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.

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 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

scholarly journals Identification of HIV-1 Genotypic Resistance in Patients on First-line Antiretroviral Therapy Using Polymerase Chain Reaction and Sequencing

2013 ◽  
Vol 2 (4) ◽  
pp. 169-175
Author(s):  
Jiang Xiao ◽  
Yan-mei Li ◽  
Ying-xiu Huang ◽  
Wen Zhang ◽  
Wen-jing Su ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Virologic Failure ◽  
Cross Resistance ◽  
Common Mutation ◽  
First Line ◽  
Chain Reaction ◽  
Hiv Viral Load ◽  
Genotypic Resistance ◽  
Pcr Products ◽  
Polymerase Chain

Abstract Objective The aim of the study was to evaluate the characteristics of HIV drug-genotypic resistance among patients taking first-line ARV regimens using polymerase chain reaction and sequencing, and guide to design optimal ARV regimens for these patients. Methods HIV reverse transcriptase-encoded gene was amplified with RT-PCR and amplified PCR products were aligned and comparatively analyzed with HIV resistance database to find drug-resistance mutations. Results Twenty-eight PCR products were amplified and sequenced successfully in 30 serum samples of recruited HIV-infected patients with virologic failure. The resistance rate was 96%, mutations in NRT region were found in 26 patients (93%), while mutations in NNRT region were found in 27 patients (96%). M184V was the most common mutation (86%), K65R was selected in 14% of recruited individuals and TAMs occurred in 50% of patients, which resulted in resistance to NRTIs. Y181C and V179D were the most common mutations in NNRTIs and prevalence was 43% (12/28) and 36% (10/28), respectively, which resulted in cross-resistance to NNRTIs due to low-genetic barrier. Conclusions Virologic failure may occur in long-term administration of first-line ARV regimens, and drugresistance mutations can be found in these patients, which resulted in resistance to first-line ARV regimens. We emphasized that HIV viral load assay and resistance assay were important tools to guide healthcare workers to design an optimal second-line ARV regimens for HAART-experienced individuals with virologic failure.

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.

DMA Amplification

1993 ◽  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cultured Cells ◽  
Profile Analysis ◽  
Single Copy ◽  
Chain Reaction ◽  
Biochemical Tests ◽  
Feasible Option ◽  
Amplification Process ◽  
Polymerase Chain

Amplification of DNA may be necessary to increase the quantity of sample available for profiling, to reduce the analysis time, or to produce probes for the hybridization process (Higuchi 1989, Li 1988, Marx 1988, Mullis 1990, Paabo 1989, Saiki 1986). Stretches of nucleotides up to at least 3,000 bp from any DNA-containing samples may be efficiently amplified by the polymerase chain reaction (PCR). Alternatively, living tissue can be placed in culture, and fibroblasts, epithelial type cells, or lymphoblasts grown. The culture process differs considerably from the PCR approach in that the total genome is reproduced. Also, tissue culture is usually at least a two-week procedure, whereas the polymerase chain reaction requires only a few hours. Cultured cells can be used for enzyme and other biochemical tests, and storage in liquid nitrogen is a standard practice for regrowth at a later time. Probe material, that is, DNA capable of hybridizing with its complementary region in the genome, must be amplified, aliquoted, and stored to provide an ongoing source for use with each profile analysis. Probe amplification has been mainly carried out in bacterial culture; however, probes can be chemically synthesized as discussed in Chapter 2 or amplified by the PCR system. At least 10 to 50 ng of high molecular weight genomic DNA are required for VNTR analysis using single-locus probes, and at least 0.5 to 1.0 μg required if multilocus probes are used. If only a small quantity of DNA is available, amplification using the PCR may be the only feasible option for obtaining sufficient material for analysis. PCR has revolutionized the approach to the recovery of DNA from a variety of sources. Microgram quantities of DNA can be produced in vitro by the amplification of picogram starting amounts. Single-copy genomic sequences greater than 2 kb in length have been amplified more than 10 millionfold in a few hours. Amplified material can also be directly sequenced without the necessity of incorporating DNA fragments into vectors such as M13 (Gyllensten 1989,1989a). Availability of oligonucleotide primers is the key to the amplification process.

TOWARDS A HIGH RELIABILITY OF THE PCR AMPLIFICATION PROCESS IN DNA COMPUTING

2002 ◽  
Vol 02 (04) ◽  
pp. 423-432
Author(s):  
MASASHI NAKATSUGAWA ◽  
SATOSHI KASHIWAMURA ◽  
AZUMA OHUCHI ◽  
MASAHITO YAMAMOTO ◽  
TOSHIKAZU SHIBA
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Calculation Result ◽  
Dna Computing ◽  
High Reliability ◽  
Pcr Amplification ◽  
Small Concentration ◽  
Chain Reaction ◽  
Amplification Process ◽  
Polymerase Chain

Polymerase Chain Reaction (PCR) is the most important experimental technique in DNA computing. When a concentration of DNA sequence is too small to investigate, PCR amplifies the DNA sequence by the addition of a polymerase. PCR is frequently used in DNA computing, because the calculation result is usually represented by a small concentration of DNA sequence. Therefore, PCR has a crucial influence on the calculation result. The reliability of PCR needs to be improved for DNA computing. In this paper, the reliability of PCR is defined by the reproducibility of the amplified concentration of DNA sequence. The PCR protocol is adjusted to improve the reliability. Quality Engineering efficiently supports this adjustment.

scholarly journals Analisis Cemaran DNA Tikus pada Bakso Daging Sapi yang Beredar di Makassar dengan Metode Polymerase Chain Reaction (PCR)

2019 ◽  
Vol 5 (1) ◽  
pp. 93-100
Author(s):  
Aminah Aminah ◽  
Ristieyen Ramadini ◽  
Tadjuddin Naid
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Analysis ◽  
Uv Light ◽  
Pcr Amplification ◽  
Polymerase Chain Reaction Method ◽  
Chain Reaction ◽  
Reaction Method ◽  
Amplification Process ◽  
Three Stages ◽  
Polymerase Chain

Analysis of rat DNA contamination in meatball meat circulating in Makassar by PCR (Polymerase Chain Reaction) method has been carried out. In this study, a polymerase chain reaction method will be developed to analyze the presence of rat meat contamination in beef meatballs. There are three stages in the PCR amplification process carried out with 30 cycles, which are 95oC temperature denaturation, 51oC attachment, and 72oC extension. DNA analysis included agarose gel electrophoresis, measurement of concentration and purification, and analysis of rat DNA using PCR. The results of PCR amplification using mouse-specific primers namely primary ND1 (NADH dehydrogenase 1) showed no bands seen in UV light. So that it can be proven that beef meatball samples in the Makassar region did not contain rat DNA. t.  

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.

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