A Simple Polymerase Chain Reaction-based Method for the Discrimination of Three Chicken Breeds - Amplified Fragment Length Polymorphism;Breed Discrimination;Chicken;Polymerase Chain Reaction;Nucleotide Polymorphism;
Seedat, Farah, Uthman, Olalekan, Robinson, Esther, Cooper, Jennifer, Takwoingi, Yemisi, Kandala, Ngianga-Bakwin, Stranges, Saverio and Taylor-Phillips, Sian (2018) Real‐time polymerase chain reaction tests versus antenatal culture tests for the screening of maternal group B Streptococcus colonisation in labour. Cochrane Database of Systematic Reviews, 2018 (5). CD013016. ISSN 1465-1858 ...
Improvements to the in situ polymerase chain reaction (PCR), a process of in vitro enzymatic amplification of specific nucleic acid sequences within the cells where they originate, can be achieved by changing the way that the enzymatic reaction is started. Reaction initiation is delayed until the start of PCR thermal cycling, either by withholding a subset of PCR reagents from the cellular preparation until the preparation has been heated to 50 C. to 80 C., immediately before thermal cycling is begun, or by adding to the PCR reagents a single-stranded DNA binding protein which blocks reaction at temperatures below about 50 C. If the in situ PCR is performed on cellular preparations already attached to a microscope slide, thermal cycling also is facilitated by use of a thermal cycler sample block or compartment designed optimally to hold the microscope slide and any vapor barrier covering the slide.
The feasibility of using a sensitive polymerase chain reaction (PCR) to evaluate malaria vaccines in small group sizes was tested in 102 adult Gambian volunteers who received either the malaria vaccine regimen FP9 ME-TRAP/MVA ME-TRAP or rabies vaccine. All volunteers received the antimalarial drugs primaquine and Lapdap plus artesunate to eliminate malaria parasites. Volunteers in a further group received an additional single treatment with sulfadoxine-pyrimethamine (SP) to prevent new infections. There was substantially lower T-cell immunogenicity than in previous trials with this vaccine regimen and no protection against infection in the malaria vaccine group. Using the primary endpoint of 20 parasites per mL, no difference was found in the prevalence of low-level infections in volunteers who received SP compared with those who did not, indicating that SP did not reduce the incidence of very low-density infection. However, SP markedly reduced the incidence of higher density infections. These findings
Lab Reagents Dna Extraction Laboratories manufactures the genomic dna extraction protocol phenol chloroform reagents distributed by Genprice. The Genomic Dna Extraction Protocol Phenol Chloroform reagent is RUO (Research Use Only) to test human serum or cell culture lab samples. To purchase these products, for the MSDS, Data Sheet, protocol, storage conditions/temperature or for the concentration, please contact DNA Extraction. Other Genomic products are available in stock. Specificity: Genomic Category: Dna Group: Extraction Protocol. Extraction Protocol information ...
TY - CONF. T1 - A novel temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device.. AU - Bu, Minqiang. AU - R. Perch-Nielsen, Ivan. AU - Sørensen, Karen Skotte. AU - Skov, Julia AU - Yi, Sun. AU - Bang, Dang Duong. AU - E. Pedersen, Michael AU - Hansen, Mikkel Fougt. AU - Wolff, Anders. PY - 2012. Y1 - 2012. N2 - We present a new temperature control method capable of effectively shortening the thermal cycling time of polymerase chain reaction (PCR) in a disposable polymer microfluidic device with external heater and temperature sensor. The method employs optimized temperature overshooting and undershooting steps to achieve a rapid ramping between the temperature steps for DNA denaturation, annealing and extension. The temperature dynamics within the microfluidic PCR chamber was characterized and the overshooting and undershooting parameters were optimized using the temperature dependent ...
The touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just above this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will swamp out any specific sequences because of the exponential nature of polymerase amplification. The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles (the number of ...
[110] Global Polymerase Chain Reaction Technologies market study by product, technology and application. The study provides an in-depth analysis of the market current and future trends.
David-Neto E, Triboni AHK, Paula FJ, Vilas Boas LS, Machado CM, Agena F, Latif AZA, Alencar CS, Pierrotti LC, Nahas WC, Caiaffa-Filho HH, Pannuti CS. A double-blinded, prospective study to define antigenemia and quantitative real time polymerase chain reaction cutoffs to start preemptive therapy in low-risk, seropositive, renal transplanted recipients [Internet]. Clinical and Transplantation Research. 2014 ; 98( 10): 1077-1081.Available from: doi: 10.1097/TP. ...
The earliest thermal cyclers were designed for use with the Klenow fragment of DNA polymerase I. Since this enzyme is destroyed during each heating step of the amplification process, new enzyme had to be added every cycle. This led to a cumbersome machine based on an automated pipettor, with open reaction tubes. Later, the PCR process was adapted to the use of thermostable DNA polymerase from Thermus aquaticus, which greatly simplified the design of the thermal cycler. While in some old machines the block is submerged in an oil bath to control temperature, in modern PCR machines a Peltier element is commonly used. Quality thermal cyclers often contain silver blocks to achieve fast temperature changes and uniform temperature throughout the block. Other cyclers have multiple blocks with high heat capacity, each of which is kept at a constant temperature, and the reaction tubes are moved between them by means of an automated process. Miniaturized thermal cyclers have been created in which the ...
AIMS: To attempt to detect p53 gene mutations in the plasma of patients with large bowel carcinoma. METHODS: Plasma was collected from 20 control patients with no history of cancer and from 17 patients with large bowel carcinoma. Corresponding tumour and benign lymph node (control) samples for each of the carcinoma patients were obtained from paraffin blocks. A Dukes stage was determined for each tumour. DNA was extracted from the plasma samples and the paraffin embedded tissue using previously described methods. A nested primer polymerase chain reaction protocol was used for the amplification of exons 5 to 8 of the p53 gene. Cold single strand conformational polymorphism (SSCP) was performed on mini gels and silver stained. Abnormal bands were excised, the DNA eluted, and reamplified for automated dye termination sequencing. Any sample showing an apparent mutation was rechecked from the original extracted DNA sample at least three times. RESULTS: p53 gene mutations were not found in the ...
