scholarly journals Antimony-resistant Leishmania donovani in eastern Sudan: incidence and in vitro correlation

2003 ◽  
Vol 9 (4) ◽  
pp. 837-843 ◽  
Author(s):  
M. G. Abdo ◽  
W. M. El Amin ◽  
E. A. G. Khalil ◽  
M. M. Mukhtar
Keyword(s):  
Leishmania Donovani ◽  
Pcr Amplification ◽  
Sodium Stibogluconate ◽  
Chain Reaction ◽  
Endemic Region ◽  
Testing Method ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Eastern Sudan

A longitudinal study was done in a leishmaniasis -endemic region in eastern Sudan during the period November 2001-February 2003 to determine the incidence of failure of sodium stibogluconate treatment. We studied 820 confirmed visceral leishmaniasis patients. All were treated with sodium stibogluconate, 20 mg/kg body weight for at least 28 days. Parasites were isolated from lymph node aspirates from 22 participants identified as relapsed patients. All isolates were typed as Leishmania donovani based on polymerase chain reaction [PCR] amplification of parasite kDNA. Six parasites showed in vitro resistance to sodium stibogluconate using murine J774 macrophage amastigote testing method. The resistant isolates showed different restriction profiles when the amplified kDNA PCR products were digested with ALU1 restriction enzyme, indicating that resistance was mediated by different parasite clones

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

1993 ◽  
Vol 39 (9) ◽  
pp. 1927-1933 ◽  
Author(s):  
J B Findlay ◽  
S M Atwood ◽  
L Bergmeyer ◽  
J Chemelli ◽  
K Christy ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Automated System ◽  
Closed Vessel ◽  
Chain Reaction ◽  
Panel Testing ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

scholarly journals DESAIN PRIMER UNTUK AMPLIFIKASI REGIO PROMOTER GEN inhA ISOLAT P016 MULTIDRUG RESISTANCE Mycobacterium tuberculosis DENGAN METODE POLYMERASE CHAIN REACTION

2018 ◽  
pp. 34
Author(s):  
Eka Nata Sari
Keyword(s):  
Polymerase Chain Reaction ◽  
Promoter Region ◽  
Pcr Amplification ◽  
Promoter Sequence ◽  
Cross Resistance ◽  
Chain Reaction ◽  
Pcr Products ◽  
Amplification Process ◽  
Polymerase Chain

Mutations in the inhA promoter region are responsible for isoniazid resistance and cross-resistance to ethionamide. To identify mutations with polymerase chain reaction (PCR), a pair of primers that can amplify the target region. This aim of this study was to obtain the best primer pair to amplify the inhA promoter region using the Clone Manager Suite 6 program. The inhA promoter sequence (Genbank: U66801) obtained from the www.ncbi.nlm.nih.gov was used as a template. The design results obtained the best primer pair tested in vitro using Polymerase Chain Reaction (PCR) method. The PCR amplification process was performed for 40 cycles with the following conditions: predenaturase (95oC for 15 minutes), denaturation (94 oC for 1 minute), annealing (56 oC for 1 minute 30 seconds), elongation (72 oC for 2 minutes), and final elongation (72 oC for 10 minutes). Detection of PCR products was performed in agarose gel electrophoresis 1.3% w / v and visualized by UV transluminator tool. The results obtained were forward primers of 5'-GGTCGAAGTGTGCTGAGTC-3 'and reverse primer 5'-TGCTCTTCTACCGCCGTGA-3' which met the good primary criterion based on Clone Manager Suite 6. The pair of primers has been able to amplify the inhA promoter region by the length of product produced at 373 bp.

Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of a Short Fragment of the Cytochrome b Gene for Identification of Flatfish Species

1998 ◽  
Vol 61 (12) ◽  
pp. 1684-1685 ◽  
Author(s):  
ANA CÉSPEDES ◽  
TERESA GARCÍA ◽  
ESTHER CARRERA ◽  
ISABEL GONZÁLEZ ◽  
BERNABÉ SANZ ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cytochrome B ◽  
Pcr Amplification ◽  
Cytochrome B Gene ◽  
Polymorphism Analysis ◽  
Chain Reaction ◽  
Greenland Halibut ◽  
Universal Primer ◽  
Pcr Products ◽  
Polymerase Chain

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the specific identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides). PCR amplification of the cytochrome b gene using a universal primer together with a primer specifically designed as a part of this study produced a 201-bp fragment in all species analyzed. Digestions of the PCR products with Sau3Al, BsmAl, Rsal, and Mn/l endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of each species analyzed.

