall infection rates of T. vaginalis were higher compared with those of C. trachomatis and N. gonorrhea. Detection rates of T. vaginalis were found to be highest among women age 46 to 55 years and may be due to T. vaginalis infiltrating the subepithelial glands and being detected only during hormone-induced or antibiotic-induced changes in the vaginal flora.

  18. Early polymerase chain reaction detection of Chagas disease reactivation in heart transplant patients.

    Science.gov (United States)

    da Costa, Priscilla Almeida; Segatto, Marcela; Durso, Danielle Fernandes; de Carvalho Moreira, Wagson José; Junqueira, Lucas Lodi; de Castilho, Fábio Morato; de Andrade, Silvio Amadeu; Gelape, Cláudio Léo; Chiari, Egler; Teixeira-Carvalho, Andréa; Junho Pena, Sergio Danilo; Machado, Carlos Renato; Franco, Gloria Regina; Filho, Geraldo Brasileiro; Vieira Moreira, Maria da Consolação; Mara Macedo, Andréa

    2017-07-01

    Heart transplantation is a valuable therapeutic option for Chagas disease patients with severe cardiomyopathy. During patient follow-up, the differential diagnosis between cardiac transplant rejection and Chagas disease infection reactivation remains a challenging task, which hinders rapid implementation of the appropriate treatment. Herein we investigate whether polymerase chain reaction (PCR) strategies could facilitate early detection of Trypanosoma cruzi (T cruzi) in transplanted endomyocardial biopsies (EMBs). In this study we analyzed 500 EMB specimens obtained from 58 chagasic cardiac transplant patients, using PCR approaches targeted to nuclear (rDNA 24Sα) and kinetoplastid (kDNA) markers, and compared the efficiency of these approaches with that of other tests routinely used. T cruzi DNA was detected in 112 EMB specimens derived from 39 patients (67.2%). The first positive result occurred at a median 1.0 month post-transplant. Conventional histopathologic, blood smear and hemoculture analyses showed lower sensitivity and higher median time to the first positive result. Patient follow-up revealed that 31 of 39 PCR-positive cases presented clinical reactivation of Chagas disease at different time-points after transplantation. PCR techniques showed considerable sensitivity (0.82) and specificity (0.60), with area under the receiver operating characteristic (ROC) curves of 0.708 (p = 0.001). Moreover, PCR techniques anticipated the clinical signs of Chagas disease reactivation by up to 36 months, with a median time of 6 months and an average of 9.1 months. We found a good association between the PCR diagnosis and the clinical signs of the disease, indicating that the PCR approaches used herein are suitable for early diagnosis of Chagas disease reactivation, with high potential to assist physicians in treatment decisions. For this purpose, an algorithm is proposed for surveillance based on the molecular tests. Copyright © 2017 International Society for the

  19. DETECTION OF PHENOL DEGRADING BACTERIA AND PSEUDOMONAS PUTIDA IN ACTIVATED SLUDGE BY POLYMERASE CHAIN REACTION

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    H. Movahedyan ، H. Khorsandi ، R. Salehi ، M. Nikaeen

    2009-04-01

    Full Text Available Phenol is one of the organic pollutants in various industrial wastewaters especially petrochemical and oil refining. Biological treatment is one of the considerable choices for removing of phenol present in these wastewaters. Identification of effective microbial species is considered as one of the important priorities for production of the biomass in order to achieve desirable kinetic of biological reactions. Basic purpose of this research is identification of phenol-degrading Pseudomonas Putida in activated sludge by polymerase chain reaction (PCR that has high speed and specificity. In this research, 10 various colonies of phenol-degrading bacteria were isolated from municipal activated sludge and the rate of phenol removal and growth rate of these bacteria were assessed in different concentrations of phenol (200 – 900 mg/L. Confirmation of the largest subunit of multicomponent phenol hydroxylase (LmPH gene and gene coding the N fragment in Pseudomonas Putida-derived methyl phenol operon (DmpN gene through PCR were used for general identification of phenol-degrading bacteria and Pseudomonas Putida, respectively. Presence of a 600 bp (base pairs bond in all of isolated strains indicated that they contain phenol hydroxylase gene. 6 of 10 isolated bacteria were Pseudomonas Putida because they produced a 199 bp PCR product by DmpN primers. According to PCR results in this study, the best phenol-degrading bacteria that can utilize 500 – 600 mg/L phenol completely after 48 hours incubation, belong to Pseudomonas Putida strains. It is clear that use of isolated bacteria can lead to considerable decrease of treatment time as well as promotion of phenol removal rate.

  20. Enteroviral RNA sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome.

    Science.gov (United States)

    Gow, J W; Behan, W M; Clements, G B; Woodall, C; Riding, M; Behan, P O

    1991-03-23

    To determine the presence of enteroviral sequences in muscle of patients with the postviral fatigue syndrome. Detection of sequences with the polymerase chain reaction in a well defined group of patients with the syndrome and controls over the same period. Institute of Neurological Sciences, Glasgow. 60 consecutive patients admitted to the institute with the postviral fatigue syndrome who had undergone extensive investigation to exclude other conditions. 41 controls from the same catchment area without evidence of fatigue, all undergoing routine surgery. Routine investigations, serological screen for antibodies to a range of viruses, and presence of enteroviral RNA sequences in muscle biopsy specimens. 15 (25%) patients and 10 (24.4%) controls had important serological findings. 12 patients had neutralising antibody titres of greater than or equal to 256 to coxsackieviruses B1-5 (six positive for enteroviral RNA sequences, six negative); three were positive for Epstein-Barr virus specific IgM (two positive, one negative). Six controls had similar neutralising antibody titres to coxsackieviruses (all negative); one was positive for Epstein-Barr virus specific IgM (negative); and three had titres of complement fixing antibody greater than or equal to 256 to cytomegalovirus (all negative). Overall, significantly more patients than controls had enteroviral RNA sequences in muscle (32/60, 53% v 6/41, 15%; odds ratio 6.7, 95% confidence interval 2.4 to 18.2). This was not correlated with duration of disease, patient and age, or to raised titres of antibodies to coxsackieviruses B1-5. Persistent enteroviral infection of muscle may occur in some patients with postviral fatigue syndrome and may have an aetiological role.

  1. Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Arakawa, C.K.; Deering, R.E.; Higman, K.H.; Oshima, K.H.; O'Hara, P.J.; Winton, J.R.

    1990-01-01

    The polymerase chain reaction [PCR) was used to amplify a portion of the nucleoprotein [NI gene of infectious hematopoietic necrosis virus (IHNV). Using a published sequence for the Round Butte isolate of IHNV, a pair of PCR pnmers was synthesized that spanned a 252 nucleotide region of the N gene from residue 319 to residue 570 of the open reading frame. This region included a 30 nucleotide target sequence for a synthetic oligonucleotide probe developed for detection of IHNV N gene messenger RNA. After 25 cycles of amplification of either messenger or genomic RNA, the PCR product (DNA) of the expected size was easily visible on agarose gels stained with ethidium bromide. The specificity of the amplified DNA was confirmed by Southern and dot-blot analysis using the biotinylated oligonucleotide probe. The PCR was able to amplify the N gene sequence of purified genomic RNA from isolates of IHNV representing 5 different electropherotypes. Using the IHNV primer set, no PCR product was obtained from viral hemorrhagic septicemia virus RNA, but 2 higher molecular weight products were synthesized from hirame rhabdovirus RNA that did not hybridize with the biotinylated probe. The PCR could be efficiently performed with all IHNV genomic RNA template concentrations tested (1 ng to 1 pg). The lowest level of sensitivity was not determined. The PCR was used to amplify RNA extracted from infected cell cultures and selected tissues of Infected rainbow trout. The combination of PCR and nucleic acid probe promises to provide a detection method for IHNV that is rapid, h~ghly specific, and sensitive.

  2. Polymerase chain reaction targeting insertion sequence for the diagnosis of extrapulmonary tuberculosis

    Science.gov (United States)

    Makeshkumar, V.; Madhavan, Radha; Narayanan, Sujatha

    2014-01-01

    Background & objectives: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult using conventional diagnostic methods. This study was conducted to evaluate the use of polymerase chain reaction (PCR) in diagnosis of definitive and probable extrapulmonary tuberculosis patients, and to assess the performance of insertion sequence (IS) 6110 based PCR assay as compared to conventional culture by Lowenstein-Jensen (LJ) method for the diagnosis of EPTB. Methods: A total of 178 non repeated clinical specimens were collected from clinically suspected extrapulmonary tuberculosis patients. The specimens included 59 ascitic fluid, 54 pleural fluid, 25 cerebrospinal fluid (CSF), 12 fine needle aspiration (FNA), 8 urine, 7 pus, 6 synovial fluid, 2 skin tissue, one pericardial fluid, one liver abscess, one pancreatic cyst fluid, one omental biopsy and one semen sample. All these clinical samples were subjected to Ziehl-Neelsen staining (ZN) for acid fast bacilli (AFB) and culture on LJ medium. PCR was performed by targeting 123bp fragment of insertion sequence IS6110 of Mycobacterium tuberculosis (MTB). Results: Of the 178 specimens, 10 (5.61%) were ZN smear positive for AFB, six (3.37%) were L-J culture positive from 10 AFB smear positive cases and 48 (26.96%) were PCR IS 6110 positive for M. tuberculosis. Interpretation & conclusions: PCR using IS6110 primer was able to pick up more EPTB patients compared to conventional L-J culture method for detection of M. tuberculosis. False positive PCR IS6110 in three CSF samples may be due to latent TB infection which was limitation in this study. PMID:24604051

  3. Enhancing tuberculosis diagnosis by polymerase chain reaction: An experience at a tertiary hospital

    Directory of Open Access Journals (Sweden)

    Fernanda de-Paris

    2016-05-01

    Full Text Available Introduction: Tuberculosis (TB persists as a severe global public health issue. The aim of the present study was to evaluate the performance of an in-house TB PCR (polymerase chain reaction in sputum. Methods: DNA from sputum specimens were submitted to a nested-PCR protocol for the IS6110 region detection. PCR results were compared to those of the traditional methods for TB diagnosis, i.e., acid-fast bacilli (AFB smear microscopy and culture. We analyzed sputum samples obtained from 133 patients. Results: A total of 48 (36% cultures yielded indeterminate results due to contamination. This high contamination rate may be explained by the fact that samples from fibrocystic patients were included in this study. Additionally, other five samples were positive for nontuberculous mycobacteria (NTM. Therefore, it was possible to compare 80 patients for M. tuberculosis detection. We found 14 positive samples: five presented positive results in the three methods (5/14; 35.7%, two were positive in culture and PCR (2/14; 14.3%, one was positive in AFB and PCR (1/14; 7.1%, five were positive only in PCR (5/14; 35.7% and 1 was positive only in culture (1/14; 7.1%. Thus, positivity rates for each technique were: 7.5% for AFB (6/80, 10% for culture (8/80 and 16.25% for PCR (13/80. Among the 48 patients who had indeterminate results in sputum culture, two samples were positive in PCR. Conclusion: Considering the limitations of the traditional methods, the use of PCR as a molecular technique could be advantageous for TB diagnosis.

  4. Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method.

    Science.gov (United States)

    Hartung, J S; Daniel, J F; Pruvost, O P

    1993-04-01

    pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and other strains of X. campestris associated with Citrus spp. as templates in the PCR. Four primer pairs directed the amplification of target DNA from X. campestris pv. citri strains but not from strains of X. campestris associated with a different disease, citrus bacterial spot. Primer pair 2-3 directed the specific amplification of target DNA from pathotype A but not other pathotypes of X. campestris pv. citri. A pH 9.0 buffer that contained 1% Triton X-100 and 0.1% gelatin was absolutely required for the successful amplification of the target DNA, which was 61% G+C. Limits of detection after amplification and gel electrophoresis were 25 pg of purified target DNA and about 10 cells when Southern blots were made after gel electrophoresis and probed with biotinylated pFL1. This level of detection represents an increase in sensitivity of about 100-fold over that of dot blotting with the same hybridization probe. PCR products of the expected sizes were amplified from DNA extracted from 7-month-old lesions from which viable bacteria could not be isolated. These products were confirmed to be specific for X. campestris pv. citri by Southern blotting. This PCR-based detection protocol will be a useful addition to current methods of detection of this pathogen, which is currently the target of international quarantine measures.

  5. Detection of nanobacteria in patients with chronic prostatitis and vaginitis by reverse transcriptase polymerase chain reaction.

    Science.gov (United States)

    Kim, Tae-Hyoung; Kim, Hye Ryoun; Myung, Soon-Chul

    2011-03-01

    We aimed to investigate the detection of nanobacteria (NB) from expressed prostatic secretions (EPS) in patients with category III chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and from vaginal swabs in patients with vaginitis by reverse transcriptase polymerase chain reaction (RT-PCR) and to evaluate the association between NB and Neisseria gonorrhea, Chlamydia trachomatis, Ureaplasma urealyticum (U. urealyticum), Mycoplasma hominis, Trichomonas vaginalis, and Mycoplasma genitalium. A group of 11 men attending a specialized CP/CPPS clinic and a group of 157 women who reported symptoms of lower genital tract infection were enrolled in this study. NB were detected by RT-PCR. A Seeplex Sexually Transmitted Disease Detection assay (Seegene Inc., Seoul, Korea) was used that could detect DNA for 6 types of sexually transmitted pathogens. In EPS samples, the detection rate of NB in patients with CP/CPPS was 9.1%, and 9 (5.7%) of 157 vaginitis patients showed positive results in RT-PCR for NB in vaginal swabs. Associations observed among the 7 microorganisms included 6 (54.5%) patients who tested positive on EPS and 75 (47.8%) patients who tested positive on vaginal swabs. Five patients with vaginitis were found to have monoinfection of NB (6.7%). We found that conventional RT-PCR for NB was rapid, simple, low in cost, and easily available for the detection of NB, and that NB may be a possible etiological factor for vaginitis and CP/CPPS. The prevalence of U. urealyticum among the four patients with NB coinfection was 75%; the presence of U. urealyticum might therefore raise suspicion for nanobacterial infection.

  6. Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in central Africa, by the polymerase chain reaction

    OpenAIRE

    Penchenier, Laurent; Simo, G.; Grébaut, Pascal; Nkinin, S.; Laveissière, Claude; Herder, Stéphane

    2000-01-01

    During a mass screening of sleeping sickness conducted in 1998 and 1999, and involving 27,932 persons in Cameroon and the Central African Republic, we tested the polymerase chain reaction (PCR) on whole blood for the diagnosis of human African trypanosomiasis due to #Trypanosoma brucei gambiense$. The 1858 samples obtained were from 4 groups : 155 infected patients, 1432 serological suspects detected by the card agglutination test for trypanosomiasis (CATT), 222 negative controls living in th...

  7. Diagnosis of foot-and mouth disease by real time reverse transcription polymerase chain reaction under field conditions in Brazil

    OpenAIRE

    Clarke Neville P; Martin Barbara M; Beckham Tammy R; Silva René R; Serra Claudia V; Barbosa Meirivan S; Reis Jorge R; Paiva Naimes O; Neta Alcina; Paixão Tatiane A; Adams L Garry; Santos Renato L

    2008-01-01

    Abstract Background Foot-and-mouth disease (FMD) is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. Virus isolation and enzyme-linked immunosorbent assay (ELISA) are the gold standard tests for diagnosis of FMD. As these methods are time consuming, assays based on viral nucleic acid amplification have been developed. Results A previously described real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay with h...

  8. [The use of the polymerase chain reaction in analyzing ancient DNAs (exemplified by that of the Enmynveem mammoth)].

    Science.gov (United States)

    Maliarchuk, B A; Derevenko, M V; Lapinskiĭ, A G; Solovenchuk, L L

    1996-01-01

    DNA was isolated from the Enmynveem mammoth muscles, and the control region and cytochrome b gene of the mitochondrial genome were analyzed by polymerase chain reaction (PCR). The mammoth DNA was amplified by both the classical PCR (two primer system) and the single-primer PCR (spPCR) resulting in DNA fragments up to 1600 bp long. Restriction analysis of the mitochondrial cytochrome b gene was carried out. Cytochromes b genes in three Elephantidae genera were compared.

  9. Impact of toxigenic Clostridium difficile polymerase chain reaction testing on the clinical microbiology laboratory and inpatient epidemiology.

    Science.gov (United States)

    Napierala, Maureen; Munson, Erik; Skonieczny, Patrice; Rodriguez, Sonia; Riederer, Nancy; Land, Gayle; Luzinski, Mary; Block, Denise; Hryciuk, Jeanne E

    2013-08-01

    Conversion from Clostridium difficile toxin A/B EIA to tcdB polymerase chain reaction for diagnosis of C. difficile infection (CDI) resulted in significant decreases in laboratory testing volume and largely unchanged C. difficile toxin detection rates. Decreases in healthcare-associated CDI rates (P ≤ 0.05) reflected a clinical practice benefit of this conversion. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Analysis of clinical information and reverse transcriptase-polymerase chain reaction for early diagnosis of enteroviral meningitis

    OpenAIRE

    Jin, Dahee; Heo, Tae Hoon; Byeon, Jung Hye; Kim, Gun-Ha; Kim, Mi Kyung; Eun, So-Hee; Eun, Baik-Lin

    2015-01-01

    Purpose Meningitis is among the most common infections affecting the central nervous system. It can be difficult to determine the exact pathogen responsible for the infection and patients are often treated with empiric antibiotics. This study was conducted to identify the most common clinical characteristics of enteroviral meningitis in children and evaluate the diagnostic efficacy of reverse transcriptase-polymerase chain reaction (RT-PCR) for early detection of an enterovirus. Methods We an...

  11. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix gene...

  12. Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

    OpenAIRE

    Valdivia, Angel; Savón, Clara; Chacón, Danay; Sarmiento, Luis; Morier, Luis; Otero, Anselmo; Soto, Yudira; Oropesa, Suset; Goyenechea, Angel

    1997-01-01

    The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with vir...

  13. Peripheral Blood Leukocytes and Serum Nested Polymerase Chain Reaction Are Complementary Methods for Monitoring Active Cytomegalovirus Infection in Transplant Patients

    Directory of Open Access Journals (Sweden)

    PD Andrade

    2013-01-01

    Full Text Available BACKGROUND: Human cytomegalovirus is an important cause of morbidity and mortality in immunocompromised patients. Qualitative polymerase chain reaction (PCR has proven to be a sensitive and effective technique in defining active cytomegalovirus infection, in addition to having low cost and being a useful test for situations in which there is no need for quantification. Real-time PCR has the advantage of quantification; however, the high cost of this methodology makes it impractical for routine use.

  14. Interlaboratory validation data on real-time polymerase chain reaction detection for unauthorized genetically modified papaya line PRSV-YK

    Directory of Open Access Journals (Sweden)

    Kosuke Nakamura

    2016-06-01

    Real-time polymerase chain reaction (PCR detection method for unauthorized genetically modified (GM papaya (Carica papaya L. line PRSV-YK (PRSV-YK detection method was developed using whole genome sequence data (DDBJ Sequenced Read Archive under accession No. PRJDB3976. Interlaboratory validation datasets for PRSV-YK detection method were provided. Data indicating homogeneity of samples prepared for interlaboratory validation were included. Specificity and sensitivity test data for PRSV-YK detection method were also provided.

  15. Evaluation of gold nanoparticles as the additive in real-time polymerase chain reaction with SYBR Green I dye

    Energy Technology Data Exchange (ETDEWEB)

    Yang Wenchao; Cao Xueyan; Zhang Xiaodong; Hu Jun [Nanobiology Laboratory, Bio-X Life Science Research Center, College of Life Science and Biotechnology, Shanghai JiaoTong University, Shanghai 200240 (China); Mi Lijuan; Fan Chunhai [Shanghai Institute of Applied Physics, Chinese Academy of Sciences, PO Box 800-204, Shanghai 201800 (China)], E-mail: xdzhang@sjtu.edu.cn

    2008-06-25

    Gold nanoparticles (AuNPs) have been proven to be able to improve the specificity or increase the efficiency of a polymerase chain reaction (PCR) when a suitable amount of AuNPs was used. However, there is still a lack of systematic evaluation of AuNPs in real-time PCR. In this study, DNA degradation and the fluorescence quenching effect of AuNPs were first tested in real-time PCR. Then two different kinds of Taq DNA polymerase, native and recombinant Taq polymerase, were employed to evaluate the AuNPs' effect on the threshold cycle (C{sub T}) values, standard curves and melting curves in real-time PCR. Different ratios of the amount of native Taq DNA polymerase to the amount of AuNPs were also tested. It was found that AuNPs could be applied in real-time PCR with correlation coefficient R{sup 2}>0.989. The combination of 2.09 nM AuNPs with 3.75 U of native Taq DNA polymerase could make the amplification curves shift to the left and enhance the efficiency of the real-time PCR (0.628 39 without AuNPs compared with 0.717 89 with 2.09 nM AuNPs), thus enabling faster detection in comparison with those of control samples. However, no improvement ability of AuNPs was found in real-time PCR based on recombinant rTaq DNA polymerase. Besides, the results suggest that a complex interaction exists between AuNPs and native Taq DNA polymerase.

  16. Dispersion quality of amine functionalized multiwall carbon nanotubes plays critical roles in polymerase chain reaction enhancement

    Energy Technology Data Exchange (ETDEWEB)

    Yuce, Meral, E-mail: meralyuce@sabanciuniv.edu; Budak, Hikmet [Sabanci University, Nanotechnology Research and Application Centre (Turkey)

    2014-12-15

    Impact of dispersion quality of NH{sub 2}-MWCNTs (13–18 nm in diameter with a length between 1 and 12 µm, >99 % purity) in the amplification efficiency of a random DNA oligonucleotide library (96 bp) was investigated. Amplification yield in the presence of non-filtered NH{sub 2}-MWCNT dispersion, filtered NH{sub 2}-MWCNT dispersion and surface-attached NH{sub 2}-MWCNTs was explored, and physical interactions between NH{sub 2}-MWCNTs and major PCR reagents including DNA template, wild type Taq DNA polymerase enzyme and primers were determined using high resolution polyacrylamide gel electrophoresis, dynamic light scattering, UV–Vis-NIR spectroscopy and scanning electron microscopy techniques. The results revealed that presence of NH{sub 2}-MWCNT dispersion which was sonicated, centrifuged and filtered, enhanced the total PCR efficiency up to 70 % while the presence of NH{sub 2}-MWCNT only centrifuged after sonication, inhibited the reaction significantly at similar concentrations. Furthermore, the NH{sub 2}-MWCNTs coupled covalently onto magnetic microspheres, contributed for the specificity enhancement whilst decreasing the amplification efficiency by 30 % at the maximum concentration, which suggests a removable enhancement system for sensitive applications. On the other hand, the relative hydrodynamic size distribution measurements displayed a clear difference between the filtered NH{sub 2} and non-filtered NH{sub 2}-MWCNT water dispersions, which justifies the inhibition of the amplification by the non-filtered NH{sub 2}-MWCNTs containing big agglomerates and bundles. Finally, we demonstrated that major PCR components adsorb onto the NH{sub 2}-MWCNTs with diverse affinities, and maintain their functions after adsorption, which provides a good framework to further develop tunable NH{sub 2}-MWCNT-carriers to be utilized in various nanobiotechnology and material science applications.

  17. Dispersion quality of amine functionalized multiwall carbon nanotubes plays critical roles in polymerase chain reaction enhancement

    Science.gov (United States)

    Yuce, Meral; Budak, Hikmet

    2014-12-01

    Impact of dispersion quality of NH2-MWCNTs (13-18 nm in diameter with a length between 1 and 12 µm, >99 % purity) in the amplification efficiency of a random DNA oligonucleotide library (96 bp) was investigated. Amplification yield in the presence of non-filtered NH2-MWCNT dispersion, filtered NH2-MWCNT dispersion and surface-attached NH2-MWCNTs was explored, and physical interactions between NH2-MWCNTs and major PCR reagents including DNA template, wild type Taq DNA polymerase enzyme and primers were determined using high resolution polyacrylamide gel electrophoresis, dynamic light scattering, UV-Vis-NIR spectroscopy and scanning electron microscopy techniques. The results revealed that presence of NH2-MWCNT dispersion which was sonicated, centrifuged and filtered, enhanced the total PCR efficiency up to 70 % while the presence of NH2-MWCNT only centrifuged after sonication, inhibited the reaction significantly at similar concentrations. Furthermore, the NH2-MWCNTs coupled covalently onto magnetic microspheres, contributed for the specificity enhancement whilst decreasing the amplification efficiency by 30 % at the maximum concentration, which suggests a removable enhancement system for sensitive applications. On the other hand, the relative hydrodynamic size distribution measurements displayed a clear difference between the filtered NH2 and non-filtered NH2-MWCNT water dispersions, which justifies the inhibition of the amplification by the non-filtered NH2-MWCNTs containing big agglomerates and bundles. Finally, we demonstrated that major PCR components adsorb onto the NH2-MWCNTs with diverse affinities, and maintain their functions after adsorption, which provides a good framework to further develop tunable NH2-MWCNT-carriers to be utilized in various nanobiotechnology and material science applications.

  18. Prognostic significance of bi/oligoclonality in childhood acute lymphoblastic leukemia as determined by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Carlos Alberto Scrideli

    2001-09-01

    Full Text Available CONTEXT: The CDR-3 region of heavy-chain immunoglobulin has been used as a clonal marker in the study of minimal residual disease in children with acute lymphoblastic leukemia. Southern blot and polymerase chain reaction studies have demonstrated the occurrence of bi/oligoclonality in a variable number of cases of B-lineage acute lymphoblastic leukemia, a fact that may strongly interfere with the detection of minimal residual disease. Oligoclonality has also been associated with a poorer prognosis and a higher chance of relapse. OBJECTIVES: To correlate bi/oligoclonality, detected by polymerase chain reaction in Brazilian children with B-lineage acute lymphoblastic leukemia with a chance of relapse, with immunophenotype, risk group, and disease-free survival. DESIGN: Prospective study of patients’ outcome. SETTING: Pediatric Oncology Unit of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo. PARTICIPANTS: 47 children with acute lymphoblastic leukemia DIAGNOSTIC TEST: Polymerase chain reaction using consensus primers for the CDR-3 region of heavy chain immunoglobulin (FR3A, LJH and VLJH for the detection of clonality. RESULTS: Bi/oligoclonality was detected in 15 patients (31.9%. There was no significant difference between the groups with monoclonality and biclonality in terms of the occurrence of a relapse (28.1% versus 26.1%, presence of CALLA+ (81.2% versus 80% or risk group (62.5% versus 60%. Disease-free survival was similar in both groups, with no significant difference (p: 0.7695. CONCLUSIONS: We conclude that bi/oligoclonality was not associated with the factors investigated in the present study and that its detection in 31.9% of the patients may be important for the study and monitoring of minimal residual disease.

  19. Antiadenoviral effects of N-chlorotaurine in vitro confirmed by quantitative polymerase chain reaction methods

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    Eiichi Uchio

    2010-11-01

    Full Text Available Eiichi Uchio1, Hirotoshi Inoue1, Kazuaki Kadonosono21Department of Ophthalmology, Fukuoka University School of Medicine, Fukuoka, Japan; 2Department of Ophthalmology, Yokohama City University Medical Center, Yokohama, JapanPurpose: Adenoviral keratoconjunctivitis is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. N-Chlorotaurine (Cl–HN–CH2–CH2–SO3H, NCT is the N-chloro derivative of the amino acid taurine, which is an oxidant produced by human granulocytes and monocytes during inflammatory reactions. Using conventional viral plaque assay, it was previously shown that NCT causes inactivation of several human adenovirus (HAdV serotypes. In this study, we evaluated the antiadenoviral effect of NCT by quantitative polymerase chain reaction (PCR methods.Methods: A549 cells were used for viral cell culture, and HAdV serotypes 3, 4, 8, 19, and 37 were used. After calculating 50% cytotoxic concentration (CC50 of NCT by MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium method, HAdV was cultured with NCT for 7 days, and extracted adenoviral DNA was quantitatively measured by real-time PCR.Results: A statistically significant (P < 0.05 dose-dependent inhibition was indicated for all serotypes except HAdV type 4 (HAdV4, which was maximally inhibited by only ~50%. Among the serotypes, NCT was particularly effective against HAdV8, HAdV19a, and HAdV37. The 50% effective concentration (EC50 obtained by real-time PCR of NCT ranged between 49 and 256 µM. EC50 of NCT against HAdV3 was slightly higher than that against serotypes of species D. The selective index (CC50/EC50 ranged between 41 and 60 except for HAdV4 (11.5.Conclusions: These results show that NCT has an antiviral effect against most serotypes of human HAdV inducing keratoconjunctivitis, indicating its possible therapeutic use.Keywords: adenovirus, N-chlorotaurine, epidemic keratoconjunctivitis, antiviral

  20. Detection of Food Hazards in Foods: Comparison of Real Time Polymerase Chain Reaction and Cultural Methods.

    Science.gov (United States)

    Bonilauri, Paolo; Bardasi, Lia; Leonelli, Roberto; Ramini, Mattia; Luppi, Andrea; Giacometti, Federica; Merialdi, Giuseppe

    2016-01-18

    Foodstuffs should not contain microorganisms or their toxins or metabolites in quantities suggesting an unacceptable risk for human health. The detection of food hazards in foods is performed by several tests that produce results dependent on the analytical method used: an analytical reference method, defined as standard, is associated with each microbiological criterion laid down in Regulation 2073/2005/EC, but, analytical methods other than the reference ones, in particular more rapid methods, could be used. Combined screening methods performed by real time-polymerase chain reaction (RT-PCR) are currently validated as alternative methods according to the ISO 16140:2003 and certified by the Association Française de Normalisation. However, the positive results obtained with these alternative methods, the investigated molecular relations that resulted positive have to be confirmed with cultural methods using the same enrichment media in which the molecular screening was performed. Since it is necessary to assess if these testing schemes provide equivalent guarantees of food safety, the aim of this retrospective study is to analyse the data collected, from 2012 to 2014 by Emilia Romagna Region in the field of Piano Regionale Alimenti (Food Regional Plan) during official controls monitoring food samples of animal and other than animal origin. Records performed by combined methods of molecular screening of Salmonella spp., Listeria monocytogenes and thermophilic Campylobacter and cultural confirmation results were gathered together and the results were compared in order to assess the sensitivity of the methods. A total of 10,604 food samples were considered in this study: the comparison of the data revealed that the RT-PCR method detected Salmonella, L. monocytogenes, and thermophilic Campylobacter in 2.18, 3.85 and 3.73% of the samples, respectively, whereas by using cultural method these pathogens were isolated in 0.43, 1.57 and 1.57% of samples, respectively. In

  1. Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.