TY - JOUR. T1 - Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. AU - Anderson, Ian C. AU - Campbell, C. D.. AU - Prosser, James Ivor. PY - 2003. Y1 - 2003. N2 - Four fungal 18S rDNA and internal transcribed spacer (ITS) polymerase chain reaction (PCR) primer pairs were tested for their specificity towards target fungal DNA in soil DNA extracts, and their ability to assess the diversity of fungal communities in a natural grassland soil was compared. Amplified PCR products were cloned, and approximate to 50 clones from each library were sequenced. Phylogenetic analysis and database searches indicated that each of the sequenced cloned DNA fragments was of fungal origin for each primer pair, with the exception of the sequences generated using the 18S rDNA primers nu-SSU-0817 and nu-SSU-1196, where 35 of the 50 sequenced clones represented soil invertebrates. Although some of the primers have previously ...
Abstract. Twenty-four male patients grafted for various pathologies with the marrow of a female donor and presenting a complete donor-type hematopoiesis when a
Quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol ...
Jadack, R.A., Yuenger, J., Ghanem, K.G., et al. (2006) Polymerase chain reaction detection of Y-chromosome sequences in vaginal fluid of women accessing a sexually transmitted disease clinic. Sexually Transmitted Diseases, 33, 22-25. doi10.1097/01.olq.0000194600.83825.81
TY - JOUR. T1 - Atomic force microscopic detection enabling multiplexed low-cycle-number quantitative polymerase chain reaction for biomarker assays. AU - Mikheikin, Andrey. AU - Olsen, Anita. AU - Leslie, Kevin. AU - Mishra, Bud. AU - Gimzewski, James K.. AU - Reed, Jason. PY - 2014/7/1. Y1 - 2014/7/1. N2 - Quantitative polymerase chain reaction is the current golden standard for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, ...
Fingerprint Dive into the research topics of Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction. Together they form a unique fingerprint. ...
The common 4977 by deletion in mitochondrial DNA (Δ4977) is commonly used as an indicator of tissue deterioration in ageing and bioenergetic diseases. Deletion levels are normally measured by a serial dilution polymerase chain reaction (PCR) approach, where test reactions are compared with dilutions of control amplifications of DNA from a similar sized stable region of the mitochondrial genome. The end-point of this assay is the dilution that can just detect any PCR product; however, this is an inherently unstable measure. We constructed a chimaeric DNA construct that binds to both control and deletion primers with similar annealing properties. This was used in a competitive PCR assay to quantify Δ4977 in human testicular tissues that had been well-characterized using the serial dilution approach. We found the competitive assay to be highly replicable as it compares the PCR product of the construct with that of test DNA samples during the linear growth phase of the PCR reaction. Moreover, ...
Background: As one of the most common bloodstream infections worldwide, Staphylococcus aureus bacteremia places a major burden on health care. Implementation of a rapid, genetic-based diagnostic test may have important implications in the clinical management of patients with S. aureus bacteremia.. Objectives: The primary objective was to assess concordance between testing based on polymerase chain reaction (PCR) and the current gold standard, culture and sensitivity testing; the secondary objective was to assess the impact of this technology on patient care.. Methods: A pre-post intervention retrospective chart review was used to document the hospital course of patients with a diagnosis of S. aureus bacteremia before and after implementation of the PCR-based diagnostic system. Laboratory results from all patient samples subjected to PCR-based analysis following implementation of this system were compared with culture and sensitivity data for the same samples to determine accuracy of the new ...
Background: As one of the most common bloodstream infections worldwide, Staphylococcus aureus bacteremia places a major burden on health care. Implementation of a rapid, genetic-based diagnostic test may have important implications in the clinical management of patients with S. aureus bacteremia.. Objectives: The primary objective was to assess concordance between testing based on polymerase chain reaction (PCR) and the current gold standard, culture and sensitivity testing; the secondary objective was to assess the impact of this technology on patient care.. Methods: A pre-post intervention retrospective chart review was used to document the hospital course of patients with a diagnosis of S. aureus bacteremia before and after implementation of the PCR-based diagnostic system. Laboratory results from all patient samples subjected to PCR-based analysis following implementation of this system were compared with culture and sensitivity data for the same samples to determine accuracy of the new ...
The Magnetic Beads Genomic DNA Extraction Kit Blood was designed specifically for efficient genomic DNA purification from blood and buffy coat. DNA is bound to the surface of the magnetic beads and released using a proprietary buffer system.
Understand how the DNA polymerase chain reaction works and is used in forensic science. Polymerase chain reaction (PCR) can be used in disease diagnosis, for example the diagnosis of avian influenza (bird flu)
We have evaluated the polymerase chain reaction for the detection of viral DNA sequences in paraffin-embedded archival tissues. In 63 frozen cervical biopsy specimens that were taken from premalignant and invasive lesions, Southern blotting detected human papillomavirus (HPV) type 16 DNA in 28 (44%) of the samples. In the polymerase chain reaction analysis of the formalin-fixed, paraffin-embedded mirror biopsy specimens, 46 (73%) of the tissues were found to be positive for HPV type 16. In three Southern blotting-positive cases, the DNA of the paraffin-embedded sections was too scant or too degraded to allow the detection of HPV DNA by the polymerase chain reaction. In 21 Southern blotting-negative cases, HPV type 16 DNA could be demonstrated in the archival sections by the polymerase chain reaction technique--a sensitivity improvement of more than 80% over the standard method of HPV detection in tissues.