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio

Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 229-235 ◽  
Author(s):  
Ninoska Madrid-Bury ◽  
Raúl Fernández ◽  
Adela Jiménez ◽  
Sonia Pérez-Garnelo ◽  
Pedro Nuno Moreira ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Preparation Method ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Initial Experiment ◽  
X Chromosomes ◽  
Primary Sex Ratio ◽  
Polymerase Chain ◽  
Offspring Gender

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.

scholarly journals Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

1997 ◽  
Vol 87 (12) ◽  
pp. 1192-1196 ◽  
Author(s):  
M. Sato ◽  
K. Watanabe ◽  
M. Yazawa ◽  
Y. Takikawa ◽  
K. Nishiyama
Keyword(s):  
Pseudomonas Syringae ◽  
Fragment Length Polymorphism ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Enzyme Gene ◽  
Indigenous Plasmid ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Primer Sets

Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the “kudzu strain”) and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.

scholarly journals Molecular Bird Sexing on Fischeri Lovebird (Agapornis fischeri) by Using Polymerase Chain Reaction

2020 ◽  
Vol 20 ◽  
pp. 04003
Author(s):  
Azalea Dyah Argarini ◽  
Herjuno Ari Nugroho ◽  
Medania Purwaningrum ◽  
Aris Haryanto
Keyword(s):  
Sex Chromosome ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Total Sample ◽  
Z Chromosome ◽  
Agarose Gel Electrophoresis ◽  
Chain Reaction ◽  
Male And Female ◽  
Pcr Products ◽  
Polymerase Chain

Fischeri Lovebird (Agapornis fischeri) found originally in Africa which has spread to many countries. In Indonesia, Fischeri Lovebird is popular as a pet animal. This lovebird is a monomorphic bird, so it is difficult to differentiate morphologically between male and female birds. In general, a male lovebird has ZZ homozygotes, whereas females' lovebird has ZW heterozygous of their sex chromosome. These sex chromosomes set used as study targets for molecular bird sexing of many species of birds because this method is effective and simple to perform. This method targeted to amplify the Chromodomain Helicase DNA-binding (CHD) gene, which found into the sex chromosome of male and female birds. The objective of this study was to rapid molecular bird sexing of Fischeri Lovebird by using PCR methods. Research samples were collected from feather calamus of A. fischeri. The total sample was 11 feathers from A. fischeri. which were collected three to six feathers for each lovebird. Then the research was followed by DNA extraction from calamus feathers, DNA amplification by PCR and agarose gel electrophoresis of PCR products and visualization of PCR predicts by UV-Transilluminator in darkroom. It concluded that PCR amplification using NP, MP and P2 primers produced double DNA bands in size of 400 bp on Z chromosome and bp on W chromosome for female Fischeri Lovebird, whereas for male Fischeri Lovebird only produced a single DNA band in size of 400 bp on Z chromosome. From eleven samples of Fischeri Lovebird showed a total of five females and six male Fischeri Lovebirds.

Mapping maize microsatellites and polymerase chain reaction confirmation of the targeted repeats using a CT primer

Genome ◽  
1993 ◽  
Vol 36 (5) ◽  
pp. 884-889 ◽  
Author(s):  
M. Lynn Senior ◽  
Manfred Heun
Keyword(s):  
Polymerase Chain Reaction ◽  
Simple Sequence Repeats ◽  
Genome Mapping ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Expected Size Range ◽  
Pcr Products ◽  
Mammalian Genomes ◽  
Polymerase Chain ◽  
Simple Sequence

Microsatellites, also called simple sequence repeats (SSRs), have yielded an important class of DNA markers most notable for mapping mammalian genomes. To study the occurrence of microsatellites and their inheritance in maize, a search was made of 280 maize GenBank® sequences. Six SSRs were chosen and unique flanking primers were designed for polymerase chain reaction (PCR) amplification. Eight different maize inbreds were studied with these six primer pairs and a mean of 3.5 polymorphic patterns occurred within the expected size range. For five of these putative microsatellites, the segregation in a maize restriction fragment length polymorphism mapping population was analyzed. Four of the microsatellites cosegregated with the Adh1, Gpc1, Pdk1, and Tpi genes from which the primer sequences were derived. The fifth primer pair (MZEGPA1) showed segregating polymorphisms, but the products were larger than expected. To verify the existence of the original SSRs in the segregating PCR products, a CT primer, containing a CT SSR and an arbitrary leader sequence, was used to reamplify these products. The four microsatellites that cosegregated with the original gene were reamplified as anticipated, whereas a suspicious 230-bp product obtained when using the MZEGPA1 primers could not be reamplified. Based on these results it is concluded that microsatellites can be a valuable tool for maize mapping.Key words: maize, microsatellites, simple sequence repeats, genome mapping.

scholarly journals Polymerase Chain Reaction for Detection of Guinea Pig Adenovirus