    Science.gov (United States)

    Rocha-Silva, Fabiana; Gomes, Luciana I; Gracielle-Melo, Cidiane; Goes, Alfredo M; Caligiorne, Rachel B

    2017-01-01

    Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological

  2. One-heater flow-through polymerase chain reaction device by heat pipes cooling.

    Science.gov (United States)

    Chen, Jyh Jian; Liao, Ming Huei; Li, Kun Tze; Shen, Chia Ming

    2015-01-01

    This study describes a novel microfluidic reactor capable of flow-through polymerase chain reactions (PCR). For one-heater PCR devices in previous studies, comprehensive simulations and experiments for the chip geometry and the heater arrangement were usually needed before the fabrication of the device. In order to improve the flexibility of the one-heater PCR device, two heat pipes with one fan are used to create the requisite temperature regions in our device. With the integration of one heater onto the chip, the high temperature required for the denaturation stage can be generated at the chip center. By arranging the heat pipes on the opposite sides of the chip, the low temperature needed for the annealing stage is easy to regulate. Numerical calculations and thermal measurements have shown that the temperature distribution in the five-temperature-region PCR chip would be suitable for DNA amplification. In order to ensure temperature uniformity at specific reaction regions, the Re of the sample flow is less than 1. When the microchannel width increases and then decreases gradually between the denaturation and annealing regions, the extension region located in the enlarged part of the channel can be observed numerically and experimentally. From the simulations, the residence time at the extension region with the enlarged channel is 4.25 times longer than that without an enlarged channel at a flow rate of 2 μl/min. The treated surfaces of the flow-through microchannel are characterized using the water contact angle, while the effects of the hydrophilicity of the treated polydimethylsiloxane (PDMS) microchannels on PCR efficiency are determined using gel electrophoresis. By increasing the hydrophilicity of the channel surface after immersing the PDMS substrates into Tween 20 (20%) or BSA (1 mg/ml) solutions, efficient amplifications of DNA segments were proved to occur in our chip device. To our knowledge, our group is the first to introduce heat pipes into

  3. Results of Multiplex Polymerase Chain Reaction Assay to Identify Urethritis Pathogens

    Directory of Open Access Journals (Sweden)

    Mehmet Sarıer

    2017-03-01

    Full Text Available Objective: The purpose of this study was to evaluate the results of multiplex polymerase chain reaction (PCR test applied to identify the pathogens in male patients who attended our urology clinic with a pre-diagnosis of urethritis related with sexual intercourse. Materials and Methods: In this study, we included a total of 91 male patients, who sought medical advice in our clinic between August 2015 and October 2016 due to complaints of urethral discharge, dysuria and urethral itching, having a visible urethral discharge during the physical examination or a positive leukocyte esterase test (Combur-Test®-Roche in the first urine sample. In the urethral swab samples of these patients, urethritis pathogens were searched with a multiplex PCR test. The multiplex PCR kit, which is able to identify nine pathogens and produced by PathoFinder® (Holland, was used in the process. The pathogens that could be detected by the kit were Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Gardnerella vaginalis, Trichomonas vaginalis, Treponema pallidum, and Candida albicans. Results: The average age of the subjects was 35.1 (19-57 years. Sixty one out of 91 patients (67% were found to have a pathogen in the urethral swab sample. In 45 patients (49.4%, only one pathogen, in 12 (13.1% - two different pathogens and in 4 (4.3% patients, 3 different pathogens were detected. The pathogens found were as follows: Ureaplasma urealyticum in 22 patients (27.1%, Gardnerella vaginalis in 15 (18.6%, Neisseria gonorrhoeae in 13 (16.1%, Mycoplasma genitalium (10 patients; 12.3%, Mycoplasma hominis (8 patients; 9.9%, Chlamydia trachomatis (8 patients; 9.9%, Trichomonas vaginalis (3 patients; 3.8%, and Candida albicans (2 patients; 2.4%. None of the patients were identified with Treponema pallidum. None of the pathogens were identified in 30 patients (32.9% whose samples were examined by PCR method. Conclusion

  4. Optimizing polymerase chain reaction testing for the diagnosis of pertussis: current perspectives

    Directory of Open Access Journals (Sweden)

    Arbefeville S

    2015-09-01

    Full Text Available Sophie Arbefeville, Patricia Ferrieri Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN, USA Abstract: Nucleic acid testing has revolutionized the diagnosis of pertussis in the clinical microbiology laboratory and has become the main avenue of testing for pertussis infection. Real-time polymerase chain reaction (RT-PCR is an important tool for timely diagnosis of pertussis and is more sensitive than culture. The most commonly amplified targets are the insertion-sequence (IS genes, which are found in multiple copies in the genome of Bordetella species. Some strains of Bordetella pertussis have more than 200 copies of IS481 in their genome. This high number of repeats allows RT-PCR assays to be very sensitive and makes nucleic acid testing two to three times more sensitive than culture. Despite these advantages, RT-PCR can give inaccurate results due to contamination or lack of specificity. Contamination can easily happen during specimen collection, DNA extraction, or nucleic acid amplification steps. To avoid contamination, laboratories need to have quality controls and good workflows in place. The poor specificity of the nucleic acid assays amplifying the IS genes is because they are found in various Bordetella species and, thus, not unique to a specific species. Bordetella holmesii, a more recently described Bordetella species found to be responsible for respiratory symptoms similar to pertussis in adolescents and adults, can be misidentified as B. pertussis in RT-PCR assays that amplify only the IS481 target. Use of multiple targets may improve specificity of RT-PCR assays for pertussis. In the past few years, the US Food and Drug Administration has cleared three commercial assays for the detection of B. pertussis in respiratory specimens. Several commercial assays and analyte-specific reagents, which are not US Food and Drug Administration cleared, are available for the detection of one

  5. An exploratory study to evaluate Clostridium difficile polymerase chain reaction ribotypes and infection outcomes

    Directory of Open Access Journals (Sweden)

    Thabit AK

    2016-06-01

    Full Text Available Abrar K Thabit,1,2 David P Nicolau1,3 1Center for Anti-Infective Research and Development, Hartford Hospital, Hartford, CT, USA; 2Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 3Division of Infectious Diseases, Hartford Hospital, Hartford, CT, USA Background: Clostridium difficile infection ranges from mild to severe prolonged diarrhea with systemic symptoms. Previous studies have assessed the correlation of some disease severity parameters to C. difficile ribotypes. However, certain clinical parameters of interest have not yet been evaluated.Aim: We conducted an exploratory study to evaluate the correlation of C. difficile ribotypes to parameters not assessed previously, notably days to diarrhea resolution (in terms of days to formed stools and days to less than three stools per day, length of hospital stay, 30-day recurrence rates, and 30-day readmission rates. Additional severity parameters evaluated include leukocytosis, serum creatinine, fever, and nausea/vomiting.Methods: Polymerase chain reaction ribotyping was performed on C. difficile isolates from baseline stool samples of 29 patients. A retrospective chart review was conducted to assess the parameters of interest.Results: The most common ribotypes were 027 (38%, 014/020 (21%, and 106/174 (21%. Numerically, 027 ribotype patients required more days to less than three stools per day versus 014/020 and 106/174 ribotype patients (P=0.2. The three ribotypes were similar regarding time to formed stools, duration of hospitalization, and 30-day readmission rate (P=0.2, 0.6, and 0.8, respectively. Recurrence within 30 days occurred in two patients with 027 and two patients with 014/020 (P=0.6. Leukocytosis and fever were more prominent with 027 than with 014/020 and 106/174 (P=0.04 for both parameters, although the degree of nausea/vomiting did not differ between the three groups (P=0.3. A serum creatinine level ≥1.5 times the premorbid level was seen in only three

  6. Integrated continuous flow polymerase chain reaction and micro-capillary electrophoresis system with bioaffinity preconcentration.

    Science.gov (United States)

    Njoroge, Samuel K; Witek, Magorzata A; Battle, Katrina N; Immethun, Vicki E; Hupert, Mateusz L; Soper, Steven A

    2011-11-01

    An integrated and modular DNA analysis system is reported that consists of two modules: (i) A continuous flow polymerase chain reaction (CFPCR) module fabricated in a high T(g) (150°C) polycarbonate substrate in which selected gene fragments were amplified using biotin and fluorescently labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones as opposed to heating and cooling large thermal masses typically performed in batch-type thermal reactors. (ii) μCE (micro-capillary electrophoresis) module fabricated in poly(methylmethacrylate) (PMMA), which utilized a bioaffinity selection and purification bed (2.9  μL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to electrophoretic sorting. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed, where they were extracted onto the surface of micropillars. The affinity bed was also fabricated in PMMA and was populated with an array of microposts (50  μm width; 100  μm height) yielding a total surface area of ∼117  mm(2). This solid-phase extraction (SPE) process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (K(d) = 10(-15)  M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single-stranded DNA released for injection into a 7-cm-long μCE channel for size-based separations and fluorescence detection. The utility of the system was demonstrated using Alu DNA typing for gender and ethnicity determinations as a model. Compared with the traditional cross-T injection procedure typically used for μCE, the affinity pre-concentration and injection procedure generated signal enhancements of 17- to 40-fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. A microfluidic device providing continuous-flow polymerase chain reaction heating and cooling

    Science.gov (United States)

    Harandi, A.; Farquhar, T.

    2014-11-01

    The objective of this study is to describe a new type of microfluidic device that could be used to manipulate fluid temperature in many microfluidic applications. The key component is a composite material containing a thermally conductive phase placed in a purposeful manner to manipulate heat flow into and out of an embedded microchannel. In actual use, the device is able to vary temperature along a defined flow path with remarkable precision. As a demonstration of capability, a functional prototype was designed and fabricated using four layers of patterned copper laminated between alternating layers of polyimide and acrylic. The key fabrication steps included laser micromachining, acid etching, microchannel formation, and hot lamination. In order to achieve the desired temperature variations along the microchannel, an outer optimization loop and an inner finite element analysis loop were used to iteratively obtain a near-optimal copper pattern. With a minor loss of generality, admissible forms were restricted to comb-like patterns. For a given temperature profile, the pattern was found by refining a starting guess based on a deterministic rubric. Thermal response was measured using fine thermocouples placed at critical locations along the microchannel wall. At most of these points, the agreement between measured and predicted temperatures was within 1 °C, and temperature gradients as high as ±45 °C mm-1 (equivalent to ±90 °C s-1 at 2 μl min-1 flow rate) were obtained within the range of 59-91 °C. The particular profile chosen for case study makes it possible to perform five cycles of continuous-flow polymerase chain reaction (PCR) in less than 15 s, i.e. it entails five successive cycles of cooling from 91 to 59 °C, rapid reheating from 59 to 73 °C, slow reheating from 73 to 76 °C, and a final reheating from 73 to 91 °C, using a resistively heated source at 100 °C at and a thermoelectrically cooled sink at 5 °C.

  8. Pelacakan Kasus Flu Burung pada Ayam dengan Reverse Transcriptase Polymerase Chain Reaction* (DETECTION OF AVIAN INFLUENZA IN CHICKENS BY REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION

    Directory of Open Access Journals (Sweden)

    Gusti Ayu Yuniati Kencana

    2013-07-01

    Full Text Available Avian Influenza (AI or Bird Flu is a fatal zoonotic disease caused by highly pathogenic avian influenza(HPAI virus of H5N1 sub-type. The disease is still endemic in Indonesia. This study was conducted toinvestigate AI cases in chickens in Bali. Virus isolation was performed in 9 day-old embryonated chickeneggs, and then followed by serologic testing by haemaglutination (HA and Haemaglutination Inhibition(HI assay using standard microtiter procedure. All of the samples were further tested with reversetrancriptasepolymerase chain reaction (RT-PCR. All work has been done in the Biomedical and MolecularBiology Laboratory, Faculty of Veterinary Medicine, Udayana University, Denpasar, during the period2009-2011. A total of ten samples were examined A total of ten chicken samples consisting of 6 fieldsamples and 4 meat samples have been confirmed to be AIV H5N1. All field cases showed clinical signsand gross pathology that were typical to the infection of avian influenza. The result indicates that AI casesare still prevalent among chickens in Bali.

  9. A noncommercial polymerase chain reaction-based method to approach one hundred percent recombinant clone selection efficiency.

    Science.gov (United States)

    Shareef, Mohammed M; Dancea, Horatiu C; Gross, Jessica L; Myers, Tamara T; Griggs, Wendy W; Ahmed, Mansoor M; Sheldon, David G

    2008-11-01

    Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones.

  10. Entanglement dynamics in critical random quantum Ising chain with perturbations

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yichen, E-mail: ychuang@caltech.edu

    2017-05-15

    We simulate the entanglement dynamics in a critical random quantum Ising chain with generic perturbations using the time-evolving block decimation algorithm. Starting from a product state, we observe super-logarithmic growth of entanglement entropy with time. The numerical result is consistent with the analytical prediction of Vosk and Altman using a real-space renormalization group technique. - Highlights: • We study the dynamical quantum phase transition between many-body localized phases. • We simulate the dynamics of a very long random spin chain with matrix product states. • We observe numerically super-logarithmic growth of entanglement entropy with time.

  11. [Development of a real-time polymerase chain reaction method for the identification of Candida species].

    Science.gov (United States)

    Ağca, Harun; Dalyan Cilo, Burcu; Özmerdiven, Gülşah Ece; Sağlam, Sezcan; Ener, Beyza

    2015-01-01

    Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 μl of extracted DNA, 2 μl of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 μl of MgCl(2) (5 mmol), 2 μl of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 μl of each primer (0.01 nmol/μl) and 1 μl of each probe (0.1 μmol/μl) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95°C for 10 mins and 50 cycles of denaturation at 95°C for 10 secs, annealing at 62°C for 10 secs and polymerisation at 72°C for 20 secs. A melting curve was

  12. Mechanism of ganciclovir-induced chain termination revealed by resistant viral polymerase mutants with reduced exonuclease activity.

    Science.gov (United States)

    Chen, Han; Beardsley, G Peter; Coen, Donald M

    2014-12-09

    Many antiviral and anticancer drugs are nucleoside analogs that target polymerases and cause DNA chain termination. Interestingly, ganciclovir (GCV), the first line of therapy for human cytomegalovirus (HCMV) infections, induces chain termination despite containing the equivalent of a 3'-hydroxyl group. Certain HCMV GCV resistance (GCV(r)) mutations, including ones associated with treatment failures, result in substitutions in the 3'-5' exonuclease (Exo) domain of the catalytic subunit of the viral DNA polymerase (Pol). To investigate how these mutations confer resistance, we overexpressed and purified wild-type (WT) HCMV Pol and three GCV(r) Exo mutants. Kinetic studies provided little support for resistance being due to effects on Pol binding or incorporation of GCV-triphosphate. The mutants were defective for Exo activity on all primer templates tested, including those with primers terminating with GCV, arguing against the mutations increasing excision of the incorporated drug. However, although the WT enzyme terminated DNA synthesis after incorporation of GCV-triphosphate and an additional nucleotide (N+1), the Exo mutants could efficiently synthesize DNA to the end of such primer templates. Notably, the Exo activity of WT Pol rapidly and efficiently degraded N+2 primer templates to N+1 products that were not further degraded. On N+1 primer templates, WT Pol, much more than the Exo mutants, converted the incoming deoxynucleoside triphosphate to its monophosphate, indicative of rapid addition and removal of incorporated nucleotides ("idling"). These results explain how GCV induces chain termination and elucidate a previously unidentified mechanism of antiviral drug resistance.

  13. Development of sensitive crop-specific polymerase chain reaction assays using 5S DNA: applications in food traceability.

    Science.gov (United States)

    Doveri, Silvia; Lee, David

    2007-06-13

    The 5S intergenic spacers were amplified using a common pair of primers and sequenced from four species (Brassica napus, Zea mays, Helianthus annuus, and Glycine max). Crop-specific assays were developed from primers designed from the spacers and tested to amplify corresponding DNAs in both conventional end-point and real-time polymerase chain reactions (PCRs). The high copy numbers of the 5S DNA in plants make it possible to detect very small amounts of DNA using this marker. This sensitivity made it possible to compare different DNA extraction methods for highly processed food products using 5S spacers, even allowing dilution of templates to overcome PCR inhibition.

  14. Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

    Science.gov (United States)

    Koh, Chung-Yan; Light, Yooli Kim; Piccini, Matthew Ernest; Singh, Anup K.

    2017-10-31

    Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required for PCR.

  15. Rapid detection of active cytomegalovirus infection by in situ polymerase chain reaction on MRC5 cells inoculated with blood specimens.

    Science.gov (United States)

    Bettinger, D; Mougin, C; Lab, M

    1994-08-01

    An in situ polymerase chain reaction was developed to amplify immediate early genes of human cytomegalovirus in cells cultured in a 96 well plate and infected with leukocytes. The technical parameters enabling optimal detection of the DNA sequences were defined. The key to this method is the fixation of cells, which facilitates the access of the PCR mixture into the cell nuclei and preserves cell morphology. Such a technique could have wide application for the detection and identification of other infectious viruses in cultured cells very early after inoculation of clinical samples.

  16. Investigation of an outbreak of varicella in Chandigarh, North India, using a real-time polymerase chain reaction approach

    Directory of Open Access Journals (Sweden)

    Mini P Singh

    2017-01-01

    Full Text Available Outbreaks of varicella are reported when susceptible population accumulates. This study reports a chickenpox outbreak in Burail in August 2014, wherein 20 laboratory-confirmed cases were identified by the detection of varicella zoster virus (VZV DNA and VZV IgM antibodies. The viral load between vesicular swabs and serum samples from 8 patients with active lesions was found to have good correlation and further also related with disease severity. Real-time polymerase chain reaction can be useful for early diagnosis of an outbreak and vesicular swab can be used as a less invasive sample for assessing the disease severity.

  17. Susceptibility of Biomphalaria tenagophila and Biomphalaria straminea to Schistosoma mansoni infection detected by low stringency polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    JANNOTTI-PASSOS Liana Konovaloff

    2000-01-01

    Full Text Available In order to determine Schistosoma mansoni infection rates in Biomphalaria tenagophila and B. straminea, low stringency polymerase chain reaction (LS-PCR technique was used as a complementary method to light exposure technique. LS-PCR has already been standardized in our laboratory to detect the trematode DNA in B. glabrata. Higher S. mansoni infection rates were detected using conventional method and LS-PCR. The parasite DNA profile was detected in both species after 7-day exposure to miracidia, using LS-PCR. This technique enables early detection of schistosomiasis transmission focuses, in endemic areas, before the beginning of cercariae shedding.

  18. [Influence of different DNA extractions on the identification of streptococcus sanguis group by arbitrary primed polymerase chain reaction

    Science.gov (United States)

    Zhang, Wei-Dong; Chen, Hui; Yu, Zhong-Sheng

    2002-08-01

    OBJECTIVE: To assess the influence of different DNA extractions on the identification of streptococcus sanguis group (SSG) species by arbitrary primed polymerase chain reaction (AP-PCR). METHODS: AP-PCR was used to distinguish SSG species by designing 25bp arbitrary primer 5'AAG AGA GGA GCT AGC TCT TCT TGG A 3', and the genomic DNA was extracted by 3 methods. RESULTS: There were great differences in the main band of DNA polymorphism among SSG species. The similar band could be got from the different DNA extractions in the same species. CONCLUSION: Different DNA extractions have no influence on the identification of SSG.

  19. Lyapunov exponent for pure and random Fibonacci chains

    Science.gov (United States)

    Velhinho, M. T.; Pimentel, I. R.

    2000-01-01

    We study the Lyapunov exponent for electron and phonon excitations, in pure and random Fibonacci quasicrystal chains, using an exact real-space renormalization-group method, which allows the calculation of the Lyapunov exponent as a function of the energy. It is shown that the Lyapunov exponent on a pure Fibonacci chain has a self-similar structure, characterized by a scaling index that is independent of the energy for the electron excitations, ``diagonal'' or ``off-diagonal'' quasiperiodic, but is a function of the energy for the phonon excitations. This scaling behavior implies the vanishing of the Lyapunov exponent for the states on the spectrum, and hence the absence of localization on the Fibonacci chain, for the various excitations considered. It is also shown that disordered Fibonacci chains, with random tiling that introduces phason flips at certain sites on the chain, exhibit the same Lyapunov exponent as the pure Fibonacci chain, and hence this type of disorder is irrelevant, either in the case of electron or phonon excitations.

  20. Entanglement dynamics in critical random quantum Ising chain with perturbations

    Science.gov (United States)

    Huang, Yichen

    2017-05-01

    We simulate the entanglement dynamics in a critical random quantum Ising chain with generic perturbations using the time-evolving block decimation algorithm. Starting from a product state, we observe super-logarithmic growth of entanglement entropy with time. The numerical result is consistent with the analytical prediction of Vosk and Altman using a real-space renormalization group technique.

  1. Water Bridging Dynamics of Polymerase Chain Reaction in the Gauge Theory Paradigm of Quantum Fields

    Directory of Open Access Journals (Sweden)

    L. Montagnier

    2017-05-01

    Full Text Available We discuss the role of water bridging the DNA-enzyme interaction by resorting to recent results showing that London dispersion forces between delocalized electrons of base pairs of DNA are responsible for the formation of dipole modes that can be recognized by Taq polymerase. We describe the dynamic origin of the high efficiency and precise targeting of Taq activity in PCR. The spatiotemporal distribution of interaction couplings, frequencies, amplitudes, and phase modulations comprise a pattern of fields which constitutes the electromagnetic image of DNA in the surrounding water, which is what the polymerase enzyme actually recognizes in the DNA water environment. The experimental realization of PCR amplification, achieved through replacement of the DNA template by the treatment of pure water with electromagnetic signals recorded from viral and bacterial DNA solutions, is found consistent with the gauge theory paradigm of quantum fields.

  2. [Detection of fungi in liquor workers with tinea corporis and tinea cruris using arbitrarily primed polymerase chain reaction].

    Science.gov (United States)

    Yan, Dan; Li, Liao; Chen, De-yu; Zhang, Yu-hua; Hu, Chao-hui; Deng, Zheng-hua

    2007-03-01

    To explore the method of rapid detection of skin fungi and the significance of conventional diagnosis liquor worker tinea corporis and tinea cruris using arbitrarily primed polymerase chain reaction AP-PCR. Among liquor workers who were 50 tinea corporis patients, 58 tinea cruris patients and 50 health persons, we amplified the DNAs of the dermatophytes were amplified using AP-PCR and random primers OPD18 5'-GAGAGCCAAC-3' and OPAA11 5'-ACCCGACCTG-3', at the same time, the dermatophytes with microscope were detected and cultured. AP-PCR analysis detected fungal DNA in 45 patients(90.00%) among 50 liquor worker patients with tinea corporis, 31 patients(62.00%) had the positive results of microscope detection, and 41 patients(82.00%) had the positive results of standard culture. Among these workers who suffered from tinea corporis, T.rubrum, T.mentagrophyte, M. canis and E.floccosum were detected by AP-PCR. T.rubrum, T.mentagrophyte and M.canis were detected by standard culture. AP-PCR analysis detected fungal DNA in 53 patients(91.38%) among 58 liquor worker patients with tinea cruris, 37 patients(63.79%) had the positive results of microscope detection, and 48(82.76%) had the positive results of standard culture. Among the 58 workers who had tinea cruris, T.rubrum, E.floccosum and T.mentagrophyte were detected by AP-PCR and standard culture. Among 50 health persons, AP-PCR analysis detected fungal DNA in 3 persons(6.00%). The detection result with AP-PCR indicated that the kinds of fungi were T.rubrum and T.mentagrophyte. No one health person had the positive result in detection of fungi using microscope detection. Only one(2.00%) health person was detected to be infected by fungus with cultural way. The kind of fungus was T.rubrum. AP-PCR is a rapid, sensitive and specific detection method for occupational dermatophyte patients. It can be used to detect and diagnose professional dermatophytosis.

  3. Detection of Escherichia coli Enteropathogens by Multiplex Polymerase Chain Reaction from Children's Diarrheal Stools in Two Caribbean–Colombian Cities

    Science.gov (United States)

    Arzuza, Octavio; Urbina, Delfina; Bai, Jing; Guerra, Julio; Montes, Oscar; Puello, Marta; Mendoza, Ketty; Castro, Gregorio Y.

    2010-01-01

    Abstract Acute diarrheal disease is a leading cause of childhood morbidity and mortality in the developing world and Escherichia coli intestinal pathogens are important causative agents. Information on the epidemiology of E. coli intestinal pathogens and their association with diarrheal disease is limited because no diagnostic testing is available in countries with limited resources. To evaluate the prevalence of E. coli intestinal pathogens in a Caribbean–Colombian region, E. coli clinical isolates from children with diarrhea were analyzed by a recently reported two-reaction multiplex polymerase chain reaction (Gomez-Duarte et al., Diagn Microbiol Infect Dis 2009;63:1–9). The phylogenetic group from all E. coli isolates was also typed by a single-reaction multiplex polymerase chain reaction. We found that among 139 E. coli strains analyzed, 20 (14.4%) corresponded to E. coli diarrheagenic pathotypes. Enterotoxigenic, shiga-toxin–producing, enteroaggregative, diffuse adherent, and enteropathogenic E. coli pathotypes were detected, and most of them belonged to the phylogenetic groups A and B1, known to be associated with intestinal pathogens. This is the first report on the molecular characterization of E. coli diarrheogenic isolates in Colombia and the first report on the potential role of E. coli in childhood diarrhea in this geographic area. PMID:19839760

  4. Detection of Escherichia coli enteropathogens by multiplex polymerase chain reaction from children's diarrheal stools in two Caribbean-Colombian cities.

    Science.gov (United States)

    Gómez-Duarte, Oscar G; Arzuza, Octavio; Urbina, Delfina; Bai, Jing; Guerra, Julio; Montes, Oscar; Puello, Marta; Mendoza, Ketty; Castro, Gregorio Y

    2010-02-01

    Acute diarrheal disease is a leading cause of childhood morbidity and mortality in the developing world and Escherichia coli intestinal pathogens are important causative agents. Information on the epidemiology of E. coli intestinal pathogens and their association with diarrheal disease is limited because no diagnostic testing is available in countries with limited resources. To evaluate the prevalence of E. coli intestinal pathogens in a Caribbean-Colombian region, E. coli clinical isolates from children with diarrhea were analyzed by a recently reported two-reaction multiplex polymerase chain reaction (Gomez-Duarte et al., Diagn Microbiol Infect Dis 2009;63:1-9). The phylogenetic group from all E. coli isolates was also typed by a single-reaction multiplex polymerase chain reaction. We found that among 139 E. coli strains analyzed, 20 (14.4%) corresponded to E. coli diarrheagenic pathotypes. Enterotoxigenic, shiga-toxin-producing, enteroaggregative, diffuse adherent, and enteropathogenic E. coli pathotypes were detected, and most of them belonged to the phylogenetic groups A and B1, known to be associated with intestinal pathogens. This is the first report on the molecular characterization of E. coli diarrheogenic isolates in Colombia and the first report on the potential role of E. coli in childhood diarrhea in this geographic area.

  5. Search for viral nucleic acid sequences in brain tissues of patients with schizophrenia using nested polymerase chain reaction.

    Science.gov (United States)

    Taller, A M; Asher, D M; Pomeroy, K L; Eldadah, B A; Godec, M S; Falkai, P G; Bogert, B; Kleinman, J E; Stevens, J R; Torrey, E F

    1996-01-01

    We used polymerase chain reaction to search for nucleic acid sequences of several viruses in DNA and RNA extracted from brain tissues of schizophrenic and control subjects. We extracted DNA and RNA templates from frozen brain specimens of 31 patients with schizophrenia and 23 nonschizophrenic control patients with other diseases. The extracts were subjected to polymerase chain reaction with oligonucleotide primers for 12 different viruses (cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1, human herpesvirus type 6, varicellazoster virus, measles virus, mumps virus, rubella virus, the picornavirus group, influenza A virus, human T-cell lymphotropic virus type I, and St Louis encephalitis virus), several of which have been suspected of involvement in schizophrenia. Nested primers were used to increase the sensitivity of the method. No amplified nucleic acid sequences encoded by the selected viral genomes were detected in extracts of any brain specimens from either schizophrenic or control patients. These data agree with previous studies that failed to find sequences of a number of viruses in the cerebrospinal fluid or selected areas of the brains of schizophrenic patients. Additional efforts should be undertaken to identify other known and unknown pathogens in schizophrenia, sampling more areas of the brain from subjects with a variety of clinical types of schizophrenia.

  6. Real-time polymerase chain reaction for diagnosing infectious mononucleosis in pediatric patients: A systematic review and meta-analysis.

    Science.gov (United States)

    Jiang, Sha-Yi; Yang, Jing-Wei; Shao, Jing-Bo; Liao, Xue-Lian; Lu, Zheng-Hua; Jiang, Hui

    2016-05-01

    In this meta-analysis, we evaluated the diagnostic role of Epstein-Barr virus deoxyribonucleic acid detection and quantitation in the serum of pediatric and young adult patients with infectious mononucleosis. The primary outcome of this meta-analysis was the sensitivity and specificity of Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) detection and quantitation using polymerase chain reaction (PCR). A systematic review and meta-analysis was performed by searching for articles that were published through September 24, 2014 in the following databases: Medline, Cochrane, EMBASE, and Google Scholar. The following keywords were used for the search: "Epstein-Barr virus," "infectious mononucleosis," "children/young adults/infant/pediatric," and "polymerase chain reaction or PCR." Three were included in this analysis. We found that for detection by PCR, the pooled sensitivity for detecting EBV DNA was 77% (95%CI, 66-86%) and the pooled specificity for was 98% (95%CI, 93-100%). Our findings indicate that this PCR-based assay has high specificity and good sensitivity for detecting of EBV DNA, indicating it may useful for identifying patients with infectious mononucleosis. This assay may also be helpful to identify young athletic patients or highly physically active pediatric patients who are at risk for a splenic rupture due to acute infectious mononucleosis. © 2015 Wiley Periodicals, Inc.

  7. [Detection of the genetically modified organisms in genetically modified soybean and maize by polymerase chain reaction method].

    Science.gov (United States)

    Mao, Deqian; Mu, Weipeng; Yang, Xiaoguang

    2002-06-01

    A method for the detection of the (genetically modified organism GMOs) in genetically modified soybean (Round-up Ready soybean, RR soybean) and maize(Bt-176 maize) is described. The polymerase chain reaction (PCR) method is discussed with the genetically modified soybean and maize whose contents are known. The detection limit can be 0.1%, that is to say, we can detect the GMO in the food whose content is only 0.1%, the detection method is just a screening method. The procedure includes: (1) extraction of genomic DNA of maize and soybean, (2) amplification of the inserted genes, CaMV35S promoter and the NOS terminator inserted by means of the polymerase chain reaction (PCR) method, (3) amplification of the specific genes of maize and soybean in order to determine that the samples are maize and soybean, (4) characterization and confirmation of the PCR products by restriction enzyme analysis and the electrophoresis on agarose gel. The RR soybean contains CaMV35S promoter and NOS terminator, and the Bt-176 maize contains only CaMV35S promoter. Due to the high content of the starch in maize, the effect of the electrophororesis is not so good as of the soybean's.