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MagListoTM 5M Plant Genomic DNA Extraction Kit 는 magnetic nano bead와 MagListoTM를 이용하여 Plant sample (leaf, root, seed) 에서 Genomic DNA를 빠르게 추출할 수 있는 획기적인 제품입니다. Magnetic nano bead와 자석을 이용해 세포 분쇄물 중 Genomic DNA만을 분리시키고 농축 및 정제하는 과정을 거치기 때문에 원심분리기를 사용하는 방법에 비해 빠르게 DNA를 분리 할 수 있습니다. 본 제품은 mini, midi, maxi scale의 prep을 위해 별도의 kit를 구매하지 않고 한 가지의 kit를 이용해 모두 prep 할 수 있으며 midi나 maxi prep을 위해 별도의 vacuum system이나 air pressure system을 구비 할 필요가 없는 것이 장점입니다.
Cy0 is a new method in Real-time polymerase chain reaction quantification (PCR) analysis that does not require the assumption of equal efficiency between unknowns and standard curve.
The study of Polymerase Chain Reaction (PCR) Products market is a compilation of the market of Polymerase Chain Reaction (PCR) Products broken down into its entirety on the basis of types, application, trends and opportunities, mergers and acquisitions, drivers and restraints, and a global outreach. The detailed study also offers a board interpretation of the Polymerase Chain Reaction (PCR) Products industry from a variety of data points that are collected through reputable and verified sources. Furthermore, the study sheds a lights on a market interpretations on a global scale which is further distributed through distribution channels, generated incomes sources and a marginalized market space where most trade occurs.. Along with a generalized market study, the report also consists of the risks that are often neglected when it comes to the Polymerase Chain Reaction (PCR) Products industry in a comprehensive manner. The study is also divided in an analytical space where the forecast is predicted ...
Polymerase chain reaction IPFS - polymerase chain reaction the many methods now used to rapid and widespread application as the polymerase chain reaction
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Background Carefully conducted, community-based, longitudinal studies are required to gain further understanding of the nature and timing of respiratory viruses causing infections in the population. However, such studies pose unique challenges for field specimen collection, including as we have observed the appearance of mould in some nasal swab specimens. We therefore investigated the impact of sample collection quality and the presence of visible mould in samples upon respiratory virus detection by real-time polymerase chain reaction (PCR) assays. Methods Anterior nasal swab samples were collected from infants participating in an ongoing community-based, longitudinal, dynamic birth cohort study. The samples were first collected from each infant shortly after birth and weekly thereafter. They were then mailed to the laboratory where they were catalogued, stored at -80àand later screened by PCR for 17 respiratory viruses. The quality of specimen collection was assessed by screening for human ...
TY - JOUR. T1 - Rapid detection of CYP2C18 genotypes by real-time fluorescence polymerase chain reaction. AU - Mizugaki, Michinao. AU - Hiratsuka, Masahiro. AU - Agatsuma, Yasuyuki. AU - Matsubara, Yoichi. AU - Fujii, Kunihiro. AU - Kure, Shigeo. AU - Narisawa, Kuniaki. PY - 2000/2. Y1 - 2000/2. N2 - In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of several drugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of the CYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located in exon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme are CYP2C18ml, located in exon 2 and CYP2C18m2, located in the 5-flanking region. We have developed an allele-specific TaqMan polymerase chain reaction (PCR) assay with which to detect CYP2C18 mutant alleles. This assay combines hybridization of the TaqMan probe and allele-specific amplification ...
Bio-Rad provides a wide range of products for use in the support of COVID-19 diagnosis and confirmation, and offers a suite of molecular testing tools including Droplet Digital PCR (ddPCR) systems and the SARS-CoV-2 ddPCR test kit.. While real-time PCR provides an accessible, high-throughput option, the high sensitivity of ddPCR makes it well suited for screening samples in patients, providing the precision needed to resolve indeterminate test results ...
The introduction of digital PCR (dPCR) represented a paradigm shift in the field of quantitative polymerase chain reaction technology. Bio-Rad brought digital PCR to another level with the introduction of the revolutionary Droplet Digital PCR (ddPCR) technology. Today, with more than 3800 published studies and growing, nowhere is the power of Droplet Digital PCR technology further apparent than Bio-Rads expansive ddPCR Publications Database.
The real-time PCR measure of I kappa B-alpha mRNA levels is a rapid, sensitive, and powerful method to quantify the transcriptional power of NF-kappa B. It can be easily used for clinical evaluation of NF-kappa B status.
Digital Polymerase Chain Reaction (dPCR) is an advancement of traditional polymerase chain reaction (PCR). The traditional PCR with its limited precision and accuracy often fail to amplify small samples of nucleic acid to a detectable level. This has evoked a need of better techniques to assess the minute quantities of DNA or RNA. dPCR is more sensitive and reliable technique with improved ability to quantify the absolute amount of nucleic acid. It divides the sample into large number of fragments, each containing either one or no template nucleic acid sequence. After DNA amplification, scoring is done with the help of fluorescence, counting the score as positive for the fraction containing template sequence and negative for the sample without the template sequence. The major factors driving the global digital polymerase chain reaction market are increasing demand for innovative diagnostic techniques, increasing disease awareness, need for early diagnosis of viral, infectious and genetic ...
This ready-to-refer market intelligence report on global Polymerase Chain Reaction (PCR) market entails a detailed analysis of the industrial ecosystem, followed by a highly reliable segment overview evaluated on multi-factor analysis, market size and dimensions in terms of volumetric gains and returns.. Get sample copy of Polymerase Chain Reaction (PCR) Market report @ https://www.orbispharmareports.com/sample-request/78970. Competitive Landscape Detailed Analysis: * Followed by constant and thorough research initiatives in data unraveling process pertaining to global Polymerase Chain Reaction (PCR) market, stringent curation processes have been directed to understand growth prognosis and development spanning across regional hubs and their respective performance and evaluation in terms of various macro and micro elements that decide further growth prognosis in global Polymerase Chain Reaction (PCR) market ...