1997 ◽  
Vol 9 (3) ◽  
pp. 232-236 ◽  
Author(s):  
Patricia Pring-Åkerblom ◽  
Karel Blažek ◽  
Jana Schramlová ◽  
Ivo Kunstýr
Keyword(s):  
Polymerase Chain Reaction ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Diagnostic Procedures ◽  
In Vitro Cultivation ◽  
Chain Reaction ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Pcr Techniques

Lack of in vitro cultivation methods has inhibited the development of rapid, reliable diagnostic procedures for adenovirus-associated necrotizing bronchopneumonia in guinea pigs. Because polymerase chain reaction (PCR) techniques are well established for human adenoviruses, primers for the amplification of guinea pig adenovirus DNA were evaluated. The DNA for PCR was purified from the lung tissue of spontaneously infected and healthy guinea pigs. Adenovirus DNA could only be detected in the lungs of the infected animals. Subsequent sequence analysis of PCR products revealed that the guinea pig adenovirus is a distinct adenovirus.

Single-step method for rapid detection of Brucella spp. in soft cheese by gene-specific polymerase chain reaction

1999 ◽  
Vol 66 (2) ◽  
pp. 313-317 ◽  
Author(s):  
LUIGI SERPE ◽  
PASQUALE GALLO ◽  
NICOLETTA FIDANZA ◽  
ALFREDO SCARAMUZZO ◽  
DOMENICO FENIZIA
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Single Step ◽  
Detection Sensitivity ◽  
Chain Reaction ◽  
Step Method ◽  
Polymerase Chain

Brucellosis can be transmitted to man by direct contact with infected animals or through contaminated meat, milk and dairy products (Nicoletti, 1989). The analysis of Brucella spp. is carried out in the laboratory by microbiological or serological assays (Alton et al. 1988). The first are more specific but are also time-consuming and expose the analyst to the risk of infection (López-Merino, 1991). However, the latter can result in false positives owing to cross reactivity with other Gram-negative bacteria (Diaz-Aparicio et al. 1994). Because of these limitations, the amplification in vitro of specific DNA regions by the polymerase chain reaction (PCR) could represent a powerful tool for rapid and specific diagnostic analysis. In recent years, several PCR methods have been developed to amplify specific DNA sequences of Brucella strains (Herman & de Ridder, 1992; Romero et al. 1995; Valentino et al. 1997). In addition, direct analysis of Brucella in contaminated abortive tissues (Fekete et al. 1992), milk and blood (Leal-Klevezas et al. 1995; Rijpens et al. 1996) has been reported.In this paper we describe a method for gene-specific PCR amplification of a 443 base pair (bp) fragment of Brucella DNA that belongs to a gene encoding for a 31 kDa outer membrane protein. This protein (BCSP-31) is a membrane antigen characteristic of the Brucella genus (Mayfield et al. 1988). The PCR method was developed for the analysis of soft cheeses. We focused our attention on Mozzarella, Pecorino and ricotta samples, because such products are not subjected to the natural microbial autopurification process of maturing. They are widely consumed in Italy and a relationship between infected foods and the areas where brucellosis is a human zoonosis is a possibility.The analysis was performed without purification of DNA from bacteria. Indeed, after homogenization, the sample was subjected to thermal shock by freeze–thaw cycles that lysed bacteria and solubilized nucleic acids for subsequent PCR amplification. Amplified DNA fragments were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Several brands of soft cheeses and ricotta contaminated at different levels with Brucella cells were analysed by our procedure to evaluate the detection sensitivity and the repeatability of the method.

scholarly journals Qualitative detection of genetically modified organisms in plant samples

2007 ◽  
pp. 309-313
Author(s):  
Csilla Uri ◽  
József Prokisch ◽  
Erzsébet Sándor ◽  
Zoltán Győri
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetically Modified Organisms ◽  
Chain Reaction ◽  
Cdna Sequences ◽  
Qualitative Detection ◽  
Mgcl2 Concentration ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Negative Controls

We analysed the GMO content of corn samples by polymerase chain reaction following the appropriate optimization of the reaction. The analysis included two main steps: extraction of DNA from the sample, and detection of the GMO content by polymerase chain reaction. The polymerase chain reaction is an in vitro method to multiply chromosomatic or cloned DNA (cDNA) sequences through the enzymatic pathway. The reaction is sensitive enough to produce DNA in sufficient amount for the analysis from a single DNA. We identified the PCR products by agarose gel electrophoresis. When optimizing the reaction, the MgCl2 concentration, reaction time and temperature have to be taken into consideration. The temperature of the anellation has to be increased until the highest specificity and yield is reached. If the temperature of the anellation is too high, the primer is linked to non-specific sites as well; in the gel visualization, more lines can be seen at one sample. If the temperature of the anellation is too high, the primer is insufficiently linked or is not linked at all (too few lines in the gel visualization). After optimization, the GMO content in the unknown sample can be determined along with the appropriate positive and negative controls.

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