  8. Detection of Mycoplasma pneumoniae and Chlamydia trachomatis in Iranian children with acute lower respiratory infections by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Mostafa Behpour Oskooee

    2014-02-01

    Full Text Available Objective: To determine the frequency of Mycoplasma pneumoniae (M. pneumoniae and Chlamydia trachomatis (C. trachomatis in young children with community acquired pneumonia (CAP and detect C. trachomatis in the subgroup of infants under 1 year of age in Tehran, Iran. Methods: This cross-sectional study was designed to detect M. pneumoniae from all children (<5 years of age presenting with CAP, admitted to a tertiary care children ’s hospital affiliated with the Shaheed Beheshti University of Medical Sciences in Tehran during a period of 14 months, from November 2010 to December 2011. Nasopharyngeal and oropharyngeal swabs were collected from 102 children during the study period. Pathogens were detected using polymerase chain reaction and confirmed with real-time polymerase chain reaction. Results: Only one case of M. pneumoniae was isolated from 102 children (1%. C. trachomatis was not detected in any of the 69 infants (<1 year of age. Conclusions: According to our findings, M. pneumoniae is an uncommon cause of CAP in children under 5 years old and C. trachomatis could not be listed as causing CAP in infants in our study population. However, more studies with a larger sample size are needed to confirm this observation.

  9. KNOTS AND RANDOM WALKS IN VIBRATED GRANULAR CHAINS

    Energy Technology Data Exchange (ETDEWEB)

    E. BEN-NAIM; ET AL

    2000-08-01

    The authors study experimentally statistical properties of the opening times of knots in vertically vibrated granular chains. Our measurements are in good qualitative and quantitative agreement with a theoretical model involving three random walks interacting via hard core exclusion in one spatial dimension. In particular, the knot survival probability follows a universal scaling function which is independent of the chain length, with a corresponding diffusive characteristic time scale. Both the large-exit-time and the small-exit-time tails of the distribution are suppressed exponentially, and the corresponding decay coefficients are in excellent agreement with the theoretical values.

  10. Comparison between qualitative and real-time polymerase chain reaction to evaluate minimal residual disease in children with acute lymphoblastic leukemia.

    Science.gov (United States)

    Paula, Francisco Danilo Ferreira; Elói-Santos, Silvana Maria; Xavier, Sandra Guerra; Ganazza, Mônica Aparecida; Jotta, Patricia Yoshioka; Yunes, José Andrés; Viana, Marcos Borato; Assumpção, Juliana Godoy

    2015-01-01

    Minimal residual disease is an important independent prognostic factor that can identify poor responders among patients with acute lymphoblastic leukemia. The aim of this study was to analyze minimal residual disease using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements by conventional polymerase chain reaction followed by homo-heteroduplex analysis and to compare this with real-time polymerase chain reaction at the end of the induction period in children with acute lymphoblastic leukemia. Seventy-four patients diagnosed with acute lymphoblastic leukemia were enrolled. Minimal residual disease was evaluated by qualitative polymerase chain reaction in 57 and by both tests in 44. The Kaplan-Meier and multivariate Cox methods and the log-rank test were used for statistical analysis. Nine patients (15.8%) were positive for minimal residual disease by qualitative polymerase chain reaction and 11 (25%) by real-time polymerase chain reaction considering a cut-off point of 1×10(-3) for precursor B-cell acute lymphoblastic leukemia and 1×10(-2) for T-cell acute lymphoblastic leukemia. Using the qualitative method, the 3.5-year leukemia-free survival was significantly higher in children negative for minimal residual disease compared to those with positive results (84.1%±5.6% versus 41.7%±17.3%, respectively; p-value=0.004). There was no significant association between leukemia-free survival and minimal residual disease by real-time polymerase chain reaction. Minimal residual disease by qualitative polymerase chain reaction was the only variable significantly correlated to leukemia-free survival. Given the difficulties in the implementation of minimal residual disease monitoring by real-time polymerase chain reaction in most treatment centers in Brazil, the qualitative polymerase chain reaction strategy may be a cost-effective alternative. Copyright © 2015 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier

  11. Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood.

    Science.gov (United States)

    Mühl, Helge; Kochem, Anna-Julia; Disqué, Claudia; Sakka, Samir G

    2010-01-01

    Universal 16S rRNA gene polymerase chain reaction (PCR) is a promising means of detecting bacteremia. Among other factors, the PCR reagents play a prominent role for obtaining a high sensitivity of detection. The reagents are ideally optimized with respect to the amplifying activity and absence of contaminating DNA. In this study, it was shown in a universal 16S rDNA real-time PCR assay that commercial PCR reagents can vary greatly among each other in these characters. Only 1 of the 5 reagents tested met the criteria of sensitive detection of pathogen DNA with a minimum of false-positive results. The reagent was validated by the detection of pathogens at low titers using bacterial DNA extracted from blood that was spiked with various Gram-positive and Gram-negative bacteria.

  12. Polymerase chain reaction in diagnosis of Borrelia burgdorferi infections and studies on taxonomic classification

    DEFF Research Database (Denmark)

    Lebech, Anne-Mette

    2002-01-01

    not regarded as a suitable sample source for B. burgdorferi PCR. The reason may be the variable presence of Taq polymerase inhibitors. Based on a semi-quantitative detection system for amplicons, reflecting the input amount of specific DNA and thus the density of spirochetes in the clinical samples high...... a PCR assay for direct detection of B. burgdorferi DNA and to evaluate the diagnostic utility of PCR in clinical specimens from patients with Lyme borreliosis and (ii) to study the taxonomic classification of B. burgdorferi isolates and its implications for epidemiology and clinical presentation...... for PCR amplification and subsequent identification of B. burgdorferi specific sequences were established and used. For all assays the analytical sensitivity was a few genome copies using purified DNA as template. The efficacy of PCR was initially evaluated using tissue samples from experimentally...

  13. A FIELD STUDY USING THE POLYMERASE CHAIN REACTION (PCR TO SCREEN FOR BRUGIA MICROFILARIAE IN HUMAN AND ANIMAL BLOOD

    Directory of Open Access Journals (Sweden)

    Janet Glover

    2012-09-01

    Full Text Available Blood samples from 43 humans and 14 cats positive with Brugia microfilariae were analyzed in a field study in Tanjung Pinang, Indonesia. The study used the polymerase chain reaction (PCR to compare the sensitivity of radioactive and biotinylated species-specific oligonuleotide probes. The cloning char­acterization of the Hha I repeat DNA family found in filarial parasites of the genus Brugia, and the development of species-specific probes for B.malayi and B.pahangi based on these repeats has been described elsewhere (PNAS USA 83: 797-801; Mol.Biochem. Parasitol. 2$: 163-170. The use of radioisotopes for labelling DNA probes is both expensive and inconvenient. To replace these probes, biotinylated DNA probes have been designed for non- radioactive detection of B.malayi and B.pahangi. These oligonucleotide probes have long tails of biotinylated uridine residues added to their 5' end. As little as 100 pg of Brugia DNA can be detected on dot blot with these probes. Detection of the probes is based on an avidin-alkaline phosphatase colorimetric assay. In order to distinguish between infected from uninfected individuals, it is necessary to detect the amount of DNA in one microfilaria (about 60 pg. The polymerase chain reaction (PCR is a procedure in which a small amount of DNA can be amplified up to 1 million-fold. A part of each sample in this study was PCR amplified and compared with the unamplified portion using both the radioactive and biotinylated DNA probe. The PCR amplified samples were accurately identified by both the radioactive and biotinylated B.malayi and B.pahangi probes. Even samples with as few as two microfilariae per lOOul of blood were easily detected. The samples that were not PCR amplified were accurately identified after only long exposures (greater than one week to the radioactive probes. The biotinylated probes, were not sensitive enough for accurate identification of the non-PCR amplified samples. The polymerase chain

  14. Mitochondrial DNA variation in chinook salmon and chum salmon detected by restriction enzyme analysis of polymerase chain reaction products

    Science.gov (United States)

    Cronin, M.; Spearman, R.; Wilmot, R.; Patton, J.; Bickman, J.

    1993-01-01

    We analyze intraspecific mitochondrial DNA variation in chinook salmon from drainages in the Yukon River, the Kenai River, and Oregon and California rivers; and chum salmon from the Yukon River and vancouver Island, and Washington rivers. For each species, three different portions of the mtDNA molecule were amplified seperately using the polymerase chain reaction and then digested with at least 19 restrictions enzymes. Intraspecific sequence divergences between haplotypes were less than 0.01 base subsitution per nucleotide. Nine chum salmon haplotypes were identified. Yukon River chum salmon stocks displayed more haplotypes (8) occurred in all areas. Seven chinook salmon haplotypes were identified. Four haplotypes occurred in the Yukon and Kenai rviers and four occured in the Oregon/California, with only one haplotype shared between the regions. Sample sizes were too small to quantify the degree of stock seperation among drainages, but the patterns of variation that we observed suggest utility of the technique in genetic stock identification.

  15. Detection of Listeria monocytogenes in ready-to-eat food by Step One real-time polymerase chain reaction.

    Science.gov (United States)

    Pochop, Jaroslav; Kačániová, Miroslava; Hleba, Lukáš; Lopasovský, L'ubomír; Bobková, Alica; Zeleňáková, Lucia; Stričík, Michal

    2012-01-01

    The aim of this study was to follow contamination of ready-to-eat food with Listeria monocytogenes by using the Step One real time polymerase chain reaction (PCR). We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Listeria monocytogenes Detection Kit for the real-time PCR performance. In 30 samples of ready-to-eat milk and meat products without incubation we detected strains of Listeria monocytogenes in five samples (swabs). Internal positive control (IPC) was positive in all samples. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in ready-to-eat food without incubation.

  16. Detection of Nesopora caninum-specific DNA from cerebrospinal fluid by polymerase chain reaction in a dog with confirmed neosporosis.

    Science.gov (United States)

    Ishigaki, Kyohei; Noya, Masahiko; Kagawa, Yumiko; Ike, Kazunori; Orima, Hiromitsu; Imai, Soichi

    2012-08-01

    A one-month male Greyhound dog presented with a swinging gait of the hindlimbs, and later developed muscular atrophy of the femoral region and hyperextension of hindlimbs. The dog had positive serum IFAT titers to Neospora caninum, but a negative titer in the cerebrospinal fluid (CSF). N. caninum-specific DNA was amplified from the CSF using a semi-nested polymerase chain reaction assay. Clusters of protozoa in biopsied muscle fibers were subsequently confirmed as N. caninum tachyzoites by immunohistochemical examination. Early recognition and treatment are necessary for effective recovery of clinical canine neosporosis, but antemortem diagnosis is difficult. We suggest that the detection of parasite deoxyribonucleic acid in the CSF is a useful antemortem diagnostic method in facilitating treatment of this disease.

  17. Detection of Chloramphenicol Resistance Genes (cat in Clinical Isolates of Pseudomonas aeruginosa with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-12-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic Gram negative bacteria, which may cause infection in eyes, ears, skin, bones, central nervous system, gastrointestinal tract, circulatory system, heart, respiratory system, and urinary tract. Recently, chloramphenicol is no longer used as the main option of the therapy due of its resistance case. The aim of this research was to detect the presence of gene which is responsible to chloramphenicol resistance in clinical isolates of P.aeruginosa. These bacteria isolated from pus of external otitis patients in Hasan Sadikin Hospital in Bandung City. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were performed to detect this resistance gene. Electropherogram from PCR products showed that the chloramphenicol resistance in clinical isolates of P. aeruginosa was caused by cat gene (317 bp. Based on this research, cat gene may be used to detect the chloramphenicol resistance in patients with external ostitis.

  18. Detection of Coxiella burnetii in Aborted Fetuses of Cattle and Sheep Using Polymerase Chain Reaction Assay in Mashhad City, Iran

    Directory of Open Access Journals (Sweden)

    Zeinab Abiri

    2016-02-01

    Full Text Available Background: Coxiella burnetii is an important intracellular pathogen that ruminants can act as primary reservoirs. Reservoirs may excrete the bacterium into the placenta, vaginal mucus and feces. Objectives: The aim of this study was to detect C. burnetii in aborted samples from ruminant flocks in Mashhad city, northeast of Iran, using the polymerase chain reaction (PCR assay. Materials and Methods: A total number of 154 fetal tissue samples of cattle, sheep and goat were subjected to nested PCR assay. Results: Sixteen (17.3% out of 92 samples from sheep and 15 (25% from 60 cattle fetuses were positive. Conclusions: The results of this study indicate the presence of C. burnetii in aborted ruminants and these can be the potential reservoirs of C. burnetii in the mentioned area.

  19. IDENTIFICATION OF MYCOBACTERIUM GENAVENSE IN A DIANA MONKEY (CERCOPITHECUS DIANA) BY POLYMERASE CHAIN REACTION AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY.

    Science.gov (United States)

    Kelly, Kathleen M; Wack, Allison N; Bradway, Dan; Simons, Brian W; Bronson, Ellen; Osterhout, Gerard; Parrish, Nicole M; Montali, Richard J

    2015-06-01

    A 25-yr-old Diana monkey (Cercopithecus diana) with a 1.5-yr history of chronic colitis and diarrhea was found to have disseminated granulomatous disease with intralesional acid fast bacilli. Bacilli were identified as Mycobacterium genavense by polymerase chain reaction, sequencing of the 16S-23S ribosomal RNA intergenic spacer (ITS) gene, and mycolic acid analysis by high-performance liquid chromatography. Mycobacterium genavense is a common cause of mycobacteriosis in free-ranging and captive birds. In addition, recognition of opportunistic infection in human immunodeficiency virus-positive patients is increasing. Disease manifestations of M. genavense are similar to Mycobacterium avium complex (MAC) and include fever, wasting, and diarrhea with disseminated disease. Similar clinical signs and lesions were observed in this monkey. Mycobacterium genavense should be considered as a differential for disseminated mycobacterial disease in nonhuman primates as this agent can mimic MAC and related mycobacteria.

  20. DETEKSI DAN SPESIASI PARASIT MALARIA SAMPEL MONITORING PENGOBATAN DIHYDROARTEMISININ-PIPERAQUINE DI KALIMANTAN DAN SULAWESI: MIKROSKOPIS VS POLYMERASE CHAIN REACTION

    Directory of Open Access Journals (Sweden)

    Reni Herman

    2012-07-01

    Full Text Available In monitoring the treatment of malaria with Dihydroartemisinin-piperaquine (DHP, microscopic cross check and Polymerase Chain Reaction (PCR performed to validate the results of laboratory examinations in the field. This study used finger prick samples from subjects with a diagnosis of malaria in monitoring the treatment of malaria with DHP in Kalimantan and Sulawesi. Samples taken at day 0, blood smears made on slides for microscopic and blood spot on filter paper for PCR examination. The PCR method used is a single-round multiplex polymerase chain reaction that has been modified, the examination of each species carried out in different tubes to distinguish the species P. falciparum or P. Vivax. Target of DNA amplification is a species-specific gene sequences in the small-subunit ribosomal RNA (SSUrRNA, 300 bp for P. falciparum and 276 bp for P.vivax.  P. falciparum and P.vivax identified in 229 samples of blood smears and blood spots. Microscopic and PCR gave the same results, positive 93.4% and negative 6.6% with a sensitivity of  99% and specificity 93.3%. P.falciparum sensitivity and specificity of 92% and 99%, P.vivax 97% and 94%, PCR as a gold standard. There are differences in the results of examination of 5 samples, ie with microscopic examination identified as P.vivax  while the PCR as P. falciparum. In this study, identification of  the microscopic parasite similar to the results of identification by PCR, but differ in determining the types of parasites. In general, the ability to microscopic diagnosis of malaria is very good, but confirmation by PCR is still needed.AbstrakPada monitoring pengobatan malaria  dengan Dihydroartemisinin-piperaquine (DHP,cek silang mikroskopis dan Polymerase Chain Reaction (PCR dilakukan untuk memvalidasi hasil pemeriksaan di laboratorium lapangan. Penelitian ini menggunakan sediaan darah jari dari subyek dengan diagnosis malaria pada monitoring pengobatan malaria dengan DHP di Kalimantan dan Sulawesi

  1. Immunohistochemistry and polymerase chain reaction for the detection of Lawsonia intracellularis in porcine intestinal tissues with proliferative enteropathy.

    Science.gov (United States)

    Kim, J; Choi, C; Cho, W S; Chae, C

    2000-07-01

    Detection method of Lawsonia intracellularis was studied in formalin-fixed paraffin-embedded intestinal tissues from 5 naturally infected pigs by immunohistochemistry with a monoclonal antibody against outer membrane protein of L. intracellularis. Warthin-Starry silver stain revealed clusters of argyrophilic, slightly curved rod-shaped organisms in the apical cytoplasm of enterocytes. Immunohistochemical staining with a L. intracellularis-specific monoclonal antibody confirmed the presence of the organism in the apical cytoplasm of hyperplastic enterocytes. The presence of L. intracellularis in the ileum of pig with proliferative enteropathy was confirmed by polymerase chain reaction (PCR) further on the basis of amplification of 319 base pair products specific for porcine L. intracellularis chromosomal DNA. Immunohistochemistry and PCR may be a complementary method to confirm the diagnosis of L. intracellularis infection in pigs.

  2. Validation and aplication of a polymerase chain reaction (PCR to detect porcine circovirus type 2 (PCV-2 in swine sera

    Directory of Open Access Journals (Sweden)

    Luisa Fernanda Villadiego Marmolejo

    2007-12-01

    Full Text Available Porcine circovirosis is an infectious-contagious syndrome caused by porcine circovirus type 2 (PCV-2 found mainly in recently weaned piglets causing dermatitis, neurological and reproductive disorders, pneumonia and encephalitis. The objectives of the present study were to validate a Polymerase Chain Reaction (PCR technique to detect PCV-2 in swine serum and to apply the validated technique in swine serum samples to detect PCV-2. After the application of two different PCRs, 100% of the surveyed animals were negative to PCV-2; furthermore, the PCR targeted to a region between ORFs 1 and 2 of the virus was found more sensitive when compared to another PCR targeted to the capsid protein gene. As a conclusion, PCR is a valid technique to detect PCV-2 in swine serum and the surveyed population was free of the virus.

  3. Impact of polymerase chain reaction testing on Clostridium difficile infection rates in an acute health care facility.

    Science.gov (United States)

    Murad, Yanal M; Perez, Justo; Nokhbeh, Reza; Ybazeta, Gustavo; Dewar, Brenda; Lefebvre, Sebastien; Diaz-Mitoma, Francisco

    2015-04-01

    Two rapid methods of Clostridium difficile infection (CDI) diagnosis were compared between June 2012 and March 2013: a GeneXpert (Cepheid, Sunnyvale, Calif) polymerase chain reaction (PCR) test and an enzyme immunoassay (EIA). The influence of these methods on the detection of hospital-acquired CDI and identification of CDI outbreaks was evaluated. We tested 1,592 stool samples for C difficile. The GeneXpert PCR test identified 211 positive samples (68 determined to be hospital-acquired infection), whereas EIA identified 105 positive samples (36 determined to be hospital-acquired infection). The GeneXpert PCR method in contrast to the EIA method increased the detection rates of nosocomial CDI cases and contributed to the declaration of CDI outbreaks. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

  4. Prevalence of mycoplasmas in eggs from birds of prey using culture and a genus-specific mycoplasma polymerase chain reaction.

    Science.gov (United States)

    Lierz, M; Hagen, N; Harcourt-Brown, N; Hernandez-Divers, S J; Lüschow, D; Hafez, H M

    2007-04-01

    Mycoplasmas are commensals and pathogens of different avian species, especially poultry and passeriforms. The role of mycoplasmas in raptors has not yet been completely determined, and especially not the possibility of vertical transmission. Therefore 424 raptor eggs were examined for the occurrence of mycoplasmas using culture, and 155 of these eggs with a Mycoplasma genus-specific polymerase chain reaction (PCR) assay. This PCR was tested for its sensitivity and specificity, especially for use in a bird population of unknown mycoplasma status (prevalence and species). The size of the amplified PCR product was large (1013 base pairs) to enable use of the product for species differentiation by sequencing. Culture and PCR yielded only one positive result, in an egg of a Northern Goshawk (Accipiter gentilis). The isolate was identified as Mycoplasma lipofaciens using an immunobinding assay, as well as by sequencing part of its 16S rRNA gene.

  5. Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

    DEFF Research Database (Denmark)

    Krøjgaard, Louise H.; Krogfelt, Karen A.; Albrechtsen, Hans-Jorgen

    2011-01-01

    Background: Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant...... temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool...... detection limit (> 10 CFU/L) to 1.6*10(6) CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively...

  6. Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.

    Science.gov (United States)

    Shyamal, G; Sowmya, P; Sudha, B; Malathi, J; Therese, L K; Madhavan, H N

    2005-10-01

    To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens. Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

  7. Comparative efficacy of conventional and taqman polymerase chain reaction assays in the detection of capripoxviruses from clinical samples.

    Science.gov (United States)

    Balamurugan, Vinayagamurthy; Jayappa, Kallesh Danappa; Hosamani, Madhusudhan; Bhanuprakash, Veerakyathappa; Venkatesan, Gnanavel; Singh, Raj Kumar

    2009-03-01

    Sheeppox and goatpox are economically important viral diseases of sheep and goats, respectively. Both diseases are reportable to the World Organization for Animal Health. To implement a control and eradication program for these diseases, a rapid and user-friendly diagnostic tool is imperative for screening. Therefore, in the present study, TaqMan quantitative polymerase chain reaction (qPCR) and conventional PCR assays targeting the DNA polymerase (DNA pol) gene were developed for the detection of Capripoxvirus DNA from clinical specimens of sheep and goats. The 2 assays used different primer sets. Conventional PCR yielded a specific product of 134 bp, whereas qPCR yielded a 180-bp product. The specificity of amplified DNA pol gene products was confirmed by their size and by sequence analysis. The 2 assays were specific for Sheeppox virus and Goatpox virus. However, in comparison to conventional PCR, the qPCR was more rapid, specific, and 100 times more sensitive, with a detection limit as low as 0.042 pg of purified DNA. The qPCR assay was more sensitive (84.05%) than conventional PCR (76.06%) when used on clinical samples (n = 71) from sheep and goats.

  8. Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates

    Directory of Open Access Journals (Sweden)

    Shyamal G

    2005-01-01

    Full Text Available Purpose: To standardize and apply a polymerase chain reaction (PCR on the glycoprotein D gene to differentiate Herpes simplex virus (HSV 1 & 2 serotypes in culture negative intraocular specimens. Methods: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV and varicella zoster virus (VZV by nucleic acid amplification methods. Results: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens, and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. Conclusions: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

  9. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    Energy Technology Data Exchange (ETDEWEB)

    Shen Cenchao; Yang Wenjuan; Ji Qiaoli; Zhang Zhizhou [HIT Bio-X Centre for Systems Biotechnology, Harbin Institute of Technology, Weihai 200642 (China); Maki, Hisaji [Department of Molecular Biology, Nara Institute of Science and Technology, Takayama-cho 8916-5, Ikoma, Nara 630-0101 (Japan); Dong Anjie, E-mail: scc20001017@gmail.co, E-mail: zhangzzbiox@gmail.co [Department of Chemical Engineering, Tianjin University, Tianjin 300072 (China)

    2009-11-11

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  10. Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: a human brain sodium channel gene is on chromosome 2q22-q23.

    Science.gov (United States)

    Han, J A; Lu, C M; Brown, G B; Rado, T A

    1991-01-01

    We have devised a general strategy for gene mapping based upon the direct amplification of a target sequence within a single microdissected Giemsa-banded chromosomal segment using the polymerase chain reaction. The usefulness of this approach was demonstrated by mapping a cloned human brain sodium channel (alpha subunit) gene sequence to chromosome 2q22-q23. When DNA from single, dissected chromosome segments 2q21-qter and 2q24-pter were used as templates, a sodium channel-specific 172-base-pair polymerase chain reaction product was obtained. This product was not synthesized when segments 2q21-pter and 2q24-qter were used. Chromosome microdissection-polymerase chain reaction is not only a simple, fast, and accurate method for gene mapping but also may offer significant advantages for other applications, such as cancer cytogenetics and linkage analysis. Images PMID:1846440

  11. Multiplex polymerase chain reaction with DNA pooling: a cost-effective strategy of genotyping rare blood types.

    Science.gov (United States)

    He, Y-L; Gao, H-H; Ye, L-Y; Guo, Z-H; Wang, P; Zhu, Z-Y

    2013-02-01

    This work aims to develop a multiplex polymerase chain reaction combined with DNA pooling for mass screening for rare blood types. The differences in most blood group antigens are associated with single-nucleotide polymorphisms (SNPs), which are used in detecting blood antigen expression at the molecular level. However, all existing sequence-specific primers polymerase chain reaction (PCR-SSP) assays for blood typing genotype one or several SNPs individually. DNA pooling is a way that reduces the amount of genotyping required. A sensitive multiplex PCR-SSP assay testing pooled DNA was established to detect the rare Fy(b) and S alleles. It was applied to screen a total of 4490 donor samples via testing 898 DNA pools. The samples in the positive pools were further tested individually. Then the positive samples, including Fy(a-b+)/Fy(a+b+) and S+s-/S+s+ genotypes, were tested via two PCR-SSP assays for alleles Fy(a) and s. The rare genotypes Fy(a-b+) and S+s- were verified using serologic tests and sequencing analysis. Two hundred and fifty-four donors were tested positive for the Fy(b) allele, whereas 101 donors were positive for the S allele. Among the 254 Fy(b+) donors, 5 were Fy(a-b+) and 249 were Fy(a+b+). Among the 101 S+ donors, 3 were S+s- and 98 were S+s+. The rare Fy(b) and S alleles comprised 2·28 and 1·16%, respectively. The PCR-SSP assays were confirmed by sequencing analysis and serological test. A multiplex PCR assay was combined with DNA pooling to reduce the number of tests required, making large-scale screening feasible. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.

  12. PssP2 is a polysaccharide co-polymerase involved in exopolysaccharide chain-length determination in Rhizobium leguminosarum.

    Directory of Open Access Journals (Sweden)

    Małgorzata Marczak

    Full Text Available Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS, displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.

  13. The Role of Multiplex Polymerase Chain Reaction in Detecting Etiological Causes of Bacterial Prostatitis Associated Benign Prostatic Hyperplasia

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    Bramastha Rosadi

    2015-01-01

    Full Text Available Background: Benign Prostatic Hyperplasia (BPH has been correlated with chronic prostatitis according recent study. Chronic pelvic pain is the chief complain of BPH followed by prostatitis. The gold standard of the etiological diagnosis is urine culture, but the negativity rate is still high. Multiplex polymerase chain reaction (PCR as a diagnostic tool in search of etiological causes could identify microorganism on DNA level. This research aims to find out the role of multiplex polymerase chain reaction as diagnostic tools on prostatitis patients. Material and Method: A total of 12 samples collected during the TURP procedure in Sanglah General Hospital Denpasar – Bali from February until May 2015. All of the samples has been diagnosed prostatitis clinically and perform urine culture test. The prostate specimen taken was sent to the Pathological anatomy for histopathology diagnostic and underwent multiplex PCR for etiologic diagnostic. Result: 12 samples have been declared as prostatitis based on histopathology examination, and then were analyzed using multiplex PCR. 10 samples were positive (6 were E. coli, 2 were C. trachomatis, the rest were N. gonorrhea and P. aeruginosa. The urine culture revealed 9 positive, within the result 6 were E. coli, and the others were P. aeruginosa, M. morganii and A. haemolyticus. Conclusion: In prostatitis patient, the etiological diagnostic was important. Multiplex PCR as diagnostic tools could detect the microorganism on a negative urine culture. The combination of the urine culture test and multiplex PCR revealed a better result on etiologic diagnosis which leads to a better management of the disease. 

  14. A Degradome-Based Polymerase Chain Reaction to Resolve the Potential of Environmental Samples for 2,4-Dichlorophenol Biodegradation.

    Science.gov (United States)

    Ibrahim, Eslam S; Kashef, Mona T; Essam, Tamer M; Ramadan, Mohammed A

    2017-12-01

    A clean way to overcome environmental pollution is biodegradation. In this perspective, at the intersection of biodegradation and metagenomics, the degradome is defined as the totality of genes related to the biodegradation of a certain compound. It includes the genetic elements from both culturable and uncultured microorganisms. The possibility of assessing the biodegradation potential of an environmental samples, using a degradome-based polymerase chain reaction, was explored. 2,4-Dichlorophenol (2,4-DCP) was chosen as a model and the use of tfdB gene as a biodegradation marker was confirmed by bioinformatics study of TfdB protein. Five primer pairs were designed for the detection of different tfdB gene families. A total of 16 environmental samples were collected from Egyptian agricultural soils and wastewaters and tested for the presence of 2,4-DCP. The biodegradation capacity of 2,4-DCP was determined, for all isolated consortia, to reach up to 350 mg/l. Metagenomic DNA was extracted directly from the soil samples while successive 2,4-DCP-degrading microbial communities were enriched, with increasing concentrations of 2,4-DCP, then their DNA was extracted. The extracted DNA was tested for the distribution of the tfdB gene using a degradome-based polymerase chain reaction. tfdB-1 and tfdB-2 were detected in 5 and 9 samples, respectively. However, the co-existence of both genes was detected only in five samples. All tfdB positive samples were capable of 2,4-DCP degradation. The developed approach of assessing the potential of different environments for degrading 2,4-DCP was successfully measured in terms of accuracy (81.25%) and specificity (100%).

  15. Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method.