Read independent reviews on High Pure PCR Template Preparation Kit from Roche Applied Science - a member of the Roche Group on SelectScience
Background: Here we examined myocardial microRNA (miRNA) expression profile in a sensory neuropathy model with cardiac diastolic dysfunction and aimed to identify key mRNA molecular targets of the differentially expressed miRNAs that may contribute to cardiac dysfunction. Methods: Male Wistar rats were treated with vehicle or capsaicin for 3 days to induce systemic sensory neuropathy. Seven days later, diastolic dysfunction was detected by echocardiography, and miRNAs were isolated from the whole ventricles. Results: Out of 711 known miRNAs measured by miRNA microarray, the expression of 257 miRNAs was detected in the heart. As compared to vehicle-treated hearts, miR-344b, miR-466b, miR-98, let-7a, miR-1, miR-206, and miR-34b were downregulated, while miR-181a was upregulated as validated also by quantitative real time polymerase chain reaction (qRT-PCR). By an in silico network analysis, we identified common mRNA targets (insulin-like growth factor 1 (IGF-1), solute carrier family 2 facilitated ...
This provides single-stranded template for the next step temperature is remain about 94 -98°C. Repeated cycles of denaturation, primer annealling and extension carried out with the heat stable enzyme, Taq polymerase, leads to exponential increases in the target DNA sequences. And this is the sketch for the polymerase chain reaction. This process uses an enzyme derived from heat-resistant bacteria. 3.7. 1. heat to denature proteins (denaturation) ~98C 2. cool to anneal primers (short … By using this method you can amplify any region of gene which you want. Performing a Polymerase Chain Reaction 3. Scientist found T. aquaticus which lived in hot springs its DNA is most active at 70 degree thats way its DNA is most stable and become suitable enzyme for PCR used. Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using ...
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Major depressive disorder (MDD) is associated with dysfunctional serotonergic and glutamatergic neurotransmission, and the genetic animal model of depression Flinders Sensitive Line (FSL) rats display alterations in these ...
prevalence of falciparum malaria measured by qPCR (quantitative real time polymerase chain reaction), 12 months after the first administration of treatment with dihydroartemisinin-piperaquine and primaquine. (1017-13 and 23-15 ...
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Is your Thermal Cycler heating and cooling properly? Achieving and maintaining the pre-programmed temperature? Giving uniform performance across the whole block? Gel Company offers Thermocycle Testing Kits which contain all the reagents needed for quick and easy performance testing on your Thermal Cycler. ...
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Discover Swift Spectrum 48 Thermal Cycler, an advanced real-time thermal cycler, which uses diode activation, PMT detection and a proprietary Peltier block design.
A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms
TY - JOUR. T1 - Use of 16S ribosomal DNA polymerase chain reaction to identify Haemophilus influenzae type B as the etiology of pericarditis in an infant [5]. AU - Benson, Crystal. AU - Gantt, Soren. AU - Zerr, Danielle M.. AU - Qin, Xuan. AU - Abe, Patrick. N1 - Copyright: Copyright 2008 Elsevier B.V., All rights reserved.. PY - 2005/3. Y1 - 2005/3. UR - http://www.scopus.com/inward/record.url?scp=14944360668&partnerID=8YFLogxK. UR - http://www.scopus.com/inward/citedby.url?scp=14944360668&partnerID=8YFLogxK. U2 - 10.1097/01.inf.0000154593.22356.e2. DO - 10.1097/01.inf.0000154593.22356.e2. M3 - Letter. C2 - 15750478. AN - SCOPUS:14944360668. VL - 24. SP - 287. EP - 288. JO - Pediatric Infectious Disease Journal. JF - Pediatric Infectious Disease Journal. SN - 0891-3668. IS - 3. ER - ...
For our initial test, we constructed the donor vector pBS-yin(B40XC), consisting of an intronless yellow gene flanked by inverted attB40 sites in the plasmid pBluescript (pBS). In constructing this donor, we subcloned the attB40 sites using complementary oligonucleotides with overhanging sticky ends that permitted ligation with restriction sites in pBS. We then used pBS-yin(B40XC) as a donor vector for RMCE via two methods. First, we co-injected the vector and mRNA encoding the ΦC31 integrase into embryos homozygous for an RMCE target in a yellow− white− background as described previously (Bateman et al. 2006). Among the progeny of surviving injectees, we were able to detect flies that had lost the white+ eye color produced by the mini-white gene in the target cassette and had gained yellow+ pigmentation, consistent with successful RMCE integration events. Although rates of integration by this method were relatively low (2-11%), they were consistent with control experiments using the ...
Rodríguez, E.; Betancourt, A.; Relova, D.; Lee, C.; Yoo, D.; Barrera, M., 2012: Development of a nested polymerase chain reaction test for the diagnosis of transmissible gastroenteritis of pigs
TY - JOUR. T1 - Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy. AU - Bruzzone, Carol M.. AU - Belcher, John D.. AU - Schuld, Nathan J.. AU - Newman, Kristal A.. AU - Vineyard, Julie. AU - Nguyen, Julia. AU - Chen, Chunsheng. AU - Beckman, Joan D.. AU - Steer, Clifford J.. AU - Vercellotti, Gregory M.. PY - 2008/12. Y1 - 2008/12. N2 - Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR ...
In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusanum solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusanum genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1 alpha gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both ...
Background. Little is known about the type-specific prevalence of anal human papillomavirus (HPV) infection and risk factors for anal high-risk (HR) HPV infection in human immunodeficiency virus (HIV)-infected women. Methods. A cross-sectional study of anal and cervical HPV infection was nested within a gynecological cohort of HIV-infected women. Specimens were tested for type-specific DNA using a polymerase chain reaction-based assay. Results. The study population consisted of 311 women with a median age of 45.3 years, of whom 42.8% originated from sub-Saharan Africa and 96.8% were receiving combination antiretroviral therapy. The median CD4 + cell count was 612/μL, and the HIV load was |50 copies/mL in 84.1%. HR-HPV types were detected in the anal canal in 148 women (47.6%) and in the cervix in 82 (26.4%). HPV-16 was the most prevalent type in both the anal canal (13.2% of women) and the cervix (5.1%). In multivariable analysis, factors associated with prevalent anal HR-HPV infection were CD4 + count
Thiopurine S-methyltransferase (TPMT) is an enzyme that converts thiopurine drugs into inactive metabolites. It is now well established that interindividual variation in sensitivity to thiopurines can be the result of the presence of genetic polymorphisms in the TPMT gene. The aim of this study was to determine the frequency and type of TPMT polymorphisms in the population of Serbia and Montenegro and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Blood samples from 100 healthy adults and 100 children with ALL were analyzed for common mutations in the TPMT gene using polymerase chain reaction-based assays. The results revealed that allelic frequencies were 0.2% for TPMT*2, 3.2% for TPMT*3A, and 0.5% for TPMT*3B. A rare TPMT*3B allele was detected in 2 families. No TPMT*3C allele was found. The general pattern of TPMT-variant allele distribution as well as their frequencies in the population of Serbia and Montenegro, is similar to those determined for ...
The need to perform gene expression profiling using next generation sequencing and quantitative real-time PCR (qPCR) on small sample sizes and single cells is rapidly expanding. However, to analyse few molecules, preamplification is required. Here, we studied global and target-specific preamplification using 96 optimised qPCR assays. To evaluate the preamplification strategies, we monitored the reactions in real-time using SYBR Green I detection chemistry followed by melting curve analysis. Next, we compared yield and reproducibility of global preamplification to that of target-specific preamplification by qPCR using the same amount of total RNA. Global preamplification generated 9.3-fold lower yield and 1.6-fold lower reproducibility than target-specific preamplification. However, the performance of global preamplification is sufficient for most downstream applications and offers several advantages over target-specific preamplification. To demonstrate the potential of global preamplification we
The alternative oxidase (AOX) of the soybean (Glycine max L.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced from cDNA analysis, were found to be 38.1 and 36.4 kD, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 kD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein ...
BIO-410 Molecular Biology Techniques I (4.00). Introduces modern molecular biology techniques utilizing nucleic acids (DNA and RNA). Includes nucleic acid purification, quantitation, cloning and restriction enzyme digests. Advanced techniques include Southern and Northern analysis, polymerase chain reaction (PCR), real-time PCR and DNA sequencing. Stresses proficiency in techniques and proper analysis of results. Lab included. Credits: 4, Hours: (1/6/0/0), Arts & Sciences Elective Code: B. ...
Blake, D P and Qin, Z and Cai, J and Smith, A L (2008) Development and validation of real-time polymerase chain reaction assays specific to four species of Eimeria. Avian Pathology, 37 (1). pp. 89-94. Full text not available from this repository ...
Affiliations: 1: Department of Biological Pest Control and Quarantine, Institute of Plant Protection, Wladyslawa Wegorka 20, Poznan, 60-318, Poland;, Email: [email protected]; 2: Department of Biological Pest Control and Quarantine, Institute of Plant Protection, Wladyslawa Wegorka 20, Poznan, 60-318, Poland, Department of Genetics, University of Valencia, Dr. Moliner 50, 46-100, Burjassot, Spain; 3: Department of Biological Pest Control and Quarantine, Institute of Plant Protection, Wladyslawa Wegorka 20, Poznan, 60-318, Poland ...
Background: DNA polymerases (Pols) represent potential candidates for cancer genes because of their central functions in DNA metabolism. Defects of some DNA Pols have shown cancer associations, but data on DNA polymerase (Pol) ε is limited. Materials and Methods: Twenty-four human breast cancer DNA samples and four control DNA samples were examined for possible mutation in the entire coding region of the 55 kDa small subunit of the human DNA Pol ε gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the DNA and sequence analysis. In addition, 20 control DNAs were studied with PCR-SSCP for the end of intron 18 and exon 19 region. Results: An AATT deletion was found at one location in intron 18 in 2 out of the 24 breast cancer cases (8%), but in none of the control cases. In addition, a single base transition was found in the cancer DNAs in intron 14, but the same changes were also found in the control DNAs, suggesting polymorphism. Conclusion: ...
Reagent Guide Applied Biosystems StepOne and StepOnePlus Real-Time PCR Systems Applied Biosystems StepOne and StepOnePlus Real-Time PCR Systems Reagent Guide Copyright 2008, 2010 Applied Biosystems. All
TY - JOUR. T1 - An accurate and rapid gender determination assay in single cells by the capillary polymerase chain reaction method. AU - Hashiba, Tsuyoshi. AU - Sueoka, Kou. AU - Kuroshima, Masako. AU - Asada, Hironori. AU - Kuji, Naoaki. AU - Yoshimura, Yasunori. N1 - Copyright: Copyright 2007 Elsevier B.V., All rights reserved.. PY - 1999. Y1 - 1999. N2 - Purpose: In preimplantation genetic diagnosis (PGD), a rapid and accurate assay has been required. We have therefore developed a capillary palymerase chain reaction (PCR) method using rapid thermal cycling programs to determine the gender of single amniocytes. Methods: Single amniocytes from each amniotic fluid sample were isolated by micromanipulation and their gender was determined by a multiplex PCR assay in a capillary tube, using primers that amplify a 308-bp DXZI and a 154-bp DYZI repeat sequence on the X and Y chromosomes, respectively. Results: All four thermal cycling programs, which took 180, 150, 120, and 90 min, were 100% accurate ...