    Science.gov (United States)

    Tie, Jian; Uchigasaki, Seisaku

    2014-11-01

    Human nail is an important forensic material for parental testing and individual identification in large-scale disasters. Detection of STR polymorphism from hard tissues generally requires DNA purification, which is technically complicated and time consuming. In the present study, we attempted to detect STR polymorphisms from untreated human nail samples by direct PCR amplification method using the primer mixture supplied with the GenePrint® SilverSTR® III System or the AmpFℓSTR® Identifiler® PCR Amplification Kit, and Tks Gflex DNA polymerase known to be effective for amplification from crude samples. A nail fragment measuring approximately 1.5 mm in breadth and 0.5 mm in length was placed directly into a PCR tube, and various PCR conditions were tested. The PCR products were analyzed by denaturing acrylamide gel electrophoresis or CE. Multiple STR polymorphisms were detected successfully. This method that detects STR polymorphisms not only from fresh human fingernails, but also from old nail fragments stored at room temperature for up to 10 years is expected to become a novel DNA analytical method in forensic medicine and genetic studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A new building block for DNA network formation by self-assembly and polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Holger Bußkamp

    2014-05-01

    Full Text Available The predictability of DNA self-assembly is exploited in many nanotechnological approaches. Inspired by naturally existing self-assembled DNA architectures, branched DNA has been developed that allows self-assembly to predesigned architectures with dimensions on the nanometer scale. DNA is an attractive material for generation of nanostructures due to a plethora of enzymes which modify DNA with high accuracy, providing a toolbox for many different manipulations to construct nanometer scaled objects. We present a straightforward synthesis of a rigid DNA branching building block successfully used for the generation of DNA networks by self-assembly and network formation by enzymatic DNA synthesis. The Y-shaped 3-armed DNA construct, bearing 3 primer strands is accepted by Taq DNA polymerase. The enzyme uses each arm as primer strand and incorporates the branched construct into large assemblies during PCR. The networks were investigated by agarose gel electrophoresis, atomic force microscopy, dynamic light scattering, and electron paramagnetic resonance spectroscopy. The findings indicate that rather rigid DNA networks were formed. This presents a new bottom-up approach for DNA material formation and might find applications like in the generation of functional hydrogels.

  17. A new building block for DNA network formation by self-assembly and polymerase chain reaction.

    Science.gov (United States)

    Bußkamp, Holger; Keller, Sascha; Robotta, Marta; Drescher, Malte; Marx, Andreas

    2014-01-01

    The predictability of DNA self-assembly is exploited in many nanotechnological approaches. Inspired by naturally existing self-assembled DNA architectures, branched DNA has been developed that allows self-assembly to predesigned architectures with dimensions on the nanometer scale. DNA is an attractive material for generation of nanostructures due to a plethora of enzymes which modify DNA with high accuracy, providing a toolbox for many different manipulations to construct nanometer scaled objects. We present a straightforward synthesis of a rigid DNA branching building block successfully used for the generation of DNA networks by self-assembly and network formation by enzymatic DNA synthesis. The Y-shaped 3-armed DNA construct, bearing 3 primer strands is accepted by Taq DNA polymerase. The enzyme uses each arm as primer strand and incorporates the branched construct into large assemblies during PCR. The networks were investigated by agarose gel electrophoresis, atomic force microscopy, dynamic light scattering, and electron paramagnetic resonance spectroscopy. The findings indicate that rather rigid DNA networks were formed. This presents a new bottom-up approach for DNA material formation and might find applications like in the generation of functional hydrogels.

  18. Tsallis thermostatics as a statistical physics of random chains

    Science.gov (United States)

    Jizba, Petr; Korbel, Jan; Zatloukal, Václav

    2017-02-01

    In this paper we point out that the generalized statistics of Tsallis-Havrda-Charvát can be conveniently used as a conceptual framework for statistical treatment of random chains. In particular, we use the path-integral approach to show that the ensuing partition function can be identified with the partition function of a fluctuating oriented random loop of arbitrary length and shape in a background scalar potential. To put some meat on the bare bones, we illustrate this with two statistical systems: Schultz-Zimm polymer and relativistic particle. Further salient issues such as the projective special linear group PSL (2 ,R ) transformation properties of Tsallis' inverse-temperature parameter and a grand-canonical ensemble of fluctuating random loops related to the Tsallis-Havrda-Charvát statistics are also briefly discussed.

  19. Tsallis thermostatics as a statistical physics of random chains.

    Science.gov (United States)

    Jizba, Petr; Korbel, Jan; Zatloukal, Václav

    2017-02-01

    In this paper we point out that the generalized statistics of Tsallis-Havrda-Charvát can be conveniently used as a conceptual framework for statistical treatment of random chains. In particular, we use the path-integral approach to show that the ensuing partition function can be identified with the partition function of a fluctuating oriented random loop of arbitrary length and shape in a background scalar potential. To put some meat on the bare bones, we illustrate this with two statistical systems: Schultz-Zimm polymer and relativistic particle. Further salient issues such as the projective special linear group PSL(2,R) transformation properties of Tsallis' inverse-temperature parameter and a grand-canonical ensemble of fluctuating random loops related to the Tsallis-Havrda-Charvát statistics are also briefly discussed.

  20. Real-time TaqMan polymerase chain reaction to quantify the effects ...

    African Journals Online (AJOL)

    Twenty-eight PIC male piglets (similar birth and parity, weaned at 21±1.5 days) were used to study the effect of the different sources of dietary starch on the number of Bifidobacteria in the digesta of the duodenum, jejunum, ileum, cecum and colon. Pigs were randomly assigned to one of four diets formulated with corn starch ...

  1. Leptospira spp detection by Polymerase Chain Reaction (PCR in clinical samples of captive black-capped Capuchin monkey (Cebus apella

    Directory of Open Access Journals (Sweden)

    Scarcelli Eliana

    2003-01-01

    Full Text Available Leptospirosis is a widely distributed zoonosis that affects domestic and wild animals, and that has the man as the end point of its epidemiological chain. Leptospirosis diagnosis in primates is more difficult than in other animal species, as clinical signs and lesions are less evident and antibody response is detected only for short periods. The aim of this article was to describe the detection of Leptospira spp using polymerase chain reaction (PCR, in clinical samples from one captive black-capped Capuchin monkey (Cebus apella, which presented characteristics compatible with leptospirosis (jaundice and haemorrhagic kdney in the macroscopic post-mortem examination. A friable kidney fragment and urine sample were cultured and submitted to experimental inoculation in guinea pigs and PCR using genus specific primer pair targeting the 16S rRNA region from Leptospira interrogans serovar canicola. Isolation of the agent was negative both in culture and experimental inoculation. The PCR amplification of the clinical samples showed a 330 pb amplified fragment that corresponds to the Leptospira genus. Based on these results PCR was considered an important tool for leptospira detection in nonhumam primates, more sensitive and specific than other techniques, especially considering that the viability of the pathogen was not possible. These advantages enable the detection of the leptospiras in urine and kidney, even when autolysed, frozen or badly conserved, which prevented the isolation and experimental inoculation from positive results.

  2. Clonality in myeloproliferative disorders: Analysis by means of polymerase chain reaction

    Energy Technology Data Exchange (ETDEWEB)

    Gilliland, D.G.; Blanchard, K.L.; Levy, J.; Perrin, S.; Bunn, H.F. (Harvard Medical School, Boston, MA (United States))

    1991-08-01

    The myeloproliferative syndromes are acquired disorders of hematopoiesis that provide insights into the transition from somatic cell mutation to neoplasia. The clonal origin of specific blood cells can be assessed in patients with X chromosome-linked polymorphisms, taking advantage of random inactivation of the X chromosome. The authors have adapted the PCR for determination of clonality on as few as 100 cells, including individual colonies grown in culture. Amplifying a polymorphic portion of the X chromosome-linked phosphoglycerate kinase (PGK) gene after selective digestion of the active X chromosome with a methylation-sensitive restriction enzyme gave results fully concordant with standard Southern blotting of DNA samples form normal (polyclonal) polymorphonuclear cells (PMN) as well as clonal PMN from patients with myelodysplastic syndrome and polycythemia vera (PCV). They have used this technique to demonstrate heterogeneity of lineage involvement in patients with PCV. The same clinical phenotype may arise from clonal proliferation of different hematopoietic progenitors.

  3. Detection of bacteria in platelet concentrates: comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing

    NARCIS (Netherlands)

    Mohammadi, Tamimount; Pietersz, Ruby N. I.; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.; Reesink, Henk W.

    2005-01-01

    BACKGROUND: Based on real-time polymerase chain reaction (PCR) technology, a broad-range 16S rDNA assay was validated and its performance was compared to that of an automated culture system to determine its usefulness for rapid routine screening of platelet concentrates (PCs). STUDY DESIGN AND

  4. Real-time polymerase chain reaction as a tool for evaluation of magnetic poly(glycidyl methacrylate)-based microspheres in molecular diagnostics

    Czech Academy of Sciences Publication Activity Database

    Trachtová, S.; Španová, A.; Horák, Daniel; Kozáková, Hana; Rittich, B.

    2016-01-01

    Roč. 22, č. 5 (2016), s. 639-646 ISSN 1381-6128 R&D Projects: GA ČR GA15-07268S Institutional support: RVO:61389013 ; RVO:61388971 Keywords : magnetic microspheres * inhibitory effect * real-time polymerase chain Subject RIV: CD - Macromolecular Chemistry; CD - Macromolecular Chemistry (MBU-M) Impact factor: 2.611, year: 2016

  5. Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique

    DEFF Research Database (Denmark)

    Handberg, Kurt; Nielsen, O.L.; Pedersen, M.W.

    1999-01-01

    Oligonucleotide pairs were constructed for priming the amplification of fragments of nucleocapsid (N) protein and spike glycoprotein (S) genes of avian infectious bronchitis virus (IBV) by reverse transcription-polymerase chain reaction (RT-PCR). One oligonucleotide pair amplified a common segment...

  6. Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes

    NARCIS (Netherlands)

    Sevilla, R.G.; Diez, A.; Noren, M.; Mouchel, O.; Jerome, M.; Verrez-Bagnis, V.; Pelt-Heerschap, van H.M.L.

    2007-01-01

    This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine

  7. Quantification of Staphylococcus aureus and Staphylococcus epidermidis on the hands of health-care workers using a real-time polymerase chain reaction method

    DEFF Research Database (Denmark)

    Horn, P; Schouenborg, P Øland; Brandslund, I

    2007-01-01

    OBJECTIVE: The objective of this study was to test a polymerase chain reaction (PCR) assay intended as a tool for monitoring hand hygiene in hospital wards. METHODS: The hands of 20 health-care workers were sampled for 10 days using real-time PCR for quantification of Staphylococcus aureus and S...

  8. Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan

    DEFF Research Database (Denmark)

    Andresen, K; Gasim, S; Elhassan, A M

    1997-01-01

    We have evaluated the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool for Leishmania donovani using blood, bone marrow and lymph node samples from Sudanese patients with a confirmed infection. Forty patients were diagnosed by microscopic examination of bone marrow or lymph...

  9. Detection of very low level Plasmodium falciparum infections using the nested polymerase chain reaction and a reassessment of the epidemiology of unstable malaria in Sudan

    DEFF Research Database (Denmark)

    Roper, C; Elhassan, I M; Hviid, L

    1996-01-01

    We have used the nested polymerase chain reaction (PCR) to assay for low level Plasmodium falciparum infections that were below the threshold of detection of blood film examination. This revealed a substantial group of asymptomatic, submicroscopically patent infections within the population of a ...

  10. Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

    DEFF Research Database (Denmark)

    Høgdall, Estrid; Vuust, Jens; Lind, Peter

    2000-01-01

    of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T...

  11. DETECTION OF HUMAN PAPILLOMAVIRUS TYPES BY THE POLYMERASE CHAIN-REACTION AND THE DIFFERENTIATION BETWEEN HIGH-RISK AND LOW-RISK CERVICAL LESIONS

    NARCIS (Netherlands)

    Cornelissen, M. T.; Bots, T.; Briët, M. A.; Jebbink, M. F.; Struyk, A. P.; van den Tweel, J. G.; Greer, C. E.; Smits, H. L.; ter Schegget, J.

    1992-01-01

    By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a

  12. Polymerase chain reaction assay to identify all immature stages of two species of the Anopheles quadrimaculatus sibling species complex (Diptera: Culicidae).

    Science.gov (United States)

    Rutledge, C R; Wesson, D M; Meek, C L

    1999-12-01

    A polymerase chain reaction assay that detects differences in the 2nd internal transcribed spacer of ribosomal DNA was tested as a means to identify all immature stages of 2 species in the Anopheles quadrimaculatus sibling species complex. The technique was successful in amplifying DNA from eggs, 1st-4th instars, and pupae of An. quadrilmaculatus Say and An. inundatus Reinert.

  13. Optimized nested polymerase chain reaction for antemortem detection of Mycobacteria in Amazon parrots (Amazona aestiva) and orange-winged Amazons (Amazona amazonica).

    Science.gov (United States)

    Baquião, Arianne Costa; Luna, Janaina Oliveira; Medina, Aziz Orro; Sanfilippo, Luiz Francisco; de Faria, Maria Jacinta; dos Santos, Manuel Armando Azevedo

    2014-03-01

    The objectives of this study were to optimize nested polymerase chain reaction (PCR) for Mycobacterium avium complex and Mycobacterium tuberculosis complex and apply them on samples from parrots. Results were negative for the presence of these Mycobacterium in the samples, and nested PCR was specific, faster, and more sensitive than other tests, thereby justifying its use in antemortem diagnosis.

  14. Presence of Helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture.

    Science.gov (United States)

    Agarwal, Sangita; Jithendra, K D

    2012-07-01

    Helicobacter pylori is an important gastrointestinal pathogen that is strongly associated with gastritis as well as peptic ulcer disease. Antimicrobial therapy frequently fails to cure H. pylori infection, which suggests there may be sanctuary sites where the organism resides. This study was aimed to assess the role of oral cavity as a reservoir of H. pylori by evaluating the occurrence of the organism in subgingival plaque of dyspeptic patients by polymerase chain reaction as well as culture. Thirty chronic periodontitis patients whose biopsy specimens were found to be H. pylori positive with rapid urease test and histopathologic examination were considered as cases and 20 chronic periodontitis patients who never had any symptoms of gastritis or peptic ulcer were taken as controls. Subgingival plaque samples were collected and sent to microbiological laboratory for detection of H. pylori by 16S rRNA based polymerase chain reaction as well as culture. 60% of the samples were found to be positive with polymerase chain reaction in the case group when compared to 15% in the controls. Also, 30% of the cases were found to be positive with culture compared to none in controls. A higher frequency of detection of H. pylori in those patients with positive antral biopsy report was seen. Also, polymerase chain reaction was found to be more sensitive than culture for detection. Thus, we conclude that detection of H. pylori in dental plaque of dyspeptic patients cannot be neglected and might represent a risk factor for recolonization of stomach after systemic eradication therapy.

  15. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients

    NARCIS (Netherlands)

    Ieven, M; Ursi, D; Van Bever, H; Quint, W; Niesters, H G; Goossens, H

    Mycoplasma pneumoniae and viruses in acute respiratory tract infections in children were studied during the winter of 1992-1993 in Antwerp, Belgium. M. pneumoniae was diagnosed in nasopharyngeal aspirates by culture and polymerase chain reaction (PCR). For this, amplification of a fragment of the PI

  16. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    Directory of Open Access Journals (Sweden)

    Pooja H. Gupta

    2015-05-01

    Full Text Available Aim: An attempt has been made to study the toll-like receptors 4 (TLR4 gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR, respectively from Kankrej (22 and Triple cross (24 cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05. Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05. Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

  17. Multiplex Reverse Transcription-Polymerase Chain Reaction untuk Deteksi Cepat Virus Flu Burung H5N1 (MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF H5N1 AVIAN INFLUENZA VIRUS

    Directory of Open Access Journals (Sweden)

    Raden Wasito

    2015-05-01

    Full Text Available Avian influenza virus subtype H5N1 (AIV H5N1 is highly pathogenic and fatal in poultry. The virusis still endemic with low virulence rate, although it may play a critical role in causing high morbidity andmortality rates in poultry in Indonesia. In general, diagnostic approach for AIV H5N1 is based onconventional serological and viral isolation methods that have the potential to produce consumings oftime and relatively expensive cost within the laboratory without compromising test utility. Thus, amolecular approach of multiplex reverse transcription-polymerase chain reaction (mRT-PCR was developedand applied for the detection of matrix gene type A influenza viruses, AIV subtype subtype H5hemagglutinin gene with simultaneous detection of N1 nucleoprotein gene. Thirty sera specimens fromthe diseased commercial chickens that were specifically amplified positive-RT-PCR for AIV H5N1 wereselected for mRT-PCR. The mRT-PCR products were visualized by agarose gel electrophoresis and consistedof DNA fragments of AIV of 245 bp, 545 bp and 343 bp for M, H5 and N1 genes, respectively. Thus, themRT-PCR that can rapidly differentiate simultaneously between these genes is very important for thecontrol and even eradication of AIV transmission in poultry in Indonesia.

  18. Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

    2014-09-01

    The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Identifikasi Brucella abortus Isolat Lokal dengan Brucella abortus Strain Specific-Polymerase Chain Reaction (IDENTIFICATION OF LOCAL ISOLATES OF BRUCELLA ABORTUS USING BRUCELLA ABORTUS STRAIN SPECIFIC-POLYMERASE CHAIN REACTION ASSAY

    Directory of Open Access Journals (Sweden)

    Susan Maphilindawati Noor

    2014-10-01

    Full Text Available Brucella abortus Strain Specific-Polymerase Chain Reaction (BaSS-PCR is a single multiplex PCRtechnique which able to identify and differentiate between Brucella abortus field strains (biovar 1, 2, and4, B. abortus vaccine strains, Brucella species, and non-Brucella species. In this study, BaSS-PCR wasapplied to identify local isolates of B. abortus in order to investigate the B. abortus strains that infectedcattle in Indonesia. Fifty local strains of B.abortus isolated from infected cattle in Java (Jakarta andBandung, South Sulawesi (Maros, East Nusa Tenggara (Kupang and Belu were used in this study. TheDNA bands were observed by agarose gel in the presence of ethidium bromide. Identification was performedbased on the size and number of DNA products amplified by PCR from each isolates. The results showedthat the 50 isolates were of B. abortus field strains. This finding showed that the cause of bovine brucellosisin Indonesia is B. abortus field strains.

  20. Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia.

    Science.gov (United States)

    Ngui, Romano; Angal, Lorainne; Fakhrurrazi, Siti Aminah; Lian, Yvonne Lim Ai; Ling, Lau Yee; Ibrahim, Jamaiah; Mahmud, Rohela

    2012-09-04

    In this study, a total of 426 human faecal samples were examined for the presence of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii infection via a combination of microscopic examination and nested polymerase chain reaction (PCR) targeting 16S ribosomal RNA of Entamoeba species. Faecal sample were collected from 426 participants in five rural villages in Peninsular Malaysia. The faecal samples were processed by direct wet smear and formalin ethyl acetate concentration technique followed by iodine staining and examined via microscopy for the presence of Entamoeba species and other intestinal parasites. Microscopically positive samples for Entamoeba species cysts were further characterized using a Nested Polymerase Chain Reaction (Nested-PCR) targeting 16S-like ribosomal RNA gene. The data entry and analysis was carried out using the SPSS software (Statistical Package for the Social Sciences) program for Windows version 17 (SPSS, Chicago, IL, USA). Based on single faecal examination, overall prevalence of Entamoeba infection was 17.6% (75/426). Females (19.1%) were more commonly infected compared to males (15.9%). Comparison by age groups showed that adults (23.9%) had higher infection rates than children (15.3%). The PCR results showed that 52 out of 75 microscopy positive samples successfully generated species-specific amplicons. The infection with E. histolytica (75.0%; 39/52) was the most common, followed by E. dispar (30.8%; 18/52) and E. moshkovskii (5.8%; 3/52). Of these, 33 (63.5%) were shown to contain only E. histolytica, 10 (19.2%) contained E. dispar and 3 (5.8%) contained only E. moshkovskii. Mixed infection with E. histolytica and E. dispar was found in 6 (11.5%) samples. The present study essentially emphasized the benefit of molecular techniques in discriminating the pathogenic Entamoeba species from the non-pathogenic for accurate diagnosis and better management of amoebiasis. The presence of E. moshkovskii is of great public health

  1. Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

    National Research Council Canada - National Science Library

    Kuwahara, Masayasu; Nagashima, Jun-ichi; Hasegawa, Masatoshi; Tamura, Takehiro; Kitagata, Rina; Hanawa, Kazuo; Hososhima, Shin-ichi; Kasamatsu, Toshiyuki; Ozaki, Hiroaki; Sawai, Hiroaki

    2006-01-01

    ...-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC...

  2. Rapid genetically modified organism (GMO screening of various food products and animal feeds using multiplex polymerase chain reaction (PCR

    Directory of Open Access Journals (Sweden)

    Lisha, V.

    2017-01-01

    Full Text Available modified crops which brought up a controversy on the safety usage of genetically modified organisms (GMOs. It has been implemented globally that all GMO products and its derived ingredients should have regulations on the usage and labelling. Thus, it is necessary to develop methods that allow rapid screening of GMO products to comply with the regulations. This study employed a reliable and flexible multiplex polymerase chain reaction (PCR method for the rapid detection of transgenic elements in genetically modified soy and maize along with the soybean LECTIN gene and maize ZEIN gene respectively. The selected four common transgenic elements were 35S promoter (35S; Agrobacterium tumefaciens nopaline synthase terminator (NOS; 5-enolypyruvylshikimate-3-phosphate synthase (epsps gene; and Cry1Ab delta-endotoxin (cry1Ab gene. Optimization of the multiplex PCR methods were carried out by using 1% Roundup ReadyTM Soybean (RRS as the certified reference material for soybean that produced fourplex PCR method detecting 35S promoter, NOS terminator, epsps gene and soybean LECTIN gene and by using 1% MON810 as the certified reference material for maize that produced triplex PCR method detecting 35S promoter, cry1Ab gene and maize ZEIN gene prior to screening of the GMO traits in various food products and animal feeds. 1/9 (11.1% of the animal feed contained maize and 1/15 (6.7% of the soybean food products showed positive results for the detection of GMO transgenic gene. None of the maize food products showed positive results for GMO transgenic gene. In total, approximately 4% of the food products and animal feed were positive as GMO. This indicated GMOs have not widely entered the food chain. However, it is necessary to have an appropriate screening method due to GMOs’ unknown potential risk to humans and to animals. This rapid screening method will provide leverage in terms of being economically wise, time saving and reliable.

  3. Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

    Science.gov (United States)

    Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

    2017-05-17

    Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

  4. Nested Polymerase Chain Reaction and Sequence- Based Detection of Leishmania Infection of Sand Flies in Recently Emerged Endemic Focus of Zoonotic Cutaneous Leishmaniasis, Southern Iran

    Directory of Open Access Journals (Sweden)

    Kourosh Azizi

    2013-06-01

    Full Text Available Background: Geographical distribution of zoonotic cutaneous leishmaniasis (ZCL has continuously been extended in recent years in Iran. The Beiza District is one of the newly-emerged endemic foci of ZCL in southern Iran. The main aim of the present study was to detect the vector(s of ZCL in this area. Methods: To detect the fauna and vectors of ZCL in this district, sand flies were caught using sticky papers. Seventy randomly selected female sand flies out of 730 were molecularly investigated for Leishmania infection using species-specific nested polymerase chain reaction (PCR assay between April and October 2010. Results: A total of 2543 sand flies were caught. The fauna was identified as 10 species (five Phlebotomus spp. and five Sergentomyia spp.. Phlebotomus papatasi was the most dominant species both indoors and outdoors (37.55% and 16.35 %, respectively. L. major was detected in 5 out of 48 investigated Phlebotomus papatasi (10.41%. Sequence-based characterization was carried out to confirm the PCR findings. The positive samples were shown to have 75-88% similarity with L. major sequences in GenBank. Conclusion: According to the findings of the present study, similar to the other foci of ZCL in Iran, P. papatasi is the proven and primary vector of CL. This study could be drawn upon for future strategy planning in this newly emerged endemic focus.

  5. Nested Polymerase Chain Reaction and Sequence- Based Detection of Leishmania Infection of Sand Flies in Recently Emerged Endemic Focus of Zoonotic Cutaneous Leishmaniasis, Southern Iran

    Science.gov (United States)

    Azizi, Kourosh; Badzohreh, Abdollah; Sarkari, Bahador; Fakoorziba, Mohammad Reza; Kalantari, Mohsen; Moemenbellah-Fard, Mohammad Djaefar; Ali-Akbarpour, Mohsen

    2013-01-01

    Background: Geographical distribution of zoonotic cutaneous leishmaniasis (ZCL) has continuously been extended in recent years in Iran. The Beiza District is one of the newly-emerged endemic foci of ZCL in southern Iran. The main aim of the present study was to detect the vector(s) of ZCL in this area. Methods: To detect the fauna and vectors of ZCL in this district, sand flies were caught using sticky papers. Seventy randomly selected female sand flies out of 730 were molecularly investigated for Leishmania infection using species-specific nested polymerase chain reaction (PCR) assay between April and October 2010. Results: A total of 2543 sand flies were caught. The fauna was identified as 10 species (five Phlebotomus spp. and five Sergentomyia spp.). Phlebotomus papatasi was the most dominant species both indoors and outdoors (37.55% and 16.35 %, respectively). L. major was detected in 5 out of 48 investigated Phlebotomus papatasi (10.41%). Sequence-based characterization was carried out to confirm the PCR findings. The positive samples were shown to have 75-88% similarity with L. major sequences in GenBank. Conclusion: According to the findings of the present study, similar to the other foci of ZCL in Iran, P. papatasi is the proven and primary vector of CL. This study could be drawn upon for future strategy planning in this newly emerged endemic focus. PMID:24031105

  6. Identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction.

    Science.gov (United States)

    Pierre, V; Drouet, M T; Deubel, V

    1994-01-01

    A reverse transcription/polymerase chain reaction (RT/PCR) protocol for the rapid detection and identification of flaviviruses was developed using a set of universal oligonucleotide primers. These primers correspond to sequences in the 3' non-coding region and in the NS5 gene which are highly conserved among the mosquito-borne flaviviruses. The sequences of the resulting amplified products were analysed for dengue 1, dengue 2, dengue 3, dengue 4, Japanese encephalitis, West Nile, yellow fever and Zika viruses, and compared with the published sequences of other flaviviruses. The 291-297 nucleotides corresponding to the C-terminus of NS5 gene showed 56 to 76% similarity, whereas the 3' non-coding region (190 to 421 nucleotides) showed only 20 to 36% similarity. Genetic classification of the Zika virus supported its traditional serological grouping. Recombinant plasmids containing the flavivirus sequences were used in a nucleic acid hybridization test to identify the RT/PCR products derived from viral RNA extracted from experimentally infected mosquitoes. The plasmids were dotted on a strip of nitrocellulose membrane and incubated with the RT/PCR product labelled with digoxigenin during the PCR step. This is a valuable method for the rapid and specific identification of mosquito-borne flaviviruses in biological specimens and for subsequent sequence analysis.

  7. Plasmodium spp. and Haemoproteus spp. infection in birds of the Brazilian Atlantic Forest detected by microscopy and polymerase chain reaction

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    Raquel Tostes

    2015-01-01

    Full Text Available In recent years haemosporidian infection by protozoa of the genus Plasmodium and Haemoproteus, has been considered one of the most important factors related to the extinction and/or population decline of several species of birds worldwide. In Brazil, despite the large avian biodiversity, few studies have been designed to detect this infection, especially among wild birds in captivity. Thus, the objective of this study was to analyze the prevalence of Plasmodium spp. and Haemoproteus spp. infection in wild birds in captivity in the Atlantic Forest of southeastern Brazil using microscopy and the polymerase chain reaction. Blood samples of 119 different species of birds kept in captivity at IBAMA during the period of July 2011 to July 2012 were collected. The parasite density was determined based only on readings of blood smears by light microscopy. The mean prevalence of Plasmodium spp. and Haemoproteus spp. infection obtained through the microscopic examination of blood smears and PCR were similar (83.19% and 81.3%, respectively, with Caracara plancus and Saltator similis being the most parasitized. The mean parasitemia determined by the microscopic counting of evolutionary forms of Plasmodium spp. and Haemoproteus spp. was 1.51%. The results obtained from this study reinforce the importance of the handling of captive birds, especially when they will be reintroduced into the wild.

  8. [DETECTION OF VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS RNA IN BIOLOGICAL SAMPLES BY REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION].

    Science.gov (United States)

    Petrov, A A; Pyshnaya, N S; Lebedev, V N; Kulish, V S; Stovba, L F; Kazantsev, A V; Borisevich, S V

    2015-01-01

    Detection-and identification of Venezuelan equine encephalomyelitis (VEE) virus RNA in biological samples by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR in real time (rRT-PCR). VEE, Sindbis, West Nile, Japanese and tick-borne encephalitis viruses were studied. Cell culture of chicken fibroblasts, outbred mice and rats, Javanese macaques were used in the experiments. Biological activity determination of the running culture of causative agents used in the experiments was carried out by negative colony method in monolayer cell culture under agar coating. and using intra-cerebral infection of mice. Reagent kits developed in the 48th Central Research Institute and Institute of Analytical Instrument Engineering were used during execution of experiments of VEE virus RNA detection by RT-PCR and rRT-PCR. VEE virus was detected in biological samples by various methods. Data from RT-PCR and rRT-PCR are in accordance with the results of virus detection in samples using sensitive animals. Use of molecular-diagnostics methods for detection in biological samples of a causative agent of a dangerous infectious disease is important for procuring biological safety of Russian Federation.

  9. Multiplex viral polymerase chain reaction testing using the FilmArray device compared with direct fluorescent antibody testing.

    Science.gov (United States)

    Wang, Jason; Simons, Debra B; Adams, James L; Jerris, Robert C; Rogers, Beverly Barton

    2014-01-01

    Advances in fluidics, electronics, and instrument design have enabled automation of complex molecular assays. The FilmArray Respiratory Panel (RP) is one such assay; it allows for rapid detection of multiple viral and bacterial respiratory targets via nested polymerase chain reaction (PCR), with all components contained within a single pouch. We performed a detailed comparison of workflow between the FilmArray RP and direct fluorescent antibody (DFA) staining. The FilmArray RP proved to be more efficient, with as few as 91 total touches needed and as little as 4 minutes 52 seconds hands-on time. This compares with as many as 502 total touches and 12 minutes 45 seconds hands-on time for DFA staining. FilmArray RP detected a greater number of organisms (20 viruses and bacteria vs 7 viruses for DFA staining) and required less training compared with DFA staining. The ease of use of the FilmArray RP makes this molecular assay amenable to operation 24 hours a day, 7 days a week in routine laboratory and point-of-care settings.