We evaluated the clinical usefulness of spoligotyping, a polymerase chain reaction-based method for simultaneous detection and typing of Mycobacterium tuberculosis strains, with acid-fast bacilli-positive slides from clinical specimens or mycobacterial cultures. Overall sensitivity and specificity were 97% and 95% for the detection of M. tuberculosis and 98% and 96% when used with clinical specimens. Laboratory turnaround time of spoligotyping was less than that for culture identification by a median of 20 days. In comparison with IS6110-based restriction fragment length polymorphism typing, spoligotyping overestimated the number of isolates with identical DNA fingerprints by ≈50%, but showed a 100% negative predictive value. Spoligotyping resulted in the modification of ongoing antimycobacterial treatment in 40 cases and appropriate therapy in the absence of cultures in 11 cases. The rapidity of this method in detection and typing could make it useful in the management of tuberculosis in a clinical
We evaluated the clinical usefulness of spoligotyping, a polymerase chain reaction-based method for simultaneous detection and typing of Mycobacterium tuberculosis strains, with acid-fast bacilli-positive slides from clinical specimens or mycobacterial cultures. Overall sensitivity and specificity were 97% and 95% for the detection of M. tuberculosis and 98% and 96% when used with clinical specimens. Laboratory turnaround time of spoligotyping was less than that for culture identification by a median of 20 days. In comparison with IS6110-based restriction fragment length polymorphism typing, spoligotyping overestimated the number of isolates with identical DNA fingerprints by approximately 50%, but showed a 100% negative predictive value. Spoligotyping resulted in the modification of ongoing antimycobacterial treatment in 40 cases and appropriate therapy in the absence of cultures in 11 cases. The rapidity of this method in detection and typing could make it useful in the management of ...
AccuPrep® Genomic DNA Extraction Kit for Biovac 96 Vacuum Manifold has been designed to quickly and conveniently extract genomic DNA from whole blood, buffy coat, lymphocytes, plasma, serum, body fluids, and cultured cells, simultaneously. The 96 samples can be handled without additional machinery such as a centrifuge. The genomic DNA is simply extracted with a vacuum pump and Biovac 96 Vacuum Manifold. This product is also available to other companies vacuum manifold system (QIAGEN, Promega and Axygen).
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
Approach and Results-The LPA null allele (rs41272114) was genotyped in the PROCARDIS case-control cohort (4073 CAD cases and 4225 controls). Lipoprotein(a) levels were measured in 909 CAD cases and 922 controls; apolipoprotein(a) isoform size was estimated using sodium dodecyl sulfate-agarose gel electrophoresis and a high-throughput quantitative polymerase chain reaction-based method. Null carriers are common (null allele frequency, 3%) and have significantly lower circulating lipoprotein(a) levels (P=2.1×10−10) and reduced CAD risk (odds ratio, 0.79 [0.66-0.97]; P=0.023) compared with noncarriers. An additive allelic model of apolipoprotein(a) isoform size, refined by null allele genotype and quantitative polymerase chain reaction values, showed a sigmoid relationship with lipoprotein(a) levels, with baseline levels for longer isoform alleles and progressively higher levels of lipoprotein(a) for shorter isoform alleles.. ...
Two rapid methods for the detection of cytomegalovirus (CMV) in saliva from congenitally and perinatally infected children were assessed by comparison with traditional virus isolation in tissue culture (TC). The polymerase chain reaction (PCR) was used to amplify a 300-bp segment of the CMV gB gene which was detected in ethidium bromide-stained agarose gels. A centrifugation-enhanced microtiter culture method with a monoclonal antibody for the detection of early-antigen fluorescent foci (DEAFF) was also used. Saliva specimens were collected with mouth swabs from children who were between the ages of 1 month and 14 years and who had either prenatal or perinatal CMV infection. One hundred sixty samples were tested by PCR and TC; 65 (40.6%) were found positive by TC, and 58 (36.8%) were found positive by PCR. Although four samples were found positive by PCR and negative by TC, saliva from seronegative and seropositive TC-negative adults were never found positive by PCR. One hundred fifty-two ...
Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL− 1 or g− 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In
Spoligotyping and Mycobacterium tuberculosis. Gori, Andrea; Bandera, Alessandra; Marchetti, Giulia; Esposti, Anna Degli; Catozzi, Lidia; Nardi, Gian Piero; Gazzola, Lidia; Ferrario, Giulio; Van Embden, Jan D. A.; Van Soolingen, Dick; Moroni, Mauro; Franzetti, Fabio // Emerging Infectious Diseases;Aug2005, Vol. 11 Issue 8, p1242 We evaluated the clinical usefulness of spoligotyping, a polymerase chain reaction-based method for simultaneous detection and typing of Mycobacterium tuberculosis strains, with acid-fast bacilli-positive slides from clinical specimens or mycobacterial cultures. Overall sensitivity and... ...
Microscopy and Polymerase Chain Reaction technique was used to evaluate 150 blood samples from cattle in three abattoirs in Kaduna State to detect the presence of Trypanosoma vivax. Out of the 150 blood samples collected and tested for the presence of T. vivax using Microscopy,13 samples from Tudun-wada, Kawo and Makera abattoirs were found to positive giving a total prevalence of 26.0%. Tudun-wada abattoir had the highest prevalence with 16.0% while 3.0% and 2.0% for Kawo and Makera abattoirs respectively. Tudun- wada abattoir at 0.016 was seen to be significantly different at p. Key words: Abattoir, DNA, Microscopy, PCR, Primer, T. vivax. ...
OBJECTIVE: Increased levels of lipoprotein(a) are a highly heritable risk factor for coronary artery disease (CAD). The genetic determinants of lipoprotein(a) levels are mainly because of genetic variation in the apolipoprotein(a) gene (LPA). We have tested the association of a relatively common null allele of LPA with lipoprotein(a) levels and CAD risk in a large case-control cohort. We have also examined how null allele genotyping complements apolipoprotein(a) isoform typing to refine the relationship between LPA isoform size and circulating lipoprotein(a) levels. APPROACH AND RESULTS: The LPA null allele (rs41272114) was genotyped in the PROCARDIS (Precocious Coronary Artery Disease) case-control cohort (4073 CAD cases and 4225 controls). Lipoprotein(a) levels were measured in 909 CAD cases and 922 controls; apolipoprotein(a) isoform size was estimated using sodium dodecyl sulfate-agarose gel electrophoresis and a high-throughput quantitative polymerase chain reaction-based method. Null carriers are
Download Free Full-Text of an article DEVELOPMENT OF TWO MULTIPLEX POLYMERASE CHAIN REACTIONS FOR THE DETECTION OF ENTEROTOXIGENIC STRAINS OF STAPHYLOCOCCUS AUREUS ISOLATED FROM FOODS
TY - JOUR. T1 - Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon. AU - Zheng, Lu. AU - Gao, Naiyun. AU - Deng, Yang. PY - 2012/2/1. Y1 - 2012/2/1. N2 - It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length ...
Article: Pourahmad F, Adams A, Thompson K & Richards R (2009) Comparative evaluation of Polymerase Chain Reaction - Restriction Enzyme Analysis (PRA) and Sequencing of Heat shock protein 65 (hsp65) gene for identification of aquatic mycobacteria. Journal of Microbiological Methods, 76 (2), pp. 128-135. http://www.sciencedirect.com/science/journal/01677012; https://doi.org/10.1016/j.mimet.2008.09.021
TY - JOUR. T1 - A consensus on fungal polymerase chain reaction diagnosis?. T2 - A United Kingdom-Ireland evaluation of polymerase chain reaction methods for detection of systemic fungal infections. AU - White, P. Lewis. AU - Barton, Richard. AU - Guiver, Malcolm. AU - Linton, Christopher J.. AU - Wilson, Steve. AU - Smith, Melvyn. AU - Gomez, Beatriz L.. AU - Carr, Michael J.. AU - Kimmitt, Patrick T.. AU - Seaton, Shila. AU - Rajakumar, Kumar. AU - Holyoake, Tessa. AU - Kibbler, Chris C.. AU - Johnson, Elizabeth. AU - Hobson, Richard P.. AU - Jones, Brian. AU - Barnes, Rosemary A.. N1 - Copyright: Copyright 2017 Elsevier B.V., All rights reserved.. PY - 2006/7. Y1 - 2006/7. N2 - The limitations of classical diagnostic methods for invasive fungal infections (IFIs) have led to the development of molecular techniques to aid in the detection of IFIs. Despite good published performance, interlaboratory reproduction of these assays is variable, and no consensus has been reached for an optimal ...
The Department of Civil and Environmental Engineering at Florida State University invites applications for a postdoctoral research associate position. The postdoc will work in a landfill leachate project. The ideal candidate should have a Ph.D. degree in Environmental Engineering or closely related fields. Candidates with previous experience in two or more of the following four areas are particularly encouraged to apply: 1) biological treatment of landfill leachate or wastewater; 2) geosynthetic clay liners; 3) quantitative real time polymerase chain reaction and next generation sequencing; 4) analytical techniques including SEM, TEM, XRD, Raman, IC, and TOC.. Review of applications will begin immediately, and will continue until the position is filled. The position is available immediately for a duration of one and a half year with the possibility of renewal based on satisfactory performance and funding. Interested applicants please contact [email protected] with a one-page cover letter, a CV, a ...
Yes, the Monarch Genomic DNA Purification Kit can be used to clean up phenol/chloroform purified gDNA by following the protocol for Genomic DNA Cleanup. However, recovery may be lower than the usual 80% because phenol/chloroform purified DNA typically yields longer fragments. Although the use of preheated elution buffer in the Monarch protocol facilitates the elution of large gDNA fragments, the fraction of gDNA that is longer than 80 kb will be eluted less efficiently from the silica matrix ...
Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, ...
TY - JOUR. T1 - Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples. AU - Ramírez, Juan David. AU - Herrera, Giovanny. AU - Hernández, Carolina. AU - Cruz-Saavedra, Lissa. AU - Muñoz, Marina. AU - Flórez, Carolina. AU - Butcher, Robert. PY - 2018/12/1. Y1 - 2018/12/1. N2 - Background: The recent development of novel Polymerase Chain Reaction (PCR) technologies that confer theoretical advantages over quantitative PCR has considerable potential in the diagnosis of low load infections, such as Trypanosoma cruzi in the chronic phase of Chagas disease. We evaluated the utility of the digital droplet (dd)PCR platform in the detection of T. cruzi infection. Methodology/Principal findings: We imported a validated qPCR assay targeting the T. cruzi satellite tandem repeat (TcSTR) region to the ddPCR platform. Following optimization, we tested and repeated a standard curve of TcI ...
This study prospectively evaluated an 18S rRNA gene-targeted real-time PCR approach in comparison with standard blood culture (BC) diagnostics for rapid diagnosis of candidaemia in a large study population of 384 patients, including 902 whole blood samples from 468 infectious episodes (IEs) of 329 adults and 55 children with haematological malignancies and various forms of immunodeficiency, and intensive care unit patients. Seven out of eight BC-proven cases (87.5 %) of candidaemia and seven out of twelve BC-positive samples (58.3 %) were positive by the Candida-specific PCR. A positive PCR result was also obtained for 28/460 BC-negative samples from IEs, including 8 patients with culture-confirmed Candida infection at primary sterile body sites. Of the PCR-positive, culture-negative patients, more than 50 % received systemic antifungal therapy. In 432/460 BC-negative IEs, the Candida specific-PCR was negative, resulting in a negative predictive value of 99.8 %. In conclusion, the Candida specific-PCR
Accuracy of genotyping of single-nucleotide polymorphisms by PCR-ELISA allele-specific oligonucleotide hybridization typing and by amplification refractory mutation ...
The Real-time Polymerase Chain Reaction is an improvised version of the original [[Polymerase Chain Reaction,Polymerase Chain Reaction]] (PCR,u,),/u, developed by Kary Mullis, who received the Nobel Prize in 1993 in Chemistry, and her coworkers during the mid-1980s. ,sup,(1),/sup ...