  10. Detection of Ampicillin Resistance Genes (bla in Clinical Isolates of Escherichia coli with Polymerase Chain Reaction Method

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    Tiana Milanda

    2014-09-01

    Full Text Available Escherichia coli is a rod negative Gram which could be pathogenic, if its value increases or located in outer gastrointestinal tract. Pathogenic E. coli will produce enterotoxin which will cause diarrhoea or infection in urine tract. Ampicilin was one of particular antibiotics to overcome infection. Ampicilin nowadays is no longer used as primary medicine, because of its resistance case. The aim of this research is to detect the presence of gene which is responsible to ampicilin resistant E. coli. We used isolated midstream urine from cystitis object in Hasan Sadikin Hospital (RSHS as samples. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were done to invenstigate the antibiotic resistency. Based on the result of antibiotic susceptibility testing to ampicillin, E. coli samples were resistant to ampicilin. Elektroforegram products of colony-PCR and DNA-PCR showed that the resistance case of ampicilin caused by bla gene (199 bp. Selective and rational antibiotic treatment is required to prevent ampicillin resistance in patients with symptoms

  11. Babesia bovis and B. bigemina DNA detected in cattle and ticks from Zimbabwe by polymerase chain reaction

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    I. Smeenk

    2000-07-01

    Full Text Available From blood collected from 94 cattle at 12 locations in the eastern and northeastern areas of Zimbabwe, DNA was extracted and analysed by polymerase chain reaction with primers previously reported to be specific for Babesia bigemina and Babesia bovis. Overall, DNA of Babesia bigemina was detected in the blood of 33/94 (35 % cattle and DNA from B. bovis was detected in 27/58 (47 % of cattle. The prevalence of DNA of B. bigemina was significantly higher in young animals (<2 years (23/46 than in animals over 2 years of age (10/48; (chi2 = 8.77; P < 0.01 %. Although tick sampling was not thorough, Boophilus decoloratus could be collected at 7/9 sites sampled and Boophilus microplus at 4/9 sites. Of the 20 B. decoloratus allowed to oviposit before PCR analysis, 1 (5 % contained DNA that could be amplified with primers for B. bigemina while 12 (60 % were positive with primers for B. bovis. Of the B. microplus allowed to oviposit, 11/16 (69 % were positive for B. bovis DNAby PCR and 2/16 (12 % were positive for B. bigemina.

  12. Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

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    Azzedine Bounamous

    2014-07-01

    Full Text Available A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b, t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

  13. Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction- restriction fragment length polymorphism

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    Caldeira Roberta L

    2003-01-01

    Full Text Available Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2 and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

  14. Morphological and polymerase chain reaction-restriction fragment lenght polymorphism characterization of Biomphalaria kuhniana and Biomphalaria amazonica from Colombia

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    Luz E Velásquez

    2002-10-01

    Full Text Available In Colombia, five Biomphalaria planorbid species are known: B. kuhniana, B. straminea, B. peregrina, B. canonica and B. oligoza(var. B. philippiana. Among them, B. straminea is intermediate host of Schistosoma mansoni and B. peregrina has been found to be experimentally susceptible to this parasite. B. straminea is commonly confused with B. kuhniana and they have been clustered together with B. intermedia in the complex named B. straminea. The difficulties involved in the specific identification, based on morphological data, have motivated the use of new techniques as auxiliary tools in cases of inconclusive morphological identification of such planorbid. In the present study, five Biomphalaria populations from the Colombian Amazon region and from Interandian Valleys were morphologically identified and characterized by polymerase chain reaction-restriction fragment lenght polymorphism directed at the internal transcribed spacer region of the rRNA gene, followed by digestion of the generated fragment with restriction enzymes (DdeI, AluI, RsaI, MvaI and HaeIII. Known profiles of the Brazilian species B. straminea, B. peregrina, B. kuhniana, B. intermedia and B. amazonica, besides B. kuhniana from Colombia, were used for comparison. The five populations under study were morphologically and molecularly identified as B. kuhniana and B. amazonica.

  15. Polymerase chain reaction-restriction fragment length polymorphism analysis: a simple method for species identification in food.

    Science.gov (United States)

    Meyer, R; Höfelein, C; Lüthy, J; Candrian, U

    1995-01-01

    The polymerase chain reaction (PCR) technique was applied to meat species identification in marinated and heat-treated or fermented products and to the differentiation of closely related species. DNA was isolated from meat samples by using a DNA-binding resin and was subjected to PCR analysis. Primers used were complementary to conserved areas of the vertebrate mitochondrial cytochrome b (cytb) gene and yielded a 359 base-pair (bp) fragment, including a variable 307 bp region. Restriction endonuclease analysis based on sequence data of those fragments was used for differentiation among species. Restriction fragment length polymorphisms (RFLPs) were detected when pig, cattle, wild boar, buffalo, sheep, goat, horse, chicken, and turkey amplicons were cut with AluI, RsaI, TaqI, and HinfI. Analysis of sausages indicates the applicability of this approach to food products containing meat from 3 different species. The PCR-RFLP analytical method detected pork in heated meat mixtures with beef at levels below 1%, and the method was confirmed with porcine- and bovine-specific PCR assays by amplifying fragments of their growth hormone genes. Inter- and intraspecific differences of more than 22 animal species with nearly unknown cytb DNA sequences, including hoofed mammals (ungulates), and poultry were determined with PCR-RFLP typing by using 20 different endonucleases. This typing method allowed the discrimination of game meats, including stag, roe deer, chamois, moose, reindeer, kangaroo, springbok, and other antelopes in marinated and heat-treated products.

  16. Tetrabutylammonium phosphate-assisted separation of multiplex polymerase chain reaction products in non-gel sieving capillary electrophoresis.

    Science.gov (United States)

    Zhang, Sheng; Jiang, Cheng; Jia, Li

    2011-01-15

    A method based on non-gel sieving capillary electrophoresis (NGS-CE) with ultraviolet (UV) detection has been developed for the separation of multiplex polymerase chain reaction (PCR) products of three pathogenic bacteria in which hydroxypropylmethylcellulose was used as the sieving medium and dynamic capillary coating. In the method, an ion pair reagent, tetrabutylammonium phosphate (TBAP), was first used in NGS-CE to improve the detection sensitivities and resolutions of DNA fragments. The interaction of TBAP and DNA was proved using the UV spectra of DNA with and without TBAP. Field-enhanced sample injection was used as an on-line preconcentration method to improve the detection sensitivity. The separation of DNA fragments ranging from 100 to 1000 bp was accomplished in 30 min. Three pairs of primers and three PCR products of bacteria were successfully separated in 25 min using the developed method. The intraday relative standard deviations (RSDs) for the migration time and peak area for each PCR product were less than 2.4% (n=5), and the interday RSDs were less than 6.1% (n=15). Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Evaluation of polymerase chain reaction using primer MPB 64 for diagnosis of clinically suspected cases of extrapulmonary tuberculosis

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    Dnyaneshwari P. Ghadage

    2014-01-01

    Full Text Available Background: Pulmonary tuberculosis can be easily diagnosed by simple techniques such as microscopy. However, extrapulmonary tuberculosis (EPTB often presents a diagnostic dilemma. Microscopy and culture have proved to be insensitive techniques for diagnosis of EPTB. There is an urgent need for rapid and sensitive diagnostic methods. Aim: The present study was conducted to evaluate the role of polymerase chain reaction (PCR in the early diagnosis of clinically suspected cases of EPTB. Materials and Methods: A total of 80 clinical specimens comprising pleural fluid, cerebrospinal fluid, ascitic fluid, fine-needle aspiration biopsy, and pus and biopsy from clinically suspected EPTB cases were processed and followed up by conventional methods and PCR using MPB64 primer. Results: Tuberculous pleural effusion (71% was found to be the most common clinical presentation of EPTB. Overall, PCR could detect EPTB in 61.2% cases. Microscopy and culture could detect 18.7% and 22.5% EPTB cases, respectively. PCR was positive in all tissue samples suggestive of tuberculosis on histopathological examination. Of the 62 EPTB patients who responded to antituberculosis treatment (ATT, 49 patients were PCR positive. Conclusion: PCR using MPB64 had a significant advantage over the conventional methods to detect the presence of M. tuberculosis in specimens of clinically suspected EPTB patients for early diagnosis of tuberculosis.

  18. Statistical uncertainty and its propagation in the analysis of quantitative polymerase chain reaction data: comparison of methods.

    Science.gov (United States)

    Tellinghuisen, Joel; Spiess, Andrej-Nikolai

    2014-11-01

    Most methods for analyzing real-time quantitative polymerase chain reaction (qPCR) data for single experiments estimate the hypothetical cycle 0 signal y0 by first estimating the quantification cycle (Cq) and amplification efficiency (E) from least-squares fits of fluorescence intensity data for cycles near the onset of the growth phase. The resulting y0 values are statistically equivalent to the corresponding Cq if and only if E is taken to be error free. But uncertainty in E usually dominates the total uncertainty in y0, making the latter much degraded in precision compared with Cq. Bias in E can be an even greater source of error in y0. So-called mechanistic models achieve higher precision in estimating y0 by tacitly assuming E=2 in the baseline region and so are subject to this bias error. When used in calibration, the mechanistic y0 is statistically comparable to Cq from the other methods. When a signal threshold yq is used to define Cq, best estimation precision is obtained by setting yq near the maximum signal in the range of fitted cycles, in conflict with common practice in the y0 estimation algorithms. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. A sample-to-answer, real-time convective polymerase chain reaction system for point-of-care diagnostics.

    Science.gov (United States)

    Shu, Bowen; Zhang, Chunsun; Xing, Da

    2017-11-15

    Timely and accurate molecular diagnostics at the point-of-care (POC) level is critical to global health. To this end, we propose a handheld convective-flow real-time polymerase chain reaction (PCR) system capable of direct sample-to-answer genetic analysis for the first time. Such a system mainly consists of a magnetic bead-assisted photothermolysis sample preparation, a closed-loop convective PCR reactor, and a wireless video camera-based real-time fluorescence detection. The sample preparation exploits the dual functionality of vancomycin-modified magnetic beads (VMBs) for bacteria enrichment and photothermal conversion, enabling cell pre-concentration and lysis to be finished in less than 3min. On the presented system, convective thermocycling is driven by a single-heater thermal gradient, and its amplification is monitored in real-time, with an analysis speed of less than 25min, a dynamic linear range from 106 to 101 copies/µL and a detection sensitivity of as little as 1 copies/µL. Additionally, the proposed PCR system is self-contained with a control electronics, pocket-size and battery-powered, providing a low-cost genetic analysis in a portable format. Therefore, we believe that this integrated system may become a potential candidate for fast, accurate and affordable POC molecular diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Semi-quantitative digital analysis of polymerase chain reactionelectrophoresis gel: Potential applications in low-income veterinary laboratories

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    John F. Antiabong

    2016-09-01

    Full Text Available Aim: The interpretation of conventional polymerase chain reaction (PCR assay results is often limited to either positive or negative (non-detectable. The more robust quantitative PCR (qPCR method is mostly reserved for quantitation studies and not a readily accessible technology in laboratories across developing nations. The aim of this study was to evaluate a semi-quantitative method for conventional PCR amplicons using digital image analysis of electrophoretic gel. The potential applications are also discussed. Materials and Methods: This study describes standard conditions for the digital image analysis of PCR amplicons using the freely available ImageJ software and confirmed using the qPCR assay. Results and Conclusion: Comparison of ImageJ analysis of PCR-electrophoresis gel and qPCR methods showed similar trends in the Fusobacterium necrophorum DNA concentration associated with healthy and periodontal disease infected wallabies (p≤0.03. Based on these empirical data, this study adds descriptive attributes (“more” or “less” to the interpretation of conventional PCR results. The potential applications in low-income veterinary laboratories are suggested, and guidelines for the adoption of the method are also highlighted.

  1. First case series of emerging Rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing

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    P Aarthi

    2013-01-01

    Full Text Available Purpose: To detect and identify the aetiological agent in the peripheral blood from the cases of neonatal sepsis. Materials and Methods: Four neonates from geographically different regions of South India presented with signs of neonatal sepsis and all the routine clinical and laboratory investigations were performed. Blood culture by Bac T Alert 3D was negative. To establish the aetiology, polymerase chain reaction (PCR for eubacterial genome and subsequent amplification with Gram positive and Gram negative primers were performed followed by deoxyribonucleic acid (DNA sequencing. Results: PCR for the detection of eubacterial genome was positive in all the four neonates and further amplification with designed Gram positive and Gram negative primers revealed the presence of Gram negative bacteria. The amplicons were identified as Orientia tsutsugamushi in three neonates and Coxiella burnetti in the other neonate. Multalin analysis was done to further characterise the strain variation among the three strains. Conclusion: PCR-based DNA sequencing is a rapid and reliable diagnostic tool to identify the aetiological agents of neonatal sepsis. This is the first case series of emerging Rickettsial neonatal sepsis in India .

  2. Helicobacter-negative gastritis: polymerase chain reaction for Helicobacter DNA is a valuable tool to elucidate the diagnosis.

    Science.gov (United States)

    Kiss, S; Zsikla, V; Frank, A; Willi, N; Cathomas, G

    2016-04-01

    Helicobacter-negative gastritis has been increasingly reported. Molecular techniques as the polymerase chain reaction (PCR) may detect bacterial DNA in histologically negative gastritis. To evaluate of Helicobacter PCR in gastric biopsies for the daily diagnostics of Helicobacter-negative gastritis. Over a 5-year period, routine biopsies with chronic gastritis reminiscent of Helicobacter infection, but negative by histology, were tested by using a H. pylori specific PCR. Subsequently, PCR-negative samples were re-evaluated using PCR for other Helicobacter species. Of the 9184 gastric biopsies, 339 (3.7%) with histological-negative gastritis and adequate material were forwarded to PCR analysis for H. pylori and 146 (43.1%) revealed a positive result. In 193 H. pylori DNA-negative biopsies, re-analysis using PCR primers for other Helicobacter species, revealed further 23 (11.9%) positive biopsies, including 4 (2.1%) biopsies with H. heilmannii sensu lato. PCR-positive biopsies showed a higher overall inflammatory score, more lymphoid follicles/aggregates and neutrophils (P gastritis. © 2016 John Wiley & Sons Ltd.

  3. An integrated CMOS quantitative-polymerase-chain-reaction lab-on-chip for point-of-care diagnostics.

    Science.gov (United States)

    Norian, Haig; Field, Ryan M; Kymissis, Ioannis; Shepard, Kenneth L

    2014-10-21

    Considerable effort has recently been directed toward the miniaturization of quantitative-polymerase-chain-reaction (qPCR) instrumentation in an effort to reduce both cost and form factor for point-of-care applications. Considerable gains have been made in shrinking the required volumes of PCR reagents, but resultant prototypes retain their bench-top form factor either due to heavy heating plates or cumbersome optical sensing instrumentation. In this paper, we describe the use of complementary-metal-oxide semiconductor (CMOS) integrated circuit (IC) technology to produce a fully integrated qPCR lab-on-chip. Exploiting a 0.35 μm high-voltage CMOS process, the IC contains all of the key components for performing qPCR. Integrated resistive heaters and temperature sensors regulate the surface temperature of the chip to an accuracy of 0.45 °C. Electrowetting-on-dielectric microfluidics are actively driven from the chip surface, allowing for droplet generation and transport down to volumes less than 1.2 nanoliter. Integrated single-photon avalanche diodes (SPADs) are used for fluorescent monitoring of the reaction, allowing for the quantification of target DNA with more than four-orders-of-magnitude of dynamic range and sensitivities down to a single copy per droplet. Using this device, reliable and sensitive real-time proof-of-concept detection of Staphylococcus aureus (S. aureus) is demonstrated.

  4. Comparison of Enterococcus quantitative polymerase chain reaction analysis results from fresh and marine waters on two real-time instruments.

    Science.gov (United States)

    Sivaganensan, Mano; Varma, Manju; Haugland, Richard A

    2012-11-01

    The U.S. Environmental Protection Agency will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for implementation of this and other qPCR methods is whether the results are comparable on different PCR instruments. In this study, quantitative estimates of Enterococcus densities from marine and freshwater samples were determined by the qPCR method from cycle threshold (Ct) measurements obtained on Applied Biosystems StepOnePlus and Cepheid SmartCycler instruments. Three variations of a comparative Ct model, differing in their sources of calibration data, were used in the estimations. Both traditional and Bayesian statistical modeling approaches were examined in the instrument comparisons. The traditional analysis of variance (ANOVA) approach indicated no significant differences (p>0.05) between mean density estimates from the instruments in two of the three model variations. The Bayesian approach indicated that the 95% Bayesian credible intervals of density estimates from the instruments overlapped in all models; however, the uncertainty of the estimates varied depending on the model. These results support the interchangeable use of the two instruments in the method and also illustrate the importance of defining the source of calibration data used in the comparative Ct model. Published by Elsevier Inc.

  5. Diagnosis of foot-and mouth disease by real time reverse transcription polymerase chain reaction under field conditions in Brazil.

    Science.gov (United States)

    Paixão, Tatiane A; Neta, Alcina V Carvalho; Paiva, Naimes O; Reis, Jorge R; Barbosa, Meirivan S; Serra, Claudia V; Silva, René R; Beckham, Tammy R; Martin, Barbara M; Clarke, Neville P; Adams, L Garry; Santos, Renato L

    2008-12-31

    Foot-and-mouth disease (FMD) is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. Virus isolation and enzyme-linked immunosorbent assay (ELISA) are the gold standard tests for diagnosis of FMD. As these methods are time consuming, assays based on viral nucleic acid amplification have been developed. A previously described real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay with high sensitivity and specificity under laboratorial and experimental conditions was used in the current study. To verify the applicability of this assay under field conditions in Brazil, 460 oral swabs from cattle were collected in areas free of FMD (n = 200) and from areas with outbreaks of FMD (n = 260). Three samples from areas with outbreaks of FMD were positive by real-time RT-PCR, and 2 of those samples were positive by virus isolation and ELISA. Four other samples were considered inconclusive by real-time RT-PCR (threshold cycle [Ct] > 40); whereas all 200 samples from an area free of FMD were real-time RT-PCR negative. real-time RT-PCR is a powerful technique for reliable detection of FMDV in a fraction of the time required for virus isolation and ELISA. However, it is noteworthy that lack of infrastructure in certain areas with high risk of FMD may be a limiting factor for using real-time RT-PCR as a routine diagnostic tool.

  6. Diagnosis of foot-and mouth disease by real time reverse transcription polymerase chain reaction under field conditions in Brazil

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    Clarke Neville P

    2008-12-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. Virus isolation and enzyme-linked immunosorbent assay (ELISA are the gold standard tests for diagnosis of FMD. As these methods are time consuming, assays based on viral nucleic acid amplification have been developed. Results A previously described real-time reverse transcriptase polymerase chain reaction (RT-PCR assay with high sensitivity and specificity under laboratorial and experimental conditions was used in the current study. To verify the applicability of this assay under field conditions in Brazil, 460 oral swabs from cattle were collected in areas free of FMD (n = 200 and from areas with outbreaks of FMD (n = 260. Three samples from areas with outbreaks of FMD were positive by real-time RT-PCR, and 2 of those samples were positive by virus isolation and ELISA. Four other samples were considered inconclusive by real-time RT-PCR (threshold cycle [Ct] > 40; whereas all 200 samples from an area free of FMD were real-time RT-PCR negative. Conclusion real-time RT-PCR is a powerful technique for reliable detection of FMDV in a fraction of the time required for virus isolation and ELISA. However, it is noteworthy that lack of infrastructure in certain areas with high risk of FMD may be a limiting factor for using real-time RT-PCR as a routine diagnostic tool.

  7. Polymerase chain reaction-based differential diagnosis of Ancylostoma duodenale and Necator americanus infections in humans in northern Ghana.

    Science.gov (United States)

    de Gruijter, Johanna M; van Lieshout, Lisette; Gasser, Robin B; Verweij, Jaco J; Brienen, Eric A T; Ziem, Juventus B; Yelifari, Lawrence; Polderman, Anton M

    2005-06-01

    We evaluated a two-step semi-nested polymerase chain reaction (PCR)-based approach for the specific detection of Ancylostoma duodenale DNA in human faeces. The test was used to determine to what extent this species of hookworm is present in the regions of Bolgatanga and Garu of northern Ghana. Initially, the sensitivity and specificity of the PCR were tested using a range of well-defined control samples. Subsequently, a total of 378 human faecal DNA samples from Bolgatanga and Garu were subjected to the PCR. The results were compared with those obtained using a previously established PCR for the specific detection of Necator americanus DNA in human faeces. Infection with A. duodenale was recorded in 74 (19.6%) samples and N. americanus in 278 (73.5%), of which 64 (16.9%), represented co-infections with both species. While A. duodenale was predominantly detected in the samples from Bolgatanga, infections in Garu related almost exclusively to N. americanus. The results showed that the present PCR approach is a valuable complementary tool for the diagnosis of A. duodenale infection in humans in Ghana, having implications for epidemiological studies and for the monitoring of the success of control programmes in regions in Africa.

  8. A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis.

    Science.gov (United States)

    Han, Jae-Ik; Chang, Dong-Woo; Na, Ki-Jeong

    2015-01-01

    Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 mL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes.

  9. Collaborative ring trial of the papaya endogenous reference gene and its polymerase chain reaction assays for genetically modified organism analysis.

    Science.gov (United States)

    Wei, Jiaojun; Li, Feiwu; Guo, Jinchao; Li, Xiang; Xu, Junfeng; Wu, Gang; Zhang, Dabing; Yang, Litao

    2013-11-27

    The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

  10. Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.

    Science.gov (United States)

    Hill, Jonathon T; Demarest, Bradley L; Bisgrove, Brent W; Su, Yi-Chu; Smith, Megan; Yost, H Joseph

    2014-12-01

    Genome editing techniques, including ZFN, TALEN, and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a polymerase chain reaction (PCR) amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of "double peaks" due to the mismatched region. To facilitate indel identification, we developed an online tool called Poly Peak Parser (available at http://yost.genetics.utah.edu/software.php) that is able to separate chromatogram data containing ambiguous base calls into wild-type and mutant allele sequences. This tool allows the nature of the indel to be determined from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning. The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals, this tool can also be used to identify heterozygous indels in many contexts. © 2014 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

  11. Efficiency of noninvasive sampling methods (swab together with Polymerase Chain Reaction (PCR for diagnosing American Tegumentary Leishmaniasis

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    Sara Macente Boni

    Full Text Available ABSTRACT Traditional diagnostic methods used to detect American Tegumentary Leishmaniasis, such as histopathology using biopsy samples, culture techniques, and direct search for parasites, have low sensitivity and require invasive collection procedures. This study evaluates the efficiency of noninvasive sampling methods (swab along with Polymerase Chain Reaction (PCR for diagnosing American Tegumentary Leishmaniasis using skin and mucous samples from 25 patients who had tested positive for leishmaniasis. The outcome of the tests performance on swab samples was compatible with PCR results on biopsy samples. The findings have also shown that PCR-kDNA test is more efficient than PCR-HSP70 and qPCR tests (sensitivity of 92.3%, 40.7%, and 41%, respectively. Given the high sensitivity of the tests and the fact that the sampling method using swabs affords greater patient comfort and safety, it could be said that this method is a promising alternative to conventional biopsy-based methods for the molecular diagnosis of leishmaniasis.

  12. Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems.

    Science.gov (United States)

    Ximenes, Camila; Brandão, Eduardo; Oliveira, Paula; Rocha, Abraham; Rego, Tamisa; Medeiros, Rafael; Aguiar-Santos, Ana; Ferraz, João; Reis, Christian; Araujo, Paulo; Carvalho, Luiz; Melo, Fabio L

    2014-12-01

    The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

  13. Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

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    Camila Ximenes

    2014-12-01

    Full Text Available The Global Program for the Elimination of Lymphatic Filariasis (GPELF aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2 and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2, which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

  14. Polymerase chain reaction optimization for the detection of Pasteurella multocida B:2, the causative agent of Haemorrhagic septicaemia

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    Lily Natalia

    2001-12-01

    Full Text Available Specific detection of Pasteurella multocida type B:2 by polymerase chain reaction (PCR, using a set of DNA primers wasoptimised. Effects of the addition of ethylene diamine tetra acetic acid (EDTA to the sample preparation, Escherichia coli contamination and the number of P. multocida on the PCR product was assessed. The PCR test was compared to the standard bacteriological method for the detection of P. multocida B:2 in tonsillar swab samples collected from slaughter houses of various regions in Indonesia. Addition of 100 mM EDTA-saline to P. multocida B:2 spiked tonsillar swab samples inhibits the production 350 base pairs (bp PCR product. The inhibitory effect of the EDT A can be eliminated by three times washing with deionised water. The PCR can detect P. multocida as low as I organism and contanimation of 100 CFU of E. coli does not effect the PCR result. The results show that the DNA primers for P. multocida B:2 is sensitive and specific. The inhibitory effect of EDTA in PCR samples can be eliminated by washings.

  15. Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction, Sierra Leone, 2014.

    Science.gov (United States)

    Liu, Licheng; Sun, Yang; Kargbo, Brima; Zhang, Chuntao; Feng, Huahua; Lu, Huijun; Liu, Wenseng; Wang, Chengyu; Hu, Yi; Deng, Yongqiang; Jiang, Jiafu; Kang, Xiaoping; Yang, Honglei; Jiang, Yongqiang; Yang, Yinhui; Kargbo, David; Qian, Jun; Chen, Weijun

    2015-09-15

    During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Utility of a rapid immunochromatographic strip test in detecting canine parvovirus infection compared with polymerase chain reaction

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    Sundaran S. Tinky

    2015-04-01

    Full Text Available Aim: The present study was undertaken to detect the presence of canine parvovirus (CPV in fecal samples of diarrheic dogs by conventional polymerase chain reaction (PCR and immunochromatographic (IC strip test and to compare the diagnostic potential of these tests. Materials and Methods: A total of 50 fecal samples collected from diarrheic dogs suspected for CPV infection were subjected to PCR using CPV-555 primer amplifying the gene coding for the VP1 protein. These samples were also tested by IC strip test using a commercial rapid Ag test kit. The results were statistically analyzed using McNemar test. Results: A total of 22 samples (44% were detected as positive by PCR, which yielded a specific amplicon of 583 bp. In IC strip test, 18 (36% samples were found to be positive. The sensitivity of the test as compared to PCR was found to be 72.22% and specificity was 92.86%. Positive predictive value and negative predictive value of IC strip test was found to be 88.89% and 81.25%, respectively. Statistical analysis of the results of PCR and IC assay using McNemar test revealed no significant difference (p>0.05. Conclusion: The IC strip test could be employed as a rapid field level diagnostic tool for the diagnosis of canine parvoviral diarrhea.

  17. Detection of African swine fever virus from formalin fixed and non-fixed tissues by polymerase chain reaction

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    P. D. Luka

    2014-10-01

    Full Text Available Aim: Formalin fixing and paraffin embedding of tissue samples is one of the techniques for preserving the structural integrity of cells for a very long time. However, extraction and analysis of genomic material from formalin fixed tissue (FFT remains a challenge despite numerous attempts to develop a more effective method. The success of polymerase chain reaction (PCR depends on the quality of DNA extract. Materials and Methods: Here we assessed the conventional method of DNA extraction from FFT for African swine fever virus (ASFV detection. The modified conventional method gave a higher quality DNA when compared with commercially available DNA extraction kits (QIAamp® DNA Mini Kit, DNeasy® Blood and Tissue Kit, and ZR Genomic DNA™ Tissue MiniPrep. Results: An average A260/A280 DNA purity of 0.86-1.68 and 3.22-5.32 μg DNA/mg for formalin fixed and non-fixed tissues, respectively using a conventional method. In a reproducible and three times repeat PCR, the ASFV DNA expected product size of 278 bp was obtained from the DNA extract of the conventional method but not from the DNA extract of the commercial kits. Conclusion: The present study has demonstrated that the conventional method extracts ASFV genome better than commercial kit. In summary, the commercial kit extraction appeared not suitable to purify ASFV DNA from FFT. We, therefore, recommend that the use of the conventional method be considered for African swine fever DNA extraction from FFT.

  18. Evaluation of four DNA extraction methods for the detection of Tritrichomonas foetus in feline stool specimens by polymerase chain reaction.

    Science.gov (United States)

    Stauffer, Stephen H; Birkenheuer, Adam J; Levy, Michael G; Marr, Henry; Gookin, Jody L

    2008-09-01

    Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect >or=10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.

  19. [Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction].

    Science.gov (United States)

    Pınar, Ahmet; Akyön, Yakut; Alp, Alpaslan; Ergüven, Sibel

    2010-07-01

    This study was aimed to adapt a sensitive DNA extraction protocol in stool samples for real-time polymerase chain reaction (PCR) detection of Entamoeba histolytica which causes important morbidity and mortality worldwide. Stool extraction is a problematic step and has direct effects on PCR sensitivity. In order to improve the sensitivity of E.histolytica detection by real-time PCR, "QIAamp DNA stool minikit (Qiagen, Germany)" was modified by adding an overnight incubation step with proteinase K and sodium dodecyl sulfate (SDS) in this study. Three different extraction methods [(1) original method, (2) cetyltrimethyl-ammonium bromide (CTAB) method, (3) modified method] were evaluated for effects on sensitivity in real-time quantitative PCR (Artus RealArt TM E.histolytica RG PCR Kit, Qiagen Diagnostics, Germany). For this purpose, several concentrations of standard E.histolytica DNA were spiked in parasite-free stool samples and three different extraction protocols were performed. Detection sensitivities of "QIAamp DNA stool minikit" was found 5000 copies/ml and of CTAB method was found 500 copies/ml. Detection sensitivity of the extraction was improved to 5 copies/mL by modified "QIAamp DNA stool minikit" protocol. Since detection sensitivities of nucleic acid extraction protocols from stool samples directly affect the sensitivity of PCR amplification, different extraction protocols for different microorganisms should be evaluated.

  20. Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples.

    Science.gov (United States)

    Espinosa, M I; Bertin, A; Squeo, F A; Cortés, A; Gouin, N

    2015-01-23

    Feces-based population genetic studies have become increasingly popular. However, polymerase chain reaction (PCR) amplification rates from fecal material vary depending on the species, populations, loci, and extraction protocols. Here, we assessed the PCR amplification success of three microsatellite markers and a segment of the mitochondrial control region of DNA extracted from field-collected feces of guanaco (Lama guanicoe) using two protocols - Qiagen DNA Stool Kit and 2 cetyltrimethylammonium bromide/phenol:chloroform:isoamyl alcohol (2CTAB/PCI) method. Chelex resin treatment to remove inhibitors was also tested. Our results show that the mitochondrial locus was the most difficult to amplify. PCR success rates improved for all markers after Chelex treatment of extracted DNA, and 2CTAB/PCI method (95.83%) appeared to perform slightly better than stool kit (91.67%) for the nuclear markers. Amplification success was significantly influenced by the extraction method, Chelex treatment, and locus (P 0.89), but they decreased slightly after treatment for amplification of nuclear markers and markedly after treatment for amplification of the mitochondrial control region. Thus, we showed that Chelex treatment gives high PCR success, especially for nuclear markers, and adequate DNA extraction rates can be achieved from L. guanicoe feces even from non-fresh fecal material. Although not significant, 2CTAB/PCI method tended to provide higher successful amplification rates on a whole set of samples, suggesting that the method could be particularly useful when using small sample sizes.