Polymerase Chain Reaction (PCR) based detection methods have received significant attention in food borne microbial pathogen detection. However, reliability and sensitivity of these methods are highly depending on the extraction of adequate amount of pure DNA using appropriate extraction method. Hence, selection of appropriate DNA extraction method is very important in PCR based detection of microbial pathogens. In this study, the extraction efficiency of five commonly used DNA extraction methods was evaluated. Salmonella enterica was used as experimental organism and five extraction methods were tested for their ability to extract DNA from spiked pork meat samples. Pork meat samples were incubated for four hours after being added a dilution series (100 - 103 CFU/mL) of Salmonella enterica culture. Then DNA was extracted from those samples by the five commonly used DNA extraction methods. Using extracted DNA, fliC gene of Salmonella was amplified by Nested PCR. Out of those five methods, the ...
Blood donations are routinely screened by multiple serologic assays for antigens/antibodies associated with infection by blood-borne viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1 and HIV-2), and human T-cell lymphotropic virus (HTLV-I and HTLV-II). A direct detection of these viruses would be more effective for the prevention of transfusion-transmitted infections than the indirect measurement of the variable host immune response to these agents. Because the polymerase chain reaction (PCR) for viral gene amplification offers the most sensitive and direct means of detecting viruses in blood, we have developed a nonisotopic PCR procedure for the detection of HBV, chosen as a prototype. The problems, common to previously described PCR methods, of nucleic acid extraction and inhibition of the PCR by plasma proteins were overcome by isolation of HBV from plasma by means of 450-microns polystyrene beads covalently coated with monoclonal antibody to
TY - JOUR. T1 - Real-time quantitative reverse transcriptase polymerase chain reaction.. AU - Fan, Hongxin. AU - Robetorye, Ryan S.. PY - 2010. Y1 - 2010. N2 - The real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) has become the method of choice for the quantification of specific mRNAs. This method is fast, extremely sensitive, and accurate, requires only very small amounts of input RNA, and is relatively simple to perform. These characteristics have made it the method of choice for minimal residual disease monitoring such as in chronic myelogenous leukemia (CML). CML comprises approximately 20% of all leukemias and is characterized by a balanced (9;22) chromosomal translocation that results in the formation of a chimeric gene comprised of the BCR (breakpoint cluster region) gene and the ABL oncogene (BCR-ABL fusion gene). The chimeric gene encodes a fusion protein with constitutively increased tyrosine kinase activity, resulting in growth factor-independent ...
Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2-deoxyribonucleoside 5-O-1-thiotriphosphates in the sequencing reactions.. ...
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Background. Although high risk HPVs are associated with an increased risk of prostate cancer it is not known if they have a causal role. The purpose of this study is to investigate the potential role of human papilloma viruses (HPVs) in prostate cancer. The aims are (i) to investigate the presence and confirm the identity of high risk HPVs in benign prostate tissues prior to the development of HPV positive prostate cancer in the same patients, and (ii) to determine if HPVs are biologically active.. Methods. We used polymerase chain reaction (PCR) to identify HPVs in specimens from 52 Australian men with benign prostate biopsies who 1 to 10 years later developed prostate cancer. Immunohistochemistry (IHC) was used to assess the expression of HPV E7 oncoproteins, cytokeratin and prostate specific antigen (PSA).. We used RNASeq data from The Cancer Genome Atlas (TCGA) to identify possible HPV RNA sequences in prostate cancer.. Results. HPV screening using standard PCR was conducted on 28 of the 52 ...
Quantitative competitive polymerase chain reaction (QC-PCR) methods were used to quantify virion-associated human immunodeficiency virus type-1 (HIV-1) RNA in plasma from 66 patients with Centers for Disease Control stage I to IVC1 infection. HIV-1 RNA, ranging from 100 to nearly 22,000,000 copies per milliliter of plasma (corresponding to 50 to 11,000,000 virions per milliliter), was readily quantified in all subjects, was significantly associated with disease stage and CD4+ T cell counts, and decreased by as much as 235-fold with resolution of primary infection or institution of antiretroviral therapy. Plasma virus levels determined by QC-PCR correlated with, but exceeded by an average of 60,000-fold, virus titers measured by endpoint dilution culture. Quantitation of HIV-1 in plasma by QC-PCR may be useful in assessing the efficacy of antiretroviral agents, especially in early stage disease when conventional viral markers are often negative. ...
The asexual root-knot nematodes (RKNs) (Meloidogyne spp.) exemplified by Meloidogyne incognita are widespread and damaging pests in tropical and subtropical regions worldwide. Comparison of amplification products of two adjacent polymorphic regions of the mitochondrial genome using DNA extracts of characterized RKN strains, including 15 different species, indicate that several species are derived from the same or closely related female lineages. Nevertheless, M. javanica, M. enterolobii, M. incognita, and other key species could each be assigned unique mitochondrial haplotypes based on polymerase chain reaction fragment size and restriction cleavage patterns. M. arenaria isolates did not group as a single haplotype, consistent with other reports of diversity within this species. To test the utility of this assay, we characterized ethanol-preserved samples from 103 single-species isolates from four countries in sub-Saharan Africa (Benin, Nigeria, Kenya, and Tanzania). Mitochondrial haplotypes ...
TY - JOUR. T1 - Absence of retroviral sequences in Graves disease. AU - Humphrey, M.. AU - Carr, F. E.. AU - Wartofsky, L.. AU - Djuh, Y. Y.. AU - Burman, K. D.. AU - Baker, J. R.. AU - Mosca, J.. AU - Drabick, J. J.. AU - Burke, D. S.. PY - 1991/1/5. Y1 - 1991/1/5. N2 - An earlier report of HIV-1 gene sequences in thyroid cell genomic DNA from patients with Graves disease prompted use of the polymerase chain reaction technique to identify such sequences in Graves disease thyroid tissue and in white blood-cells from these patients. We were unable to confirm the existence of HIV-1-related DNA sequences in Graves specimens.. AB - An earlier report of HIV-1 gene sequences in thyroid cell genomic DNA from patients with Graves disease prompted use of the polymerase chain reaction technique to identify such sequences in Graves disease thyroid tissue and in white blood-cells from these patients. We were unable to confirm the existence of HIV-1-related DNA sequences in Graves specimens.. UR - ...