  1. Evaluation of target sequences for the polymerase chain reaction-based detection of Salmonella in artificially contaminated beef.

    Science.gov (United States)

    de Almeida, Michelle Vieira; Silva, Abelardo; Nero, Luís Augusto

    2014-02-01

    Salmonella is a major cause of foodborne diseases worldwide, which has fueled the demand for the development and evaluation of sensitive, specific, and rapid detection methodologies, such as polymerase chain reaction (PCR). In this study, six primer pairs for the detection of Salmonella were evaluated by PCR with isolates of Salmonella spp. (115) and other bacteria (104). The primers designed for the sifB gene provided the best performance regarding specificity and sensitivity (100%). These primers were selected and used to develop a PCR assay for Salmonella detection during the enrichment steps of the conventional detection method in spiked beef samples. The enrichment steps were: buffered peptone water (BPW), Rappaport-Vassiliadis soya broth (RVS) and at the Müller-Kauffmann tetrathionate novobiocin broth (MKTTn), after 18 h (BPW) and 24 h (RVS and MKTTn) of incubation. The initial concentrations of the Salmonella inocula were 10¹, 10², and 10³ colony-forming units/25 g. The protocol was able to detect Salmonella at all concentrations in the enrichment steps, but not in the nonenriched samples. These results indicated that the proposed protocol was suitable to detect Salmonella in beef during the intermediate stages of the conventional isolation protocol, substantially reducing the time required to obtain the final results.

  2. Continuous Excretion ofLeptospira borgpeterseniiBallum in Mice Assessed by Viability Quantitative Polymerase Chain Reaction.

    Science.gov (United States)

    Soupé-Gilbert, Marie-Estelle; Bierque, Emilie; Geroult, Sophie; Teurlai, Magali; Goarant, Cyrille

    2017-10-01

    Rodents are the main reservoir animals of leptospirosis. In this study, we characterized and quantified the urinary excretion dynamics of Leptospira by Mus musculus infected with 2 × 10 8 virulent Leptospira borgpetersenii serogroup Ballum. Each micturition was collected separately in metabolic cages, at 12 time points from 7 to 117 days post-infection (dpi). We detected Leptospira in all urine samples collected (up to 8 per time point per mouse) proving that Leptospira excretion is continuous with ca. 90% live L. borgpetersenii Ballum, revealed by viability quantitative polymerase chain reaction. Microscopic visualization by Live/Dead fluorescence confirmed this high proportion of live bacteria and demonstrated that L. borgpetersenii Ballum are excreted, at least partly, as bacterial aggregates. We observed two distinct phases in the excretion dynamics, first an increase in Leptospira concentration shed in the urine between 7 and 63 dpi followed by a plateau phase from 63 dpi onward, with up to 3 × 10 7 Leptospira per mL of urine. These two phases seem to correspond to progressive colonization of renal tubules first, then to stable cell survival and maintenance in kidneys. Therefore, chronically infected adult mice are able to contaminate the environment via urine at each micturition event throughout their lifetime. Because Leptospira excretion reached its maximum 2 months after infection, older rodents have a greater risk of contaminating their surrounding environment.

  3. System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation.

    Science.gov (United States)

    Spiess, Andrej-Nikolai; Rödiger, Stefan; Burdukiewicz, Michał; Volksdorf, Thomas; Tellinghuisen, Joel

    2016-12-13

    Real-time quantitative polymerase chain reaction (qPCR) data are found to display periodic patterns in the fluorescence intensity as a function of sample number for fixed cycle number. This behavior is seen for technical replicate datasets recorded on several different commercial instruments; it occurs in the baseline region and typically increases with increasing cycle number in the growth and plateau regions. Autocorrelation analysis reveals periodicities of 12 for 96-well systems and 24 for a 384-well system, indicating a correlation with block architecture. Passive dye experiments show that the effect may be from optical detector bias. Importantly, the signal periodicity manifests as periodicity in quantification cycle (Cq) values when these are estimated by the widely applied fixed threshold approach, but not when scale-insensitive markers like first- and second-derivative maxima are used. Accordingly, any scale variability in the growth curves will lead to bias in constant-threshold-based Cqs, making it mandatory that workers should either use scale-insensitive Cqs or normalize their growth curves to constant amplitude before applying the constant threshold method.

  4. Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children

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    Thiago Leite Fraga

    2010-05-01

    Full Text Available The diagnosis of visceral leishmaniasis (VL generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes. This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6% that is similar to that from the PCR of bone marrow aspirates (91.1% and higher than that achieved with microscopy (80% or culture (26.7% methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

  5. "DETECTION OF BACTERIAL, METHICILLIN RESISTANCE, AND β-LACTAMASE GENES FOUND IN WOUND SWABS BY MULTIPLEX POLYMERASE CHAIN REACTION"

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    S. Sadeghian

    2004-05-01

    Full Text Available Coagulase-positive and coagulase negative, methicillin-resistant staphylococci are major causes of serious nosocomial infections and it is very important to have a reliable test to detect these bacteria. A multiplex polymerase chain reaction (mPCR was used on 100 clinical samples for simultaneous amplification of the universal bacterial, mec-A encoding the penicillin binding protein 2a, which is associated with staphylococcal methicillin resistance and TEM-1 encoding the β-lactamase, which accounts for the majority of all cases of the plasmid β-lactamase resistance worldwide. Out of 100 wound swabs tested, 99% with universal primers, 26% with TEM-1 primers and 6% with mec-A primers were positive. Dot blot Digoxigenin hybridization on the 30 samples was carried out to confirm identified bacteria with specific bacterial probes. Out of 100 wound swabs, 38% were positive with Staphylococcus aureus probe, 23% were positive with enteric bacteria probe, 7% were positive with Streptococcus agalactia probe and 1% were positive with Haemophilus influenza probe. The mPCR method used in this study, was designed to be incorporated into the workflow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.

  6. Real-Time Polymerase Chain Reaction Detection of Angiostrongylus cantonensis DNA in Cerebrospinal Fluid from Patients with Eosinophilic Meningitis.

    Science.gov (United States)

    Qvarnstrom, Yvonne; Xayavong, Maniphet; da Silva, Ana Cristina Aramburu; Park, Sarah Y; Whelen, A Christian; Calimlim, Precilia S; Sciulli, Rebecca H; Honda, Stacey A A; Higa, Karen; Kitsutani, Paul; Chea, Nora; Heng, Seng; Johnson, Stuart; Graeff-Teixeira, Carlos; Fox, LeAnne M; da Silva, Alexandre J

    2016-01-01

    Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis. Timely diagnosis of these infections is difficult, partly because reliable laboratory diagnostic methods are unavailable. The aim of this study was to evaluate the usefulness of a real-time polymerase chain reaction (PCR) assay for the detection of A. cantonensis DNA in human cerebrospinal fluid (CSF) specimens. A total of 49 CSF specimens from 33 patients with eosinophilic meningitis were included: A. cantonensis DNA was detected in 32 CSF specimens, from 22 patients. Four patients had intermittently positive and negative real-time PCR results on subsequent samples, indicating that the level of A. cantonensis DNA present in CSF may fluctuate during the course of the illness. Immunodiagnosis and/or supplemental PCR testing supported the real-time PCR findings for 30 patients. On the basis of these observations, this real-time PCR assay can be useful to detect A. cantonensis in the CSF from patients with eosinophilic meningitis. © The American Society of Tropical Medicine and Hygiene.

  7. Linear Methods for Analysis and Quality Control of Relative Expression Ratios from Quantitative Real-Time Polymerase Chain Reaction Experiments

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    Robert B. Page

    2011-01-01

    Full Text Available Relative expression quantitative real-time polymerase chain reaction (RT-qPCR experiments are a common means of estimating transcript abundances across biological groups and experimental treatments. One of the most frequently used expression measures that results from such experiments is the relative expression ratio (RE, which describes expression in experimental samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to one or more experimental or nonbaseline condition in terms of fold change relative to calibrator samples (i.e., RNA isolated from organisms, tissues, and/or cells that were exposed to a control or baseline condition. Over the past decade, several models of RE have been proposed, and it is now clear that endogenous reference gene stability and amplification efficiency must be assessed in order to ensure that estimates of RE are valid. In this review, we summarize key issues associated with estimating RE from cycle threshold data. In addition, we describe several methods based on linear modeling that enable researchers to estimate model parameters and conduct quality control procedures that assess whether model assumptions have been violated.

  8. Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents.

    Science.gov (United States)

    Añez-Lingerfelt, Mariaclara; Fox, George E; Willson, Richard C

    2009-01-01

    An important problem in measurement of messenger RNA (mRNA) levels by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, and/or are hazardous. We present a rapid, nuclease-free, and cost-effective method of eliminating contaminating DNA in RNA samples using selective precipitation by compaction agents. Compaction agents are cationic molecules that bind to double-stranded nucleic acids, driven by electrostatic interactions and steric complementarity. The effectiveness and DNA selectivity of six compaction agents were investigated: trivalent spermidine, Triquat A, and Triquat 7; tetravalent spermine and Quatro-quat; and hexavalent Quatro-diquat. Effectiveness was measured initially by supernatant UV absorbance after precipitation of salmon sperm DNA. Effectiveness and selectivity were then investigated using differences in RT-PCR C(t) values with synthetic mixtures of human genomic DNA and total RNA and with total RNA isolated from cells. With 500 microM spermidine or Triquat A, the supernatant DNA could not be detected up to 40 cycles of PCR (C(t)12.6), whereas the C(t) for the mRNA was increased by only five cycles. Therefore, spermidine and Triquat A each show strong DNA selectivity and could be used to eliminate contaminating DNA in measurements of mRNA.

  9. [The rapid specific characterization of clinical isolates of the genus Mycobacterium by the polymerase chain reaction and restriction enzyme analysis].

    Science.gov (United States)

    Cuende, J I; Jaime, M L; Gómez, M T; Del Campo, F; Alba, A; Pérez de Diego, I J

    1995-02-18

    Typing at species level of Mycobacterium is usually performed by microbiological and biochemical methods that require a long time and/or sufficient amount of bacteria. Molecular biology can avoid these problems using different techniques. A colony growth of the following mycobacteria has been analyzed: M. tuberculosis, M. kansasii, M. avium, M. intracellulare, M. gordonae, M. phlei, M. aurum, M. fortuitum, M. flavescens, M. marinum, M. xenopi, M. nonchromogenicum, M. terrae and M. chelonei. Strains were grown in Löwenstein-Jensen medium. DNA was obtained by proteolytic digestion and fenol extraction. The 16S rRNA gen was amplified by polymerase chain reaction (PCR) and the amplification was digested by HaeIII, HpaII, RsaI and AluI restriction enzymes. Restriction fragment patterns were analyzed by agarose gel electrophoresis and UV transillumination. The combination of the patterns obtained with HpaII and RsaI was sufficient to generate 13 different combined ones. The patterns of M. intracellulare and M. avium were the same. PCR and restriction enzyme analysis is an useful method for typing at species level of clinical isolates of mycobacteria.

  10. Development of a polymerase chain reaction-based assay for the detection of Alternaria fungal contamination in food products.

    Science.gov (United States)

    Zur, G; Hallerman, E M; Sharf, R; Kashi, Y

    1999-10-01

    Alternaria sp. are important fungal contaminants of vegetable, fruit, and grain products, including Alternaria alternata, a contaminant of tomato products. To date, the Howard method, based on microscopic observation of fungal filaments, has been the standard examination for inspection of tomato products. We report development of a polymerase chain reaction (PCR)-based method for detection of Alternaria DNA. PCR primers were designed to anneal to the internal transcribed regions ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria but not to other microbial or tomato DNA. We demonstrate use of the PCR assay to detect Alternaria DNA in experimentally infested and commercially obtained tomato sauce and tomato powder. Use of the PCR method offers a rapid and sensitive assay for the presence of Alternaria DNA in tomato products. The apparent breakdown of DNA in tomato sauce may limit the utility of the assay to freshly prepared products. The assay for tomato powder is not affected by storage time.

  11. Detection of Natural Toxoplasma gondii Infection in Chicken in Thika Region of Kenya Using Nested Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    John Mokua Mose

    2016-01-01

    Full Text Available The detection of Toxoplasma gondii in free-range chickens is a good indicator of possible risk to human beings. The aim of this study was to investigate the occurrence of T. gondii in free-range chicken using polymerase chain reaction (PCR. Brain samples from 105 free-range chickens from three administrative areas in Thika region, Kenya, were collected, DNA-extracted, and analyzed using PCR to detect presence of T. gondii. The overall prevalence of T. gondii in all the three areas was 79.0% (95% CI: 70.0–86.4% and the prevalence across the three areas was not significantly different (P=0.5088; χ2=1.354. Female chickens had higher (79.4% prevalence than males (78.6%, although the difference was not significant (P=0.922, χ2 = 0.01. However, chickens that were more than 2 years old had significantly (P=0.003; χ2 = 11.87 higher prevalence compared to younger ones. The study indicates that there was a high occurrence of T. gondii infection in free-range chickens from Thika region and that the infection rate is age dependent. Further studies should be carried out to determine the possible role of roaming chickens in the epidemiology of the disease among humans in the area.

  12. Detection of human papillomavirus infection in penile samples through liquid-based cytology and polymerase chain reaction.

    Science.gov (United States)

    de Lima Rocha, Maria Gabrielle; Faria, Fábio Lopes; Souza, Maria do Carmo M; Vago, Annamaria Ravara; Fernandes, Ana Paula; Fernandes, Paula Avila

    2008-12-25

    The human papillomavirus (HPV) is strongly related to cervical cancer and its precursor lesions. However, unlike in the case of women, there are limited data regarding HPV infection in men. Analysis of male HPV infection is frequently hindered by the lack of consistency in collection methods, sample adequacy, and low sensitivity of cytologic analysis. The objective of the current study was to compare the results of liquid-based cytology and HPV DNA testing through polymerase chain reaction in 99 penile samples collected from men presenting with condyloma acuminate or male partners of HPV-infected women who had attended a public health service in the city of Belo Horizonte, Minas Gerais, Brazil. Classic and nonclassic cytomorphologic signs were adopted to evaluate the presence of HPV infections in penile smears. HPV DNA was detected in 93 (93.9%) of the 99 samples analyzed. Koilocytosis was detected in 1 smear and nonclassic signs were detected in 23 smears, 22 of which were found to be positive for HPV DNA. The cytopathologic detection of HPV infection in penile samples collected for liquid-based cytology is low, even when cytologic nonclassic signs are applied, and does not appear to improve the diagnosis of HPV infection in men. (c) 2008 American Cancer Society.

  13. Development of a novel polymerase chain reaction-enzyme-linked immunosorbent assay for the diagnosis of Trichophyton rubrum onychomycosis.

    Science.gov (United States)

    Pankewitz, F; Nenoff, P; Uhrlaß, S; Bezold, G; Winter, I; Gräser, Y

    2013-06-01

    The prevalence of onychomycosis has increased steadily in the past decade. An accurate diagnosis at the outset is important for successful and cost-effective treatment of patients. However, current diagnostic tests for onychomycosis are not rapid, sensitive or specific. To develop a microsatellite-based polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (MS-ELISA) for the detection of Trichophyton rubrum, which is the most common aetiological agent of onychomycosis. An archival set of 434 nail and skin specimens from 217 patients was included as the test sample in this study. We also compared MS-ELISA with an earlier published topoisomerase PCR-ELISA (TI-ELISA) using template DNA extracted by another method. The MS-ELISA detected the highest number of positive samples (69%) followed by direct microscopy (56%), TI-ELISA (44%) and fungal culture (30%). When an identical DNA extraction method was applied to 120 specimens, the MS-ELISA proved to be twice as sensitive as the TI-ELISA. We have optimized a target gene and DNA extraction method for rapid detection of T. rubrum onychomycosis. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

  14. Molecular typing among beef isolates of Escherichia coli using consensus repetitive intergenic enterobacteria-polymerase chain reaction (ERIC-PCR)

    Science.gov (United States)

    Zoolkifli, Nurliyana Wan; Mutalib, Sahilah Abd

    2013-11-01

    Genomic DNA of Escherichia coli were characterized by enterobacterial repetitive intergenic consensus-Polymerase chain reaction (ERIC-PCR) and the presence of Shiga toxin gene-I (Stx1) and Shiga toxin gene-2 (Stx2). These isolates were originated from imported raw beef which are come from two countries namely Australia and India. The isolation of E. coli was conducted by using Eosin Methylene Blue Agar (EMBA). A total of 94 strains had been isolated from 30 samples of imported raw beefand 42 strains had been detected positively E. coli by doing biochemical tests. All strains had been tested and the results of biochemical tests showed that 3 strains were from Australia samples while the other 39 strains were from India samples. The biochemical tests used are Indole test, Methyl Red test, Voges-Proskauer test and Citrate test. All the 42 strains were examined for Shiga toxin (stx1 and stx2) gene detection by two pair primers which are stx2F (5'-TTCTTCGGTATCCTATTCCC-3'), stx2R (5'-ATGCATCTCTGGTCATTGTA-3'), stx1F (5'-CAGTTAATGTGGTGGCGAAG-3'), and stx1R (5'-CTGTCACAGTAACAACCGT-3'). The results showed that none of the strains are positive for Shiga toxin gene. Application of ERIC-PCR method towards E. coli had successfully shown the high diversity polymorphism in 21 different genome types of DNA with primers ERIC1R (5'- CACTTAGGGGTCCTCGAATGTA- 3') and ERIC2R (5'- AAGTAAGTGACTGGGGTGACGC- 3').

  15. Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection

    Directory of Open Access Journals (Sweden)

    Ronaldo Bragança Martins Júnior

    2014-09-01

    Full Text Available Viruses are the major contributors to the morbidity and mortality of upper and lower acute respiratory infections (ARIs for all age groups. The aim of this study was to determine the frequencies for a large range of respiratory viruses using a sensitive molecular detection technique in specimens from outpatients of all ages with ARIs. Nasopharyngeal aspirates were obtained from 162 individuals between August 2007-August 2009. Twenty-three pathogenic respiratory agents, 18 respiratory viruses and five bacteria were investigated using multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR and indirect immunofluorescence assay (IIF. Through IIF, 33 (20.4% specimens with respiratory virus were recognised, with influenza virus representing over half of the positive samples. Through a multiplex real-time RT-PCR assay, 88 (54.3% positive samples were detected; the most prevalent respiratory viral pathogens were influenza, human rhinovirus and respiratory syncytial virus (RSV. Six cases of viral co-detection were observed, mainly involving RSV. The use of multiplex real-time RT-PCR increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ARI cases, including the most recently described respiratory viruses [human bocavirus, human metapneumovirus, influenza A (H1N1 pdm09 virus, human coronavirus (HCoV NL63 and HCoV HKU1].

  16. Impact of polymerase chain reaction results on patient management during a viral meningitis outbreak in Tropical North Queensland.

    Science.gov (United States)

    Stonehouse, Vicki; Furyk, Jeremy; Norton, Robert

    2012-02-01

    Enterovirus is the most commonly isolated pathogen in viral meningitis. We report on the first outbreak of viral meningitis in Tropical Queensland and the effect of polymerase chain reaction (PCR) results on antibiotic use and hospital length of stay. Retrospective case series of consecutive patients presenting to the Townsville ED with viral meningitis were evaluated by examining hospital medical records. The study period was November 2008 to February 2009. Forty-three patients were available for full analysis of which 17 (40%) were female and 17 (40%) had a positive enteroviral PCR. Antibiotics were commenced on 37 (86%) of patients. There was no difference in hospital length of stay in patients with a negative versus positive PCR (2.52 vs 2.72 days, P = 0.68) or duration of antibiotic therapy (2.20 vs 1.94 days, P = 0.61). In our study a positive result on PCR was not associated with a shorter hospital length of stay or a shorter duration of antibiotic therapy. This contrasts with previous reports on this topic and requires further evaluation. © 2011 The Authors. EMA © 2011 Australasian College for Emergency Medicine and Australasian Society for Emergency Medicine.

  17. Polymerase chain reaction detection of Leishmania kDNA from the urine of Peruvian patients with cutaneous and mucocutaneous leishmaniasis.

    Science.gov (United States)

    Veland, Nicolas; Espinosa, Diego; Valencia, Braulio Mark; Ramos, Ana Pilar; Calderon, Flor; Arevalo, Jorge; Low, Donald E; Llanos-Cuentas, Alejandro; Boggild, Andrea K

    2011-04-01

    We hypothesized that Leishmania kDNA may be present in urine of patients with cutaneous leishmaniasis (CL). Urine samples and standard diagnostic specimens were collected from patients with skin lesions. kDNA polymerase chain reaction (PCR) was performed on samples from patients and 10 healthy volunteers from non-endemic areas. Eighty-six of 108 patients were diagnosed with CL and 18 (21%) had detectable Leishmania Viannia kDNA in the urine. Sensitivity and specificity were 20.9% (95% confidence interval [CI] 12.3-29.5%) and 100%. Six of 8 patients with mucocutaneous involvement had detectable kDNA in urine versus 12 of 78 patients with isolated cutaneous disease (P < 0.001). L. (V.) braziliensis (N = 3), L. (V.) guyanensis (N = 6), and L. (V.) peruviana (N = 3) were identified from urine. No healthy volunteer or patient with an alternate diagnosis had detectable kDNA in urine. Sensitivity of urine PCR is sub-optimal for diagnosis. On the basis of these preliminary data in a small number of patients, detectable kDNA in urine may identify less localized forms of infection and inform treatment decisions.

  18. Detection of Natural Toxoplasma gondii Infection in Chicken in Thika Region of Kenya Using Nested Polymerase Chain Reaction

    Science.gov (United States)

    Karanja, Simon Muturi; Ngotho, Maina; Kamau, David Muchina; Njuguna, Adele Nyambura

    2016-01-01

    The detection of Toxoplasma gondii in free-range chickens is a good indicator of possible risk to human beings. The aim of this study was to investigate the occurrence of T. gondii in free-range chicken using polymerase chain reaction (PCR). Brain samples from 105 free-range chickens from three administrative areas in Thika region, Kenya, were collected, DNA-extracted, and analyzed using PCR to detect presence of T. gondii. The overall prevalence of T. gondii in all the three areas was 79.0% (95% CI: 70.0–86.4%) and the prevalence across the three areas was not significantly different (P = 0.5088; χ 2 = 1.354). Female chickens had higher (79.4%) prevalence than males (78.6%), although the difference was not significant (P = 0.922, χ 2 = 0.01). However, chickens that were more than 2 years old had significantly (P = 0.003; χ 2 = 11.87) higher prevalence compared to younger ones. The study indicates that there was a high occurrence of T. gondii infection in free-range chickens from Thika region and that the infection rate is age dependent. Further studies should be carried out to determine the possible role of roaming chickens in the epidemiology of the disease among humans in the area. PMID:27981052

  19. Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces.

    Science.gov (United States)

    Szabo, E A; Pemberton, J M; Gibson, A M; Eyles, M J; Desmarchelier, P M

    1994-06-01

    The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.

  20. A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

    Science.gov (United States)

    Scherrer, Simone; Frei, Daniel; Wittenbrink, Max Michael

    2016-12-01

    Progressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs. In comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR. The approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.

  1. Detection of Pasteurella multocida isolates from local pigs of India by polymerase chain reaction and their antibiogram.

    Science.gov (United States)

    Rajkhowa, Swaraj; Shakuntala, Ingudam; Pegu, Seema Rani; Das, Rajib Kumar; Das, Anubrata

    2012-10-01

    Pasteurella multocida has been recognized as an important veterinary pathogen for over a century. Conventional methods for diagnosis of pasteurellosis rely on the detection of the organism by microscopy and its isolation and identification. However, as far as pasteurellosis is concerned, it is not just sufficient to know the identity of the organism. To constitute effective control measures, it is important to know the serotype of the organism. A study was undertaken to characterize the Pasteurella isolates from local pigs in India with clinical respiratory disease by determination of their capsule types and presence or absence of toxin gene. Pasteurella could be isolated from 66.70% of pigs with clinical respiratory disease. All the isolates were confirmed through biochemical characterization and P. multocida-specific polymerase chain reaction. It has also been observed that all the isolates belonged to capsular type D. All the isolates were sensitive to chloramphenicol, chlortetracycline, doxycycline, and enrofloxacin, while the rest of the antibiotics were less effective. It has also been observed that all isolates were resistant to cephalexin, penicillin G, and sulphadiazine. The study revealed the detection of P. multocida serotype D from clinical respiratory diseases of local pigs of India, which could be one of the important respiratory tract pathogens responsible for mortality of local pigs in India.

  2. Characterization of the ptfA gene of avian Pasteurella multocida strains by allele-specific polymerase chain reaction.

    Science.gov (United States)

    Sellyei, Boglárka; Bányai, Krisztián; Magyar, Tibor

    2010-07-01

    Pasteurella multocida is the causative agent of fowl cholera in domesticated and wild birds. The disease outcome is affected by various host- and pathogen-specific determinants. Several putative virulence factors have been proposed to play a key role in this interaction, including the ptfA gene, the products of which assemble to form type 4 fimbriae on the bacterial surface. One way to understand more precisely how ptfA contributes to pathogenesis is to gather molecular features of this gene in circulating avian P. multocida strains. Therefore, molecular characterization of the ptfA gene of P. multocida strains isolated from domestic poultry was performed using the combination of nucleotide sequence analysis and a newly developed allele-specific polymerase chain reaction assay. Two major ptfA alleles were identified among 31 strains, representing various serogroups and somatic serotypes. It was noteworthy that allele specificity and case severity of a subset of strains correlated with the available gross pathology data. Therefore, the acquisition of comprehensive clinical and epidemiological data together with molecular characteristics of individual strains will help to design and implement adequate preventive and intervention strategies.

  3. Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

    2013-06-01

    A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

    2009-08-01

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  5. Capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells.

    Science.gov (United States)

    He, Y; Zhang, Y H; Yeung, E S

    2001-07-27

    A miniaturized, integrated and automated system based on capillary fluidics has been developed for nanoliter DNA analysis directly from cheek cells. All steps for DNA analysis, including injecting aqueous reagents and DNA samples, mixing the solutions together, thermal cell lysis, polymerase chain reaction (PCR), transfer and injection of PCR product, separation, sizing and detection of those products are performed in a capillary-based integrated system. A small amount of cheek cells collected by a plastic toothpick is directly dissolved in the PCR cocktail in a plastic vial or mixed on-line with a small volume of PCR cocktail (125 nl) in the capillary. After thermal cell lysis and PCR in a microthermal cycler, the DNA fragments are mixed with DNA size standards and transferred to a micro-cross for injection and separation by capillary gel electrophoresis. Programmable syringe pumps, switching valves, multiposition and freeze-thaw valves are used for microfluidic control in the entire system. This work establishes the feasibility of performing all the steps of DNA analysis from real samples in a capillary-based nanoliter integrated system.

  6. A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

    Science.gov (United States)

    Soejima, Takashi; Xiao, Jin-Zhong; Abe, Fumiaki

    2016-06-23

    Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

  7. Polymerase chain reaction (PCR) identification of rodent blood meals confirms host sharing by flea vectors of plague.

    Science.gov (United States)

    Franklin, Heather A; Stapp, Paul; Cohen, Amybeth

    2010-12-01

    Elucidating feeding relationships between hosts and parasites remains a significant challenge in studies of the ecology of infectious diseases, especially those involving small or cryptic vectors. Black-tailed prairie dogs (Cynomys ludovicianus) are a species of conservation importance in the North American Great Plains whose populations are extirpated by plague, a flea-vectored, bacterial disease. Using polymerase chain reaction (PCR) assays, we determined that fleas (Oropsylla hirsuta) associated with prairie dogs feed upon northern grasshopper mice (Onychomys leucogaster), a rodent that has been implicated in the transmission and maintenance of plague in prairie-dog colonies. Our results definitively show that grasshopper mice not only share fleas with prairie dogs during plague epizootics, but also provide them with blood meals, offering a mechanism by which the pathogen, Yersinia pestis, may be transmitted between host species and maintained between epizootics. The lack of identifiable host DNA in a significant fraction of engorged Oropsylla hirsuta collected from animals (47%) and prairie-dog burrows (100%) suggests a rapid rate of digestion and feeding that may facilitate disease transmission during epizootics but also complicate efforts to detect feeding on alternative hosts. Combined with other analytical approaches, e.g., stable isotope analysis, molecular genetic techniques can provide novel insights into host-parasite feeding relationships and improve our understanding of the role of alternative hosts in the transmission and maintenance of disease. © 2010 The Society for Vector Ecology.

  8. Comparative efficacy of conventional diagnostic methods and evaluation of polymerase chain reaction for the diagnosis of bovine brucellosis

    Directory of Open Access Journals (Sweden)

    Raheela Akhtar

    2010-04-01

    Full Text Available The comparative efficacy of Rose Bengal Plate Test (RBPT and Milk Ring test (MRT was calculated in terms of sensitivity and specificity for the diagnosis of bovine brucellosis in cows (Group A and buffaloes (Group B from Lahore and Okara districts of Punjab, Pakistan. Using bacterial growth as a gold standard RBPT showed high sensitivity values of 100% in both groups. While its specificity was 96.29% (Group A and 90.62% (Group B. On the other hands MRT showed low sensitivity (80.0% in Group A; 86.6% in Group B while its specificity was 100% in all the animals of both groups. The calculated positive predictive and negative predictive values of both groups were in correspondence with their specificity and sensitivity values respectively. High sensitivity and low specificity of RBPT as compare to high specificity and low sensitivity of MRT in all groups suggested the poor efficacy of both tests used individually as compare to bacterial growth. In the continuation of this study polymerase chain reaction (PCR was evaluated for its diagnostic efficacy of quick Brucella abortus isolation from same samples. PCR conducted on serum samples gave more positive results than on milk samples. Therefore, the combination of both conventional tests alongwith serum PCR can be recommended. [Vet. World 2010; 3(2.000: 53-56

  9. Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

    Directory of Open Access Journals (Sweden)

    Angel Valdivia

    1997-05-01

    Full Text Available The aim of this study was to develop a polymerase chain reaction (PCR for the detection of respiratory syncytial virus (RSV genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

  10. Analysis of respiratory syncytial virus in clinical samples by reverse transcriptase-polymerase chain reaction restriction mapping.

    Science.gov (United States)

    Valdivia, A; Savón, C; Chacón, D; Sarmiento, L; Morier, L; Otero, A; Soto, Y; Oropesa, S; Goyenechea, A

    1997-01-01

    The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.

  11. Polymerase chain reaction species diagnostic assay for Anopheles quadrimaculatus cryptic species (Diptera: Culicidae) based on ribosomal DNA ITS2 sequences.

    Science.gov (United States)

    Cornel, A J; Porter, C H; Collins, F H

    1996-01-01

    Species-specific differences in the nucleotide sequences of the 2nd internal transcribed spacer (ITS2) of nuclear ribosomal DNA (rDNA) were used to develop a diagnostic assay based on the polymerase chain reaction (PCR) that can distinguish 4 of the 5 cryptic sibling species in the common malaria mosquito, Anopheles quadrimaculatus Say, complex. The assay requires only a small amount of tissue from an individual mosquito and a mixture of 5 PCR primers. The plus strand universal primer is derived from a sequence in the 5.8S coding region that is identical in all members of the complex. The 4 minus strand primers were selected from species-unique sequences within the ITS2 region. PCR amplification produces a different sized fragment for each of the 4 species which can be visualized readily under ultraviolet light after electrophoresis through an ethidium bromide-containing agarose gel. The assay has been developed and tested only with An. quadrimaculatus complex specimens from Florida populations.

  12. Polymerase chain reaction-based identification and genotyping of Anopheles mosquitoes with a 96-pin bacterial replicator.

    Science.gov (United States)

    Rafferty, Cristina S; Campbell, Scott R; Wirtz, Robert A; Benedict, Mark Q

    2002-03-01

    A simple method for rapid identification of large numbers of Anopheles mosquitoes was developed based on polymerase chain reaction (PCR) amplification of the rDNA intergenic spacer and internal transcribed spacer 2. By means of previously described primers for the Anopheles gambiae and An. quadrimaculatus species complexes, rDNA was amplified simultaneously from 96 whole mosquitoes or parts. No homogenization or individual DNA preparation was necessary, and transfer of 96 samples to PCR reactions was performed simultaneously with a bacterial replicator. Control reactions indicate that the level of cross-contamination is negligible, and false-negative findings are rare. The method was tested on larvae, pupae, adult heads, whole adult males and females, and single tarsi. All parts except tarsi provided satisfactory template. Fresh, ethanol-preserved, dried, and frozen adults were also tested with similar results. The method was also tested for amplification of a single-copy gene, white. Results were generally positive, although some false-negative findings were observed. This method allows rapid analysis of large numbers of mosquitoes without robotic equipment and should enable rapid and extensive PCR analysis of field-collected samples and laboratory specimens.

  13. [Quantitative detection of Streptococcus mutans in different people with dental caries by real-time polymerase chain reaction].

    Science.gov (United States)

    Zhao, Dong; Wang, Zhan-yong; Wang, Jian-qiu; Xiao, Bai; Zhou, Yan

    2010-04-01

    To establish a quantity detection method of Streptococcus mutans (Sm) and bacteria and compare the relationship between the number of these bacteria and the prevalence of dental caries in different people. With specific primers for a unique sequence in a 14 kb HaeIII restriction fragment consistently presenting during detecting Sm by chromosomal DNA fingerprints, the total number of Sm and bacteria of 99 saliva samples were detected by real-time polymerase chain reaction (PCR) and statistically analyzed. The primers were specific for Sm and the minimum detectable level by real-time PCR was 0.1 microg/L. The total number of bacteria in the dental caries and people without caries was 51.4 x 10(8) cell copies/L and 221.6 x 10(8) cell copies/L respectively, in which the ratio of Sm to bacteria was 0.0193 and 0.0059 respectively. The differences were significantly different between the people with dental caries and those without caries in the total number of bacteria and the ratio of Sm to bacteria. The primers can be used to detect the Sm by real-time PCR. The ratio of Sm to bacteria was closely associated with the prevalence of dental caries.

  14. [Differentiation of Entamoeba histolytica from Entamoeba dispar using Gal/GalNAc-lectin and polymerase chain reaction].

    Science.gov (United States)

    López, Omaira Y; López, Myriam C; Corredor, Vladimir; Echeverri, M Clara; Pinilla, Análida E

    2012-04-01

    Entamoeba histolytica and Entamoeba dispar are morphologically identical. However, the former is highly pathogenic and the latter is not. To differentiate Entamoeba histolytica from Entamoeba dispar through ELISA and PCR techniques in Colombian isolates from feces. Descriptive study of Colombian fecal samples from 53 males and 47 women, that were positive for the complex E. histolytica/E. dispar on light microscopy. Positive samples were cultured on Robinson medium to isolate trophozoites. The presence of specific Gal/ GalNAc-lectin was determined by ELISA and polymerase chain reaction in genomic DNA, using the combination of three nucleotides that recognize a variable region of 16S small subunit ribosomal RNA, generating a 166 base pair (bp) product for E. histolytica and 752 pb product for E. dispar. After verification, only eight of the 100 samples were positive for the complex E. histolytica/E. dispar and were cultivated. Isolates were obtained in six cultures, one corresponded to E. histolytica and six to E. dispar. The presence of E. histolytica/E. dispar complex was largely overestimated with light microscopy. In the few samples where isolates were obtained, the technique described differentiated between both strains.

  15. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase.

    Science.gov (United States)

    Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi; Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki; Noda, Mamoru; Igimi, Shizunobu; Ikebukuro, Kazunori

    2013-11-01

    An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268-luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF-luciferase fusion protein. By means of the automatic analyzer with ZF-luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×10(6) copies. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. A Comparative Analysis of Polymerase Chain Reaction and Direct Fluorescent Antibody Test for Diagnosis of Genital Herpes.

    Science.gov (United States)

    Patwardhan, Vrushali; Bhalla, Preena; Rawat, Deepti; Garg, Vijay Kumar; Sardana, Kabir; Sethi, Sumit

    2017-01-01

    To compare laboratory tests that can simultaneously detect and type herpes simplex virus (HSV) directly from the genital ulcer specimens in clinically suspected cases of genital herpes. A study was conducted over 10 months and 44 adult male and female patients clinically suspected with genital herpes were recruited. Genital ulcer swab specimens were subjected to glycoprotein-G gene-based conventional polymerase chain reaction (PCR) and commercially available direct fluorescent antibody (DFA) test and the results were compared. PCR for HSV was positive in 82% (36/44) cases. DFA was positive in 68.2% (30/44) cases. There was 100% agreement between HSV types detected by DFA and PCR. The strength of agreement between the results was better in primary genital herpes than recurrent cases. PCR was found to be better in the detection of HSV in recurrent genital herpes patients. It is a better modality, especially when genital herpes clinically presents with ulcerative or crusted lesions, and is also a cheaper alternative as compared to DFA.

  17. Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia

    Directory of Open Access Journals (Sweden)

    Jelena V Jovanovic

    2011-10-01

    Full Text Available Molecular detection of minimal residual disease (MRD has become established to assess remission status and guide therapy in patients with PML-RARA+ acute promyelocytic leukemia (APL. However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF-RARA and STAT5b-RARA. Despite their relative rarity (<1% of APL we identified 6 cases (PLZF-RARA, n=5; STAT5b-RARA, n=1, established the respective breakpoint junction regions and designed real-time quantitative polymerase chain reaction (RQ-PCR assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17-associated APL, affording assay sensitivities of ~1 in 104-105. Serial samples were available from 2 PLZF-RARA APL patients. One showed persistent PCR positivity, predicting subsequent relapse, and remains in CR2, ~11 years post-autograft. The other, achieved molecular remission (CRm with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b-RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RQ-PCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly-defined subsets of acute leukemia.

  18. A quantitative real time polymerase chain reaction approach for estimating processed animal proteins in feed: preliminary data

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    Maria Cesarina Abete

    2013-04-01

    Full Text Available Lifting of the ban on the use of processed animal proteins (PAPs from non-ruminants in non-ruminant feed is in the wind, avoiding intraspecies recycling. Discrimination of species will be performed through polymerase chain reaction (PCR, which is at a moment a merely qualitative method. Nevertheless, quantification of PAPs in feed is needed. The aim of this study was to approach the quantitative determination of PAPs in feed through Real Time (RT-PCR technique; three different protocols picked up from the literature were tested. Three different kind of matrices were examined: pure animal meals (bovine, chicken and pork; one feed sample certified by the European reference laboratory on animal proteins (EURL AP in feed spiked with 0.1% bovine meal; and genomic DNAs from bovine, chicken and pork muscles. The limit of detection (LOD of the three protocols was set up. All the results obtained from the three protocols considered failed in the quantification process, most likely due to the uncertain copy numbers of the analytical targets chosen. This preliminary study will allow us to address further investigations, with the purpose of developing a RT-PCR quantitative method.

  19. Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection.

    Science.gov (United States)

    Nishimori, Asami; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Nakahara, Ayako; Murata, Shiro; Ohashi, Kazuhiko

    2016-06-01

    Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.

  20. Use of an improved quantitative polymerase chain reaction assay to determine differences in human rhinovirus viral loads in different populations.

    Science.gov (United States)

    Granados, Andrea; Luinstra, Kathy; Chong, Sylvia; Goodall, Emma; Banh, Lisa; Mubareka, Samira; Smieja, Marek; Mahony, James

    2012-12-01

    Human rhinoviruses (HRV) frequently cause acute respiratory infections and chronic respiratory disease exacerbations. However, testing is not generally offered. We developed a modified HRV quantitative polymerase chain reaction (qPCR) assay to assess viral loads in the community and hospital patients. The assay had a lower limit of detection of 2 log(10) viral copies/mL and displayed linearity over 5 log(10) viral copies, with a lower limit of quantitation of 4 log(10) viral copies/mL. Mean viral loads (95% confidence interval) for hospitalized children, university students, and institutionalized elderly, were 7.08 log(10) viral copies/mL (6.7-7.5), 6.87 log(10) viral copies/mL (6.5-7.2), and 7.09 log(10) viral copies/mL (6.9-7.3), respectively (P = 0.67). Serial specimens of 14 university students showed a decrease of mean viral loads from 6.36 log(10) viral copies/mL on day 1 to 2.32 log(10) viral copies/mL 7 days past symptom onset (P < 0.001). Using an HRV qPCR, we showed that viral loads did not differ between the community and hospitalized populations and significantly decreased following symptoms onset in healthy individuals. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction, and ergosterol assay

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    Yiing Yng Chow

    2017-01-01

    Full Text Available This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb introduced into oil palm ramets (host model. The endophytes selected were Diaporthe phaseolorum (WAA02, Trichoderma asperellum (T2, and Penicillium citrinum (BTF08. Ramets were first inoculated with 100 mL of fungal cells (106 cfu mL−1 via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR detected and identified the isolates from the plant tissues. The ergosterol assay (via high-performance liquid chromatography, HPLC confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49 days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

  2. Detection of Avibacterium paragallinarum by Polymerase chain reaction from outbreaks of Infectious coryza of poultry in Andhra Pradesh

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    T. M. Nabeel Muhammad

    2015-01-01

    Full Text Available Aim: This study was carried out for the detection of Avibacterium paragallinarum from outbreaks of infectious coryza of poultry Materials and Methods: The polymerase chain reaction (PCR was standardized for the diagnosis of infectious coryza by using infectious coryza Killed vaccine, ventri biologicals, Pune as source of DNA of A. paragallinarum. Five outbreaks of infectious coryza from Andhra Pradesh were investigated in the present study. A total of 56 infra orbital sinus swabs and 22 nasal swabs were tested by PCR. Results: PCR analysis showed 56 positives (71.7% for infectious coryza out of total 78 samples tested. Of 56 infra orbital sinus swabs tested, 47 were positive (83.9% and 9 nasal swabs (40.9% out of 22 tested had given positive results for infectious coryza. Samples collected from birds at acute stage of disease and samples collected before treatment with antibiotics were given better results on PCR. Conclusion: For preventing the economic losses associated with the disease, an early, accurate and rapid diagnosis is essential. PCR is a rapid and highly sensitive diagnostic technique which can substitute conventional cultural examination.

  3. Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats.

    Science.gov (United States)

    Aktaş, M; Altay, K; Dumanli, N

    2005-11-05

    In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.

  4. Pathology and polymerase chain reaction detection of ovine progressive pneumonia (maedi cases in slaughtered sheep in India

    Directory of Open Access Journals (Sweden)

    Rahul Singh

    2017-11-01

    Full Text Available Aim: The small ruminant lentiviruses are known to cause maedi-visna (MV and caprine arthritis - encephalitis in sheep and goats, typically affecting joints, udder, lungs, and the central nervous system. The diagnosis usually involves serology, clinical signs, immunohistochemistry, and polymerase chain reaction (PCR. In the present study, the histopathologically positive pneumonia cases of MV were confirmed by PCR in lung tissue probably for the first time in India. Materials and Methods: A total of 888 lungs of adult sheep, aged between 2 and 5 years, were screened during slaughter, of which 121 were found to have pneumonic lesions. The tissues from each pneumonic lung including associated lymph nodes were collected in 10% neutral buffered formalin for histopathology. The frozen tissues of the same were also collected and stored at -20°C for PCR confirmation. Results: Three of 121 cases of pneumonic lungs of sheep revealed gross and histopathological lesions suggestive of maedi or ovine progressive pneumonia infection. These 3 cases were further confirmed by PCR technique that amplified 291-base pair DNA in the long terminal repeat sequence of MV provirus. Conclusion: This study suggests the low occurrence of MV virus (MVV infection in India in naturally affected sheep based on pathomorphological lesions and using the molecular tool of PCR detection of the virus in tissues. Further, a combination of pathomorphology or/and PCR testing might be optimal for detecting the animals infected with MVV.

  5. Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification.

    Science.gov (United States)

    Paton, A W; Paton, J C; Lawrence, A J; Goldwater, P N; Harris, R J

    1992-01-01

    A rapid method for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates, involving a combination of reverse transcription and polymerase chain reaction amplification (RT-PCR), has been developed. The RT-PCR assay employs oligonucleotide primers specific for the region of the RSV genome which encodes the F1 subunit of the fusion (F) glycoprotein. Other respiratory viruses do not give a positive reaction. The RT-PCR assay was tested on 202 nasopharyngeal aspirates collected from children with clinical signs of respiratory infection, and the results from RT-PCR were compared with those obtained from virus culture and direct detection by enzyme immunoassay (EIA). RT-PCR results were positive in 118 of 125 samples from which RSV was cultured, as well as in 4 of 7 samples which were culture negative but EIA positive. RT-PCR results were negative in 68 of 70 culture-negative, EIA-negative samples, which included 11 samples from which other respiratory viruses were isolated. The speed, sensitivity (94.6%), and specificity (greater than 97%) of the RT-PCR assay suggest that this technique could be useful for rapid detection of RSV in clinical samples. Images PMID:1374080

  6. Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka.

    Science.gov (United States)

    Denipitiya, D T H; Chandrasekharan, N V; Abeyewickreme, W; Hartskeerl, C M; Hartskeerl, R A; Jiffrey, A M; Hapugoda, M D

    2016-11-01

    Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  7. [Detection of microsatellite instability in colorectal cancer by fluorescence multiplex polymerase chain reaction and its clinical value].

    Science.gov (United States)

    Jin, Hei-ying; Lai, Ren-sheng; Ding, Yi-jiang; Xie, Ling; Yang, Bo-lin; Liu, Fei; Ding, Shu-qing; Ge, Yong-sheng

    2007-05-01

    To detect microsatellite instability(MSI) in colorectal cancer by fluorescence multiplex polymerase chain reaction(FM-PCR) and explore its clinical value. MSI of 110 colorectal cancer patients undergone surgical resection in our department from 2004 to 2005 were examined by FM-PCR, and the pathological characteristics were compared between MSI and microsatellite stable (MSS) colorectal cancer patients. Among 110 cases, the male were 66 and the female were 44. Mean age was 60.8 (26-94) yrs. All 5 microsatellite markers were amplified. Out of them, 10 cases (8.1%) were MSI-H, 13 cases (11.8%) were MSI-L and 87 cases (79.1%) were MSS. Instability of BAT-26 was found in 9 cases (8.2%), BAT-25 was in 11 cases (10.0%), D2S123 was in 11 cases (10.0%), D5S346 was in 6 cases (8.2%) and D17S250 was in 8 cases (7.3%). Age between MSI and MSS colorectal cancer patients was significant and other pathological characteristics were not significant. FM-PCR is a clinically stable method for MSI detection in colorectal cancer patients. There are no significant differences between MSI and MSS pathological characteristics of colorectal cancer patients.

  8. Rapid and sensitive method for the detection of Aeromonas caviae and Aeromonas trota by polymerase chain reaction.

    Science.gov (United States)

    Khan, A A; Cerniglia, C E

    1997-04-01

    A 16S rDNA-based polymerase chain reaction (PCR) method was developed for the detection of Aeromonas caviae and Aeromonas trota. These two species were identified from other Aeromonas spp. and closely related species by primers set (AER1 and AER2). The amplified product was 316 bp. The identity of the amplified product was confirmed by DNA-DNA hybridization. Two sets of primers (AER8 and AER9) were used for specific identification of Aer. caviae. Amplifying the 260 bp fragment of 16S rRNA gene region and digesting it with AluI restriction enzyme, yielded 180- and 80-bp fragments. For PCR assay, template DNA was released by mixing equal volumes of homogenized seeded crab meat with Aer. caviae and Chelex 100 (6%) incubated for 10 min at 56 degrees C followed by addition of an equal volume of 0.1% Triton-X-100 and boiled for 10 min. The detection limit was between 50 and 100 cells g-1 of crab meat. This method is very rapid and obviates the need for DNA isolation from complex food matrices and is specific for detecting two Aeromonas species.

  9. Identification of Aeromonas trota (hybridization group 13) by amplification of the aerolysin gene using polymerase chain reaction.

    Science.gov (United States)

    Khan, A A; Nawaz, M S; Khan, S A; Cerniglia, C E

    1999-04-01

    Aeromonas trota is recognized as an important enteropathogen, and its haemolysin (aerolysin) is purported to be one of the virulence factors. Rapid detection and identification of A. trota is important for early and specific diagnosis of the infectious diseases that it causes. Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a species-specific sequence of the aerA gene, which encodes the aerolysin of A. trota. A DNA fragment of 622 bp was amplified from both lysed cells and isolated DNA from A. trota. The identity of the amplified 622 bp fragment was confirmed by digestion with BamH I restriction endonuclease, which produced the predicted 557 and 65 bp fragments. The lower limit for detection of the aerA gene by PCR amplification was 10 pg of total DNA or 10-15 cells ml-1. Primer specificity for A. trota was determined by the PCR assay with cells of 55 strains of Aeromonas sppincluding all of the 14 currently recognized DNA hybridization groups. A strain of Aeromonas enteropelogenes that had been reclassified as A. trota was also PCR positive. The method described here can be used to detect aerolysin-producing A. trota (hybridization group 13) strains from environmental and clinical samples without the use of selective media or additional biochemical tests. Copyright 1999 Academic Press.

  10. Identification of nickel response genes in abnormal early developments of sea urchin by differential display polymerase chain reaction.

    Science.gov (United States)

    Ryu, Tae Kwon; Lee, Gunsup; Rhee, Yong; Park, Heung-Sik; Chang, Man; Lee, Sukchan; Lee, Jaean; Lee, Taek-Kyun

    2012-10-01

    Bioassays and biomarkers have been previously developed to assess the effects of heavy metal contaminants on the early life stages of the sea urchin. In this study, malformation in the early developmental processes was observed in sea urchin (Strongylocentrotus intermedius) larvae exposed to 10 ppm Ni for over 30 h. The most critical stage at which the triggering of nickel effects takes place is thought to be the blastula stage, which occurs after fertilization in larval development. To investigate the molecular-level responses of sea urchin exposed to heavy metal stress and to explore the differentially expressed genes that are induced or repressed by nickel, differential display polymerase chain reaction (DD-PCR) was used with sea urchin mRNAs. The malformation-related genes expressed in the early life stages of the sea urchin were cloned from larvae exposed to 10 ppm of nickel for 15 h, and accessed via DD-PCR. Sequence analysis results revealed that each of the genes evidenced high homology with EGF2, PCSK9, serine/threonine protein kinase, apolipophorin precursor protein, and MGC80921 protein/transcript variant 2. This result may prove useful in the development of novel biomarkers for the assessment of heavy metal stresses on sea urchin embryos. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Analysis by polymerase chain reaction of myc oncogene expression in heptachlor-induced differentiation of human leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Chuang, A.J.; Chuang, L.F.; Chuang, R.Y. (Univ. of California, Davis (United States))

    1991-03-11

    Heptachlor, a chlorinated hydrocarbon insecticide, was previously demonstrated to be able to induce human leukemia ML-1 to differentiate into monocyte- or macrophage-like cells, a phenomenon similar to that induced by the known tumor promoter TPA. By hybridization studies, TPA-induced ML-1 differentiation has been shown to associate with a decline in the steady-state myc oncogene expression. In the present study, polymerase chain reaction (PCR) was combined with primer-extension reverse transcription to detect subtle changes in RNA transcripts in both TPA- and heptachlor-treated ML-1 cells. Three sets of 5{prime} primers (P1, P2 and P3) within the region bordered by the two promoter sequences were individually paired with the 3{prime} primer sequence for the synthesis of the first exon of the c-myc oncogene. The results showed that RNA transcript was not detected from the P1 primer, suggesting a possible 5{prime} truncation of myc oncogene ML-1. TPA caused an initial increase of myc gene expression followed by a rapid decline of myc transcripts, as shown by PCR using P2 and P3 5{prime}-primers. Heptachlor, under the same assay conditions, decreased the expression of the myc oncogene without an initial stimulation. Furthermore, heptachlor was shown to stimulate the in vitro protein kinase C (PKC) activity and the binding of {sup 3}(H)PDBu to ML-1, indicating a dissociation of PKC activation and myc expression in ML-1.

  12. C-myc proto-oncogene amplification detected by polymerase chain reaction in archival human ovarian carcinomas.

    Science.gov (United States)

    Schreiber, G.; Dubeau, L.

    1990-01-01

    Polymerase chain reaction (PCR) technology was used to examine the state of amplification of the proto-oncogene c-myc in archival ovarian carcinomas. Sequences from the c-myc gene and from a control gene were amplified simultaneously by PCR and the ratios of the two products measured. The results provided an accurate measurement of the relative number of copies of the two genes in each tumor genome if the control and test sequences amplified by PCR were of equal lengths. The results were not affected by the number of PCR cycles used. This technique should facilitate gene amplification studies in clinical medicine. Increased c-myc copy number was found in 17% of the 30 cases examined when a control from the same chromosome as c-myc was used, but in 37% of cases if a control from another chromosome was used. This underlines the importance of the genetic location of the selected control genes for such studies. Images Figure 2 PMID:2205100

  13. Identification ofListeriaSpp. Strains Isolated from Meat Products and Meat Production Plants by Multiplex Polymerase Chain Reaction.

    Science.gov (United States)

    Mazza, Roberta; Piras, Francesca; Ladu, Daniela; Putzolu, Miriam; Consolati, Simonetta Gianna; Mazzette, Rina

    2015-11-02

    Listeriosis is a foodborne disease caused by Listeria monocytogenes and is considered as a serious health problem, due to the severity of symptoms and the high mortality rate. Recently, other Listeria species have been associated with disease in human and animals. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) in order to simultaneously detect six Listeria species (L. grayi , L. welshimeri , L. ivanovii , L. monocytogenes , L. seeligeri , L. innocua) in a single reaction. One hundred eighteen Listeria spp . strains, isolated from meat products (sausages) and processing plants (surfaces in contact and not in contact with meat), were included in the study. All the strains were submitted to biochemical identification using the API Listeria system. A multiplex PCR was developed with the aim to identify the six species of Listeria . PCR allowed to uniquely identify strains that had expressed a doubtful profile with API Listeria The results suggest that the multiplex PCR could represent a rapid and sensitive screening test, a reliable method for the detection of all Listeria species, both in contaminated food and in clinical samples, and also a tool that could be used for epidemiological purposes in food-borne outbreaks. A further application could be the development of a PCR that can be directly applied to the pre-enrichment broth.

  14. Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction.

    Science.gov (United States)

    Tokuno, Osamu; Hayakawa, Akira; Yanai, Tomoko; Mori, Takeshi; Ohnuma, Kenichiro; Tani, Ayumi; Minami, Hironobu; Sugimoto, Takeshi

    2015-01-01

    To evaluate broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) as a rapid screening tool to detect bacterial contamination of stem-cell products. We performed the evaluation using whole blood spiked with serially diluted bacterial-type strains. Detection sensitivity was defined as the bacterial concentration for which all replicates were positive at each concentration (100% detection). We tested the sterility of 29 bags of autologous peripheral blood stem cell (PBSC) products harvested at our facility using the 16S rDNA PCR method. The detection sensitivity of 16S rDNA PCR in spiked whole blood was 10¹ to 10² colony-forming units (CFU) per mL, depending on the bacterial strain. We detected no amplified 16S rDNA among the PBSCs we used in this study. The BacT/ALERT automated bacterial culture system that we used also showed no positive signals in any of the PBSCs tested. Our data indicate that bacterial 16S rDNA PCR is a useful alternative for rapid sterility testing, not only for blood products used in transfusion medicine but also for stem-cell products used in regenerative medicine. Copyright© by the American Society for Clinical Pathology (ASCP).

  15. Evaluation of G2677T/A polymorphism of MDR1 gene by polymerase chain reaction in Mazandaran province, Iran

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    Razieh Keshavarz-Maleki

    2015-06-01

    Full Text Available The human MDR1 gene encodes for a P-glycoprotein (PGP, which acts as an efflux pump that transports a large variety of substrates from the inside of cells to the outside until protection against xenobiotics. The G2677T/A polymorphism in exon 21 is associated with PGP expression and function in humans. The present study was aimed to determine the frequencies of this polymorphism in a healthy population from Mazandaran province of Iran. A total of 120 unrelated healthy subjects from Mazandaran province, residing in Sari, coming for blood donating at Sari Blood Transfusion Center were enrolled. Genomic DNA was extracted from the peripheral blood lymphocytes of each subject. All subjects were genotyped for G2677T/A polymorphism by polymerase chain reaction-restriction fragment length polymorphism method. The genotype frequencies were G2677G (65%, G2677T (20.83%, G2677A (14.17% and TT, AA, TA genotypes were not observed. Moreover, frequency of G allele (82.5% was significantly (p ˂ 0.05 higher than the T (10.42% and A (7.08%. This is the first study to investigate the G2677T/A polymorphism of MDR1 gene in population from Mazandaran province of Iran. These data may be relevant for dose recommendation of PGP substrate drugs and can help for individualizing drug therapy of organ transplantation and important diseases such as cancer and AIDS, congestive heart failure and etc.

  16. Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi Otimização da reação de amplificação aleatória do DNA polimórfico - reação em cadeia da polimerase para tipagem molecular de Salmonella enterica sorovar Typhi

    Directory of Open Access Journals (Sweden)

    Bianca Ramalho Quintaes

    2004-03-01

    Full Text Available Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.A otimização da reação de RAPD para a caracterização de cepas de Salmonella enterica sorovar Typhi foi estudada com o objetivo de assegurar a reprodutibilidade e o poder discriminatório desta técnica. Oito cepas de Salmonella sorovar Typhi isoladas de algumas regiões do Brasil foram usadas para examinar os padrões de fragmentação produzidos quando foram empregadas concentrações diferentes do DNA molde, do iniciador, do MgCl2 e da enzima Taq DNA polimerase. Com a utilização de dois diferentes perfis de ciclos termais de baixa estringência, foram comparados os padrões de bandeamento obtidos. Um conjunto de dezesseis iniciadores foi avaliado quanto à capacidade de produzir elevado número de fragmentos distintos. Observou-se que variações associadas a todos os parâmetros testados modificaram os padrões de bandeamento. Para as amostras de Salmonella enterica sorovar Typhi utilizadas neste experimento, definiu-se um conjunto de condições para a reação de RAPD-PCR que resultou num método de tipagem simples, rápido e

  17. Entanglement spectrum and entangled modes of random XX spin chains

    Science.gov (United States)

    Pouranvari, Mohammad; Yang, Kun

    2013-08-01

    In this paper, we study the ground-state entanglement properties of finite XX spin-1/2 chains with random couplings using the Jordan-Wigner transformation. We divide the system into two parts and study the reduced density matrices (RDMs) of its subsystems. Due to the free-fermion nature of the problem, the RDMs take the form of that of a free-fermion thermal ensemble. Finding the spectrum of the corresponding entanglement Hamiltonian and corresponding eigenvectors and comparing them with the real-space renormalization-group (RSRG) treatment, we establish the validity of the RSRG approach for entanglement in the limit of strong disorder but also find its limitations when disorder is weak. In the latter case, our paper provides a way to visualize the ``effective spins'' that form long-distance singlet pairs.

  18. Electronic properties of Fibonacci and random Si-Ge chains

    Energy Technology Data Exchange (ETDEWEB)

    Vasconcelos, M S [Escola de Ciencias e Tecnologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Azevedo, David L; Hadad, A [Departamento de Fisica, Universidade Federal do Maranhao 65080-040, Sao LuIs-MA (Brazil); Galvao, D S, E-mail: mvasconcelos@ect.ufrn.br [Instituto de Fisica Gleb Wataghin, Universidade Estadual de Campinas CP 6165, 13083-970 Campinas, SP (Brazil)

    2011-10-12

    In this paper we address a theoretical calculation of the electronic spectra of an Si-Ge atomic chain that is arranged in a Fibonacci quasi-periodic sequence, by using a semi-empirical quantum method based on the Hueckel extended model. We apply the Fibonacci substitutional sequences in the atomic building blocks A(Si) and B(Ge) through the inflation rule or a recursion relation. In our ab initio calculations we use only a single point, which is sufficient for considering all the orbitals and charge distribution across the entire system. Although the calculations presented here are more complete than the models adopted in the literature which take into account the electronic interaction only up to the second and third neighbors, an interesting property remains in their electronic spectra: the fractality (which is the main signature of this kind of system). We discuss this fractality of the spectra and we compare them with the random arrangement of the Si-Ge atomic chain, and with previous results based on the tight-binding approximation of the Schroedinger equation considering up to the nearest neighbor. (paper)

  19. Pengembangan Sejumlah Primer untuk Reverse Transcriptase Polymerase Chain Reaction Guna Melacak Virus Flu Burung di Indonesia (DEVELOPMENt OF PRIMERS FOR REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION TO DETECT AVIAN INFLUENZA VIRUS IN INDONESIA

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    Ni Luh Putu Indi Dharmayanti

    2016-07-01

    Full Text Available Until recently, two clades of of avian influenza viruses (AIVs designated as 2.3.2 and 2.2.3 havebeen circulating in Indonesia. Mutations of AIV genes have cretaed many more variants of the virus. It istherefore important to evaluate the appropriate methods used for the detection and diagnosis of AI virusin the field. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR have been used as a standardmethod for detection of AIV in many laboratories in Indonesia. The success of RT-PCR for detection ofAIV virus is dependent on the nucleotide sequences of primer that match with the circulating of AIVs. Theaims of this study was to develop RT-PCR by designing primers for H5 subtype specific to the circulatingAIVs in the field. The primers were designed using Primer Design software, and optimization andvalidation of the primer were conducted using AIVs that have been characterized in the previous study.The primers were then used RT-PCR using AIV isolates from field samples and their sensitivity andspecificity were then determined. The results showed that the H5 primers designed in this study, H5-IDand H5-NLP, was able to detect the AIVs in field samples better than the H5-specific primers have beenused previously. In conclusion, H5 primers designed based on recent viruses in the field showed betterresults in the detection of AI virus as compared to the previous primers. As AIV-H5N1 subtype in the fieldwill continue to change and evolve, the use of primers designed in this study is recommended for diagnosisof H5 AIV.

  20. Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

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    Miriam Ribas Zambenedetti

    Full Text Available BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV, hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+ plasmid, generating pET47b(+-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

  1. Gene molecular study of biofilm of Staphylococcus aureus isolated from fresh milk using multiplex polymerase chain reaction

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    Atefeh Mahmoudi-Kojedi

    2017-08-01

    Full Text Available Background: Staphylococcus aureus is one of the main causes of food poisoning in the world. This pathogen has the ability to create biofilms that can lead to food contamination. The presence of biofilm genes in bacteria is very important. The aim of this study was to identify sticky genes (eno, cna, ebp, bbp that play an important role in virulence and pathogenicity of the bacteria and even prevent the penetration of antibiotics in pathogenicity time. Materials and Methods: A total of 100 samples of fresh milk were collected from live animals and 60 isolates were selected to identify sticky genes (eno, cna, ebp, bbp in the production of biofilm of S. aureus using the multiplex polymerase chain reaction method. In addition, the frequency rates of S. aureus strains resistant and susceptible to antibiotics such as methicillin, vancomycin, and clindamycin were determined among the samples. Results: From a total of 60 isolates of fresh milk, 43.4% of the colonies had laminin-binding protein gene or eno gene. Also, 90% of the isolates were sensitive to vancomycin, 50% sensitive to clindamycin and 43.4% sensitive to methicillin. Distribution rates of other sticky genes including ebp, cna, bbp were 11.6%, 20% and 25%, respectively. Molecular study results showed that the highest and lowest percentages of genes were related to the eno and bbp genes, respectively. Conclusion: The present study shows that the maximum sensitivity of the samples (90% was related to vancomycin and the least amount of sensitivity (43.3% was related to methicillin.

  2. Development of a droplet digital polymerase chain reaction for rapid and simultaneous identification of common foodborne pathogens in soft cheese

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    Paola Cremonesi

    2016-10-01

    Full Text Available Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a third-generation PCR provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4×106 to 4×101 CFU/g of pure cultures from the American Type Culture Collection.Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay without the necessity of a standard curve.In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.

  3. Development of a Droplet Digital Polymerase Chain Reaction for Rapid and Simultaneous Identification of Common Foodborne Pathogens in Soft Cheese.

    Science.gov (United States)

    Cremonesi, Paola; Cortimiglia, Claudia; Picozzi, Claudia; Minozzi, Giulietta; Malvisi, Michela; Luini, Mario; Castiglioni, Bianca

    2016-01-01

    Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a "third-generation PCR") provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4 × 106 to 4 × 101 CFU/g) of pure cultures from the American Type Culture Collection. Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay) without the necessity of a standard curve. In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.

  4. Multiplex polymerase chain reaction assays for the detection of the zearalenone chemotype of Fusarium species in white and brown rice.

    Science.gov (United States)

    Sim, Jae Ho; Tian, Fei; Jung, Soo Yeon; Auh, Joong-Hyuck; Chun, Hyang Sook

    2018-02-03

    Early detection of the zearalenone (ZEA) chemotype of Fusarium species could be a precautionary measure for preventing ZEA contamination in rice. In this study, a multiplex polymerase chain reaction (mPCR) assay for detecting ZEA-producing fungi in rice was established using a set of four primers targeting the ZEA biosynthesis genes PKS3, PKS13, ZEB1, and ZEB2. Two mPCR approaches were used: one that amplified the DNA obtained from Fusarium isolates (conventional method) and another that directly amplified the target DNA from rice samples without time-consuming DNA isolation (direct method). The two mPCR methods showed high sensitivity in detecting ZEA-producing species, with a detection limit of 1.25 pg/μL of genomic DNA and 10 2 and 10 3 spores/g of white and brown rice, respectively. Both methods were specific for ZEA-producing species and gave four band patterns. The application of the two mPCR methods to 51 Fusarium isolates and 41 rice samples revealed that 31% (16 of 51) and 24% (10 of 41) of the samples were contaminated with ZEA-producing species, respectively. The mPCR results were further evaluated using high-performance liquid chromatography; in general, the two methods yielded similar results. These findings indicate that both mPCR methods are suitable for the detection of ZEA-producing Fusarium species in white and brown rice; however, the direct method yielded more rapid results. Copyright © 2018. Published by Elsevier B.V.

  5. Application of Polymerase Chain Reaction for High Sensitivity Detection of Roundup Ready™ Soybean Seeds and Grains in Varietal Mixtures

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    Ashok Pandey

    2011-01-01

    Full Text Available Strong increase in the production of genetically modified organisms (GMOs observed over the years has led to a consolidation of transgenic seed industries worldwide. The dichotomy between the evaluated risk and the perceived risk of transgenic use has defined their level of acceptability among different global societies. GMOs have been widely applied to agricultural commodities, among them the Roundup Ready™ (RR™ soybean line GTS 40-3-2 has become the most prevalent transgenic crop in the world. This variety was developed to confer plant tolerance against glyphosate-based agricultural herbicide Roundup Ready™. Issues related to detection and traceability of GMOs have gained worldwide interest due to their increasing global diffusion and the related socioeconomic and health implications. Also, due to the widespread use of GMOs in food production, labelling regulations have been established in some countries to protect the right of consumers and producers. Besides regulatory demand, consumer concern issues have resulted in the development of several methods of detecting and quantifying foods derived from genetically engineered crops and their raw materials. Polymerase chain reaction (PCR has been proven to be the method of choice to detect the presence or absence of the introduced genes of GMOs at DNA level. The present paper aims to verify whether the PCR technique can detect RR™ soybean seeds among conventional ones to further certification as non-GM soybean seeds and grains. This analysis could be accomplished through the development of new methodology called 'intentional contamination' of soybean conventional seeds or grains with the respective RR™ soybeans. The results show that the PCR method can be applied with high sensitivity in order to certify conventional soybean seeds and grains.

  6. Rapid and Sensitive Detection of Phytophthora sojae in Soil and Infected Soybeans by Species-Specific Polymerase Chain Reaction Assays.

    Science.gov (United States)

    Wang, Yuanchao; Zhang, Wenli; Wang, Ying; Zheng, Xiaobo

    2006-12-01

    ABSTRACT Root and stem rot caused by Phytophthora sojae is one of the most destructive diseases of soybean (Glycine max) worldwide. P. sojae can survive as oospores in soil for many years. In order to develop a rapid and accurate method for the specific detection of P. sojae in soil, the internal transcribed spacer (ITS) regions of eight P. sojae isolates were amplified using polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. The sequences of PCR products were aligned with published sequences of 50 other Phytophthora species, and a region specific to P. sojae was used to design the specific PCR primers, PS1 and PS2. More than 245 isolates representing 25 species of Phytophthora and at least 35 other species of pathogens were used to test the specificity of the primers. PCR amplification with PS primers resulted in the amplification of a product of approximately 330 bp, exclusively from isolates of P. sojae. Tests with P. sojae genomic DNA determined that the sensitivity of the PS primer set is approximately 1 fg. This PCR assay, combined with a simple soil screening method developed in this work, allowed the detection of P. sojae from soil within 6 h, with a detection sensitivity of two oospores in 20 g of soil. PCR with the PS primers could also be used to detect P. sojae from diseased soybean tissue and residues. Real-time fluorescent quantitative PCR assays were also developed to detect the pathogen directly in soil samples. The PS primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in soil and infected soybean tissue.

  7. Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction.

    Science.gov (United States)

    Gillespie, B E; Oliver, S P

    2005-10-01

    The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.

  8. Aphids preserved in propylene glycol can be used for reverse transcription-polymerase chain reaction detection of Potato virus Y.

    Science.gov (United States)

    Nie, Xianzhou; Pelletier, Yvan; Mason, Nicola; Dilworth, Andrea; Giguère, Marie-Andrée

    2011-08-01

    The effectiveness of propylene glycol on the retention of RNA target of Potato virus Y (PVY), an aphid stylet-borne virus, in Myzus persicae was investigated in comparison to ethanol and liquid nitrogen/-80°C. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the PVY targets from the propylene glycol/ethanol/liquid nitrogen preserved single aphids after a 5min acquisition period from infected potato plants. In the liquid nitrogen/-80°C and 70% ethanol treatments, 55.6% and 38.8% aphids tested PVY-positive, respectively. In the 0-75% propylene glycol treatments, 12.2-44.7% aphids tested PVY-positive. The lowest detection rate was in the 0% (positive rate, 15.2%) and the 10% propylene glycol (positive rate, 12.2%). As the propylene glycol concentration increased to 25%, 29.8% aphids tested positive. A high PVY-positive rate was also found in 35-75% propylene glycol treatments at 44.7% (35% propylene glycol), 36.7% (50% propylene glycol) and 34.8% (75% propylene glycol), which is comparable to the rate shown in 70% ethanol. No significant difference in the positive detection rate was observed in aphids preserved in 50% propylene glycol at room temperature for 2, 4 and 10 days. These results demonstrate that propylene glycol at 25-75% can retain PVY targets effectively in aphids for an extended time period, and thus can be used in aphid traps to preserve viruliferous aphids for later RT-PCR detection of PVY. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  9. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP for rapid diagnosis of neonatal sepsis

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    Anusha Rohit

    2016-01-01

    Full Text Available Background & objectives: The difficulties in diagnosis of neonatal sepsis are due to varied clinical presentation, low sensitivity of blood culture which is considered the gold standard and empirical antibiotic usage affecting the outcome of results. Though polymerase chain reaction (PCR based detection of bacterial 16S rRNA gene has been reported earlier, this does not provide identification of the causative agent. In this study, we used restriction fragment length polymorphism (RFLP of amplified 16S rRNA gene to identify the organisms involved in neonatal sepsis and compared the findings with blood culture. Methods: Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture and PCR-RFLP. Results: Bacterial DNA was detected by 16S rRNA gene PCR in 55 cases, while BacT/Alert culture was positive in 34 cases. Staphylococcus aureus was the most common organism detected with both methods. Klebsiella spp. was isolated from four samples by culture but was detected by PCR-RFLP in five cases while Acinetobacter spp. was isolated from one case but detected in eight cases by PCR-RFLP. The sensitivity of PCR was found to be 82.3 per cent with a negative predictive value of 85.7 per cent. Eighty of the 97 neonates had prior exposure to antibiotics. Interpretation & conclusions:The results of our study demonstrate that PCR-RFLP having a rapid turnaround time may be useful for the early diagnosis of culture negative neonatal sepsis.

  10. Analysis of colorectal cancer and polyp for presence herpes simplex virus and cytomegalovirus DNA sequences by polymerase chain reaction

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    Sahar Mehrabani khasraghi

    2016-05-01

    Full Text Available Introduction: In recent years, it was demonstrated that there is a clear association between the complicated course of colorectal cancer (CRC and the presence of herpes viruses. Despite a great number of published reports, the exact pathogenic role of herpes viruses remains unclear in these patients. The purpose of this study is to explore the prevalence of herpes simplex virus (HSV and cytomegalovirus (CMV in patients with CRC and polyp in comparison with healthy subjects using the polymerase chain reaction (PCR method. Methods: In this case-control study, 15 biopsies of patients with CRC and 20 colorectal polyp sample were selected. From each patient, two tissue samples were obtained: one sample from malignant tissue, and the other from normal colorectal tissue in an area located 15 cm away from the malignant tissue. Furthermore, 35 samples from healthy people as controls were selected. After DNA extraction, PCR was used to determine HSV and CMV genomes by specific primers. A statistical analysis was performed using the chi-square test. Results: Five CRC patients (33.3% had HSV DNA detected in both the malignant and the matched normal tissue. Five CRC patients (33.3% and seven polyp patients (35.0% had CMV DNA detected in both the malignant and the matched normal tissue. HSV DNA was found in 20% and CMV DNA in 37.1% of samples from healthy people as a control group. Thus, no significant association was observed between the prevalence of HSV and CMV, and an incidence of CRC and polyps according to the location of the samples as compared with the control group. Conclusion: The findings demonstrated that there is no direct molecular evidence to support the association between HSV and CMV and human colorectal malignancies. However, the results from this study do not exclude a possible oncogenic role of these viruses in the neoplastic development of colon cells.

  11. Optimization and Validation of a Real Time Reverse Transcriptase Polymerase Chain Reaction with RNA Internal Control to Detect Rubella RNA

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    Winny Xie

    2013-12-01

    Full Text Available BACKGROUND: According to a report from WHO, cases of rubella infection in Indonesia has increased up to 10-fold from 2007 to 2011. Despite no data of congenital rubella syndrome in the report, there are approximately 45,000 cases of babies born with heart failure and 0.1-0.3% live births with congenital deafness in Indonesia. Allegedly, rubella infection during pregnancy may play a role in this condition. This study aimed to optimize and validate a real-time reverse transcriptase polymerase chain reaction (RT-qPCR method to detect rubella virus RNA as an aid for the diagnosis of congenital rubella infection. METHODS: Method optimization was conducted using nucleic acids extracted from Trimovax Merieux vaccine with the High Pure Viral Nucleic Acid Kit. One step RT-qPCR was performed with Quantifast Multiplex RTPCR+R Kit. Target synthetic DNA was designed and used to determine the sensitivity of the method. RNA internal control was synthesized to control the process of extraction and amplification. RESULTS: The analytical sensitivity of this method was as low as 5 copies target synthetic DNA/μl. The mean Coefficient of Variation (CV % of the critical threshold (Ct obtained were 2.71%, 1.20%, 1.62%, and 1.59% for within run, between run, between kit lots, and between operators, respectively. Recovery of the target synthetic DNA from amniotic fluid was 100.51% (by the log copies/μl at the concentration of 1,000,000 copies/μl. CONCLUSIONS: RT-qPCR is successfully used for the detection of rubella virus RNA in vaccine and synthetic nucleic acid. With its high sensitivity, good precision and recovery, this method offers a means to improve the diagnosis of congenital rubella infection in developing countries like Indonesia. KEYWORDS: congenital rubella, RT-qPCR, prenatal diagnosis, amniotic fluid.

  12. Use of polymerase chain reaction to identify Brucella abortus strain RB51 among Brucella field isolates from cattle in Italy.

    Science.gov (United States)

    Adone, R; Ciuchini, F; La Rosa, G; Marianelli, C; Muscillo, M

    2001-03-01

    Brucella abortus strain RB51, a rough mutant of the B. abortus 2308 virulent strain, was recently approved in the United States as the official vaccine for brucellosis in cattle. Following recent evidence of unauthorized use of RB51 vaccine in Italy, where the use of vaccines for brucellosis is no longer allowed, the suitability of an RB51-specific polymerase chain reaction assay for identifying the RB51 strain among Brucella field isolates from cattle in Italy was investigated. The oligonucleotide primers used in this study, belonging to a six-primer cocktail for Brucella species previously described by other authors, allowed the amplification of a 364-base pair (bp) fragment specific for RB51 and its parent strain 2308, and a 498-bp product specific for B. abortus. In addition, unresolved bands ranging from 600 to 700 bp were observed from RB51 strain. Brucella abortus biovars 1, 2 and 4 have only one specific sensitive 498-bp band. The B. abortus biovars 3, 5 and 6 did not give any signal. The 498-bp product from a reference Brucella strain was sequenced and submitted to EMBL with the accession number AJ271969 while the 364-bp fragment from RB51 strain was submitted to EMBL database with accession number AJ271968. The sequence studies confirmed the specificity of the detected fragments. No amplification was obtained by testing DNA from strains antigenically related to Brucella, such as Yersinia enterocolitica O:9, Escherichia coli O:157, Salmonella urbana and Pasteurella multocida. The results of this study indicate that this technique, in combination with specific serological tests, could be a useful diagnostic method to verify the use of RB51 vaccine and can contribute to the creation of a databank of circulating strains.

  13. Prevalence and co-infection of haemotropic mycoplasmas in Portuguese cats by real-time polymerase chain reaction.

    Science.gov (United States)

    Martínez-Díaz, Verónica L; Silvestre-Ferreira, Ana Cristina; Vilhena, Hugo; Pastor, Josep; Francino, Olga; Altet, Laura

    2013-10-01

    The diagnosis of feline haemoplasmosis has improved over the years, with several techniques enabling a clear and specific diagnosis, and where polymerase chain reaction (PCR) is considered as the 'gold standard'. The aim of this study was to survey the prevalence of feline haemoplasmas in 320 cats from the north-central region of Portugal by the use of real-time PCR, as well as to evaluate any associations between infection, clinical presentation and risk factors. The overall prevalence of infection by feline haemoplasmas was 43.43% (139/320), where 41.56% (133/320) corresponded to Candidatus Mycoplasma haemominutum (CMhm), 12.81% (41/320) to Mycoplasma haemofelis (Mhf), 4.38% (14/320) to Candidatus Mycoplasma haematoparvum and 1.25% (4/320) to Candidatus Mycoplasma turicensis. Almost 13% (47/320) of the samples were co-infected, with the most common co-infection being CMhm and Mhf (23.74%). Infection was found statistically significant with feline immunodeficiency/feline leukaemia virus status (P = 0.034), but no significant association was found for breed, sex, fertility status (neutered/spayed/entire), age, clinical status, living conditions (in/outdoor), anaemia status, or the presence/absence of ticks or fleas. Cats from north-central Portugal are infected with all the known feline haemoplasma species, with CMhm being the most common one. Prevalence of all feline haemoplasmas was higher than that reported previously in cats from other European countries, but similar to that described in Portugal for dogs. These data provide a better perspective regarding Mycoplasma species infection in Europe, and new information that helps us better understand feline haemoplasmosis.

  14. Identification of Propionibacterium acnes by polymerase chain reaction for amplification of 16S ribosomal RNA and lipase genes.

    Science.gov (United States)

    Nakamura, Masahiko; Kametani, Ikuyo; Higaki, Shuichi; Yamagishi, Takayoshi

    2003-02-01

    Propionibacterium acnes belongs to the cutaneous flora and is present in sebaceous follicles. The fatty acids that are released from sebum triglycerides by the action of this bacterial lipase play an important role in the pathogenesis of acne vulgaris. P. acnes is also involved in postoperative disorders and opportunistic infections in immunosuppressed hosts. Recently, it has been proposed that P. acnes causes sarcoidosis. Therefore, rapid isolation and identification of P. acnes is important. This study evaluated the polymerase chain reaction (PCR) for the detection of the 16S rRNA and lipase genes of P. acnes. The PCR used to detect the 16S rRNA gene could amplify the gene of P. acnes, but not the genes of the other tested strains of P. avidum, P. granulosum, P. lymphophilum, P. jensenii, P. acidipropionici and P. thoenii. The PCR to detect the lipase gene of P. acnes, however, could amplify not only the gene of P. acnes but also that of P. avidum. The PCR product of this lipase gene was not found in the strains of the other species tested. Therefore, the organism that has both the 16S rRNA gene and lipase gene was identified as P. acnes, while the strain with the lipase gene but not the 16S rRNA gene of P. acnes was characterized as P. avidum. These findings were confirmed by the conventional biochemical tests including lipase activity. Furthermore, out of the seven clinical isolates from acne vulgaris, four were identified as P. acnes and three as P. avidum by the PCR method and biochemical tests. The combination of two PCR, one for the detection of the 16S rRNA and the other of lipase genes was shown to be an easier, faster and more accurate method to identify P. acnes and P. avidum than conventional methods.

  15. Detection of DNA from Leishmania (Viannia): accuracy of polymerase chain reaction for the diagnosis of cutaneous leishmaniasis.

    Science.gov (United States)

    Neitzke-Abreu, Herintha Coeto; Venazzi, Mateus Sabaini; Bernal, Marcos Vinicius Zandonadi; Reinhold-Castro, Kárin Rosi; Vagetti, Fernanda; Mota, Camila Alves; Silva, Naielly Rodrigues; Aristides, Sandra Mara Alessi; Silveira, Thaís Gomes Verzignassi; Lonardoni, Maria Valdrinez Campana

    2013-01-01

    Cutaneous leishmaniasis (CL) can occur in skin and mucosa, causing disfiguring lesions. The laboratory diagnosis of CL involves immunological methods and optical detection of the parasite, al of which have limitations. There is a need for more effective diagnostic methods for CL which wil allow treatment to be initiated more promptly in order to help prevent the development of severe forms of mucosal disease, and to estimate the prognosis of the infection. The polymerase chain reaction (PCR) has been widely used to diagnose CL, because of its higher sensitivity. This study estimated the accuracy and compared PCRs of samples from lesion scarification (PCR-L) and blood sample-enriched leukocytes (PCR-B) with three conventional diagnostic techniques: parasite direct search (DS), Montenegro skin test (MST), and indirect immunofluorescence reaction (IIF). The study included 276 patients under suspicion of CL. We conducted a cross-sectional study, in which patients were selected by convenience sampling. We used MP3H/MP1L primers to generate a Leishmania (Viannia) (minicircle kDNA) fragment of 70-bp. Of 106 patients with CL, 83.87%, 51.67%, 64.52%, 85.71%, or 96.10% tested positive by PCR-L, PCR-B, DS, IIF, or MST, respectively. Five patients tested positive only by PCR-L, and two other patients only by PCR-B. PCR-L is indicated for use in patients with chronic lesions or Leishmania reinfection, which may progress to mucosal lesion. PCR-B is indicated for use in patients with negative results in conventional tests or for patients with no apparent lesion. PCR is not only useful in diagnosing CL but also helps to identify the infecting species.

  16. Differential detection of Entamoeba histolytica from Entamoeba dispar by parasitological and nested multiplex polymerase chain reaction methods

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    esmaiel fallah

    2014-02-01

    Full Text Available Introduction: Amebiasis is an intestinal illness caused by a one-celled parasite (amoeba called Entamoeba (E histolytica. E histolytica and E dispar are morphologicallyundistinguishable but have genetic and functional differences. E. histolytica is invasive andcause amoebiasis, but E dispar cause an asymptomatic colonization which does not need to bemedically treated. We have performed a nested multiplex Polymerase Chain Reaction (PCRtargeting small subunit rRNA (Ribosomal ribonucleic acid gene for differential detection of Ehistolytica and E dispar directly from stool samples. Methods: All the fecal samples collected without preservation and were screened for amebiccells by parasitological methods. Fecal samples that containing amebic cells were stored at -20ºC until DNA extraction. DNA extraction was down by using a DNA extraction kit. Thegenus specific primers were designed using nucleotide sequences of 18S-rRNA gene ofEntamoeba. Results: Thirty one (4.28% stool samples out of 724 samples were positive for E histolytica/E dispar. The nested multiplex PCR illustrated that the size of diagnostic fragments of PCR products was obviously different for two Entamoeba species, the specific product size for Ehistolytica and E dispar was 439 and 174 bp. The nested multiplex PCR was positive in 25 outof 31 stool specimens that 17 (54.8% samples were positive for E dispar and 8 (25.8%samples were positive for E histolytica. Conclusion: Nested multiplex PCR was useful for the specific detection of E histolytica and Edispar in stool samples. In current study we detected that E dispar was more prevalent in our study area.

  17. Field expansion of DNA polymerase chain reaction for early infant diagnosis of HIV-1: The Ethiopian experience

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    Peter Fonjungo

    2013-05-01

    Full Text Available Background: Early diagnosis of infants infected with HIV (EID and early initiation of treatment significantly reduces the rate of disease progression and mortality. One of the challengesto identification of HIV-1-infected infants is availability and/or access to quality molecular laboratory facilities which perform molecular virologic assays suitable for accurate identificationof the HIV status of infants. Method: We conducted a joint site assessment and designed laboratories for the expansion of DNA polymerase chain reaction (PCR testing based on dried blood spot (DBS for EID insix regions of Ethiopia. Training of appropriate laboratory technologists and development of required documentation including standard operating procedures (SOPs was carried out. The impact of the expansion of EID laboratories was assessed by the number of tests performed as well as the turn-around time. Results: DNA PCR for EID was introduced in 2008 in six regions. From April 2006 to April 2008, a total of 2848 infants had been tested centrally at the Ethiopian Health and Nutrition Research Institute (EHNRI in Addis Ababa, and which was then the only laboratory with the capability to perform EID; 546 (19.2% of the samples were positive. By November 2010, EHNRI and the six laboratories had tested an additional 16 985 HIV-exposed infants, of which 1915 (11.3% were positive. The median turn-around time for test results was 14 days (range 14−21 days. Conclusion: Expansion of HIV DNA PCR testing facilities that can provide quality and reliable results is feasible in resource-limited settings. Regular supervision and monitoring for quality assurance of these laboratories is essential to maintain accuracy of testing.

  18. Two-step glutamate dehydrogenase antigen real-time polymerase chain reaction assay for detection of toxigenic Clostridium difficile.

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    Goldenberg, S D; Cliff, P R; Smith, S; Milner, M; French, G L

    2010-01-01

    Current diagnosis of Clostridium difficile infection (CDI) relies upon detection of toxins A/B in stool by enzyme immunoassay [EIA(A/B)]. This strategy is unsatisfactory because it has a low sensitivity resulting in significant false negatives. We investigated the performance of a two-step algorithm for diagnosis of CDI using detection of glutamate dehydrogenase (GDH). GDH-positive samples were tested for C. difficile toxin B gene (tcdB) by polymerase chain reaction (PCR). The performance of the two-step protocol was compared with toxin detection by the Meridian Premier EIA kit in 500 consecutive stool samples from patients with suspected CDI. The reference standard among samples that were positive by either EIA(A/B) or GDH testing was culture cytotoxin neutralisation (culture/CTN). Thirty-six (7%) of 500 samples were identified as true positives by culture/CTN. EIA(A/B) identified 14 of the positive specimens with 22 false negatives and two false positives. The two-step protocol identified 34 of the positive samples with two false positives and two false negatives. EIA(A/B) had a sensitivity of 39%, specificity of 99%, positive predictive value of 88% and negative predictive value of 95%. The two-step algorithm performed better, with corresponding values of 94%, 99%, 94% and 99% respectively. Screening for GDH before confirmation of positives by PCR is cheaper than screening all specimens by PCR and is an effective method for routine use. Current EIA(A/B) tests for CDI are of inadequate sensitivity and should be replaced; however, this may result in apparent changes in CDI rates that would need to be explained in national surveillance statistics. Copyright 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

  19. Detection of hblA and bal Genes in Bacillus cereus Isolates From Cheese Samples Using the Polymerase Chain Reaction

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    Molayi Kohneshahri

    2016-03-01

    Full Text Available Background Bacillus cereus is a Gram-positive spore-forming bacterium, which causes food poisoning. Spores enable the persistence of B. cereus in the environment, and B. cereus strains can tolerate adverse environmental conditions, such as temperature and insufficient nutrients. B. cereus causes food poisoning via the production of two enterotoxins. Most isolates produce toxins leading to diarrhea (enterotoxins and vomiting (emetic forms. Diarrhea is caused by the production of three different heat-labile enterotoxins: HBL, NHE, and cytotoxin K. A heat-stable toxin, cereulide, is responsible for emesis. Objectives This study aimed to detect enterotoxigenic B. cereus isolates in cheese samples using the polymerase chain reaction (PCR. Materials and Methods Two-hundred pasteurized (n = 100 and nonpasteurized (n = 100 cheese samples were collected. The initial isolation was performed on PEMBA specific medium. Antibiotic susceptibility testing was performed using several antibiotic disks, according to the guidelines of the Clinical Laboratory and Standards Institute. Specific primers amplifying the hblA enterotoxin-encoding gene and bal hemolysin-encoding gene were used for the molecular detection of the toxins. Results Ten samples were positive for the presence of B. cereus, with both Gram staining and biochemical reactions. All the isolates were resistant to penicillin and ampicillin but susceptible to vancomycin, erythromycin, and ciprofloxacin. Six and three isolates were resistant to tetracycline and trimethoprim-sulfamethoxazole, respectively. The hblA and bal genes were amplified in all the B. cereus isolates. Conclusions The prevalence of B. cereus among the cheese samples was low. All the isolates were positive for genes encoding the hblA enterotoxin and bal toxin.

  20. Multiple polymerase chain reaction markers for the differentiation of canine cutaneous peripheral nerve sheath tumours versus canine fibrosarcomas.

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    Meyer, A; Klopfleisch, R

    2014-01-01

    Currently canine fibrosarcomas and peripheral nerve sheath tumours (PNSTs) are differentiated by their histopathological phenotype. Preliminary global transcriptomic analysis has identified genes with significant differential expression in both tumour types that may act as potential tumour markers. The aim of the present study was to establish reverse transcriptase polymerase chain reaction (RT-PCR) assays for the differentiation of formalin-fixed and paraffin wax-embedded tumours of both types. Fifty histologically well-defined examples of canine fibrosarcomas and PNSTs were characterized immunohistochemically for the expression of S100, laminin and PGP 9.5. RT-PCR assays for the potential fibrosarcoma markers FHL2-Ex4 and FHL2-Ex9 and the PNST markers GLI1 and CLEC3B were established and tested for their specificity and sensitivity to differentiate fibrosarcomas and PNSTs by their mRNA expression. Immunohistochemical analysis challenged the value of S100, laminin and PGP 9.5 for the diagnosis of PNSTs, since both PNSTs and fibrosarcomas showed similar expression of these proteins. In contrast, a combination of the markers GLI1 and CLEC3B differentiated PNSTs from fibrosarcomas with a sensitivity of 89% and a specificity of 87%. The proposed fibrosarcoma markers FHL2-Ex4 and FHL2-Ex9 failed to separate PNSTs and fibrosarcomas (sensitivity 50%, specificity 88%). The failure of these markers to unequivocally separate fibrosarcomas and PNSTs raises questions as to whether histologically uniform PNSTs are less uniform at the molecular level than expected or if both tumour types, despite their different morphology, are more closely related in terms of their histogenesis than previously thought. Copyright © 2013 Elsevier Ltd. All rights reserved.