Partial sequence of the 24S rRNA and polymerase chain reaction based assay for the toxic dinoflagellateDinophysis acuminata

1998 ◽  
Vol 55 (3) ◽  
pp. 597-604 ◽  
Author(s):  
O Puel ◽  
F Galgani ◽  
C Dalet ◽  
P Lassus
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Pcr Product ◽  
Dinophysis Acuminata ◽  
Base Pair Fragment ◽  
Variable Domains ◽  
Polymerase Chain ◽  
Seawater Samples ◽  
Pair Fragment

We describe the polymerase chain reaction (PCR) amplification of an 805 base pair fragment of 24S rRNA from the toxic dinoflagellate Dinophysis acuminata and the sequence of this fragment. We also describe a PCR-based assay for the specific detection of D. acuminata in seawater samples. Conserved primers, starting at positions 711 and 1489 of the 24S rRNA from the dinoflagellate Prorocentrum micans, were used for the PCR. The PCR product was cloned and sequenced. The fragment was aligned with rRNA sequences from other protists. Two oligonucleotides in variable domains of the sequence from D. acuminata were chosen and a protocol was defined for PCR-based detection of D. acuminata (30 cycles, 50°C). Experiments conducted with seawater samples led to the detection of D. acuminata in naturally contaminated samples. The PCR enabled us to detect down to 30 cells/L seawater. The problem of interference from large concentrations of other phytoplankton species may be solved using nested PCR.

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 Species-Specific Polymerase Chain Reaction Amplification of Camel (Camelus) DNA Extracts

2005 ◽  
Vol 88 (5) ◽  
pp. 1394-1398 ◽  
Author(s):  
Ying Chen ◽  
Ya-Jun Wu ◽  
Bao-Liang Xu ◽  
Jing Wan ◽  
Zeng-Ming Qian
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Amplification ◽  
Chain Reaction ◽  
Pcr Product ◽  
Mitochondrial Cytochrome B ◽  
Base Pair Fragment ◽  
Sensitive Polymerase Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Species Specific

Abstract A sensitive polymerase chain reaction (PCR) method based on amplification of a specific DNA fragment was established for the identification of camel (Camelus) materials. The species-specific primer pair L183/H372 was designed based on the nucleotide sequence of the mitochondrial cytochrome b gene, and its specificity was confirmed by amplification of 3 camel (domestic double-humped camel, wild double-humped camel, wild one-humped camel) samples and 11 non-Camelus animal (sheep, goat, pig, chicken, cattle, fish, dog, horse, donkey, deer, and rabbit) materials. An expected 208 base pair fragment was amplified from camel materials; no cross-reactive or additional fragments were generated from other animal materials. Taq I restriction endonuclease digestion of the unpurified PCR product can be used routinely to confirm the camel origin of the amplified sequence.

scholarly journals Cytomegalovirus is not specifically associated with immunoglobulin A nephropathy.

1994 ◽  
Vol 4 (8) ◽  
pp. 1623-1626
Author(s):  
J S Park ◽  
J H Song ◽  
W S Yang ◽  
S B Kim ◽  
Y K Kim ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Immunoglobulin A ◽  
Renal Tissue ◽  
Early Gene ◽  
Chain Reaction ◽  
Indirect Immunofluorescence Staining ◽  
Base Pair Fragment ◽  
Polymerase Chain ◽  
Pair Fragment ◽  
Frequent Observation

Cytomegalovirus (CMV) has been suspected to be involved in the pathogenesis of IgA nephropathy (IgAN). Whether CMV is present in renal tissue of IgAN, however, remains controversial. To determine the presence of CMV in IgAN, compared with other glomerulonephritis (GN) as disease control, polymerase chain reaction amplifying a 159-base-pair fragment of the immediate early gene of CMV and indirect immunofluorescence staining with anti-CMV monoclonal antibody were performed on 10 IgAN and 14 non-IgAN GN renal tissues. CMV DNA was detected in 6 of 10 IgAN tissues and 10 of 14 other GN by polymerase chain reaction, whereas no CMV antigen was detected in all renal tissues by immunofluorescence. This frequent observation of CMV DNA in various types of GN as well as in IgAN would suggest that CMV is not specifically associated with the pathogenesis of IgAN seen in endemic areas of CMV infection.

scholarly journals Sex Identification in Cattle, based on Amelogenin Gene

2021 ◽  
Author(s):  
Aftab Ahmad ◽  
Muhammad Israr ◽  
Murad Ali Rahat ◽  
Adnan Wahab ◽  
Subhan Uddin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Base Pair ◽  
Sex Identification ◽  
Polymerase Chain Reaction Test ◽  
Chain Reaction ◽  
Reliable Technique ◽  
Amelogenin Gene ◽  
Base Pair Fragment ◽  
Polymerase Chain ◽  
Pair Fragment

Sex identification is considered an important step in forensic sciences, wildlife and livestock breeding management. In the current experiment we used Amelogenin gene as a biological marker for polymerase chain reaction test to identify the sex of cattle from blood remnants, collected at slaughter house. Due to the conserved region of the gene on both sex chromosomes (X and Y) a single primers pair was employed to amplify the gene in a single polymerase chain reaction. In case of band patterns, a 178 base pair fragment for AMELY and a 241 base pair fragment for AMELX genes were produced. The primer competence and exactness was initially checked on known gender cattle samples and then applied to unknown cattle samples for the validation of the experiment. PCR amplicons of unknown gender showed only one band (241-bp) for female DNA and two bands (241-bp, 178-bp) for male DNA, on the platform of agarose gel upon electrophoresis. Our findings showed that the PCR protocol based on AMELX or Y gene is a reliable technique for the identification of cattle sex.

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

scholarly journals A Self-Contained Portable Polymerase-Chain-Reaction System Integrated with Electromagnetic Mini-Actuators for Bi-Directional Fluid Transport

2011 ◽  
Vol 27 (3) ◽  
pp. 357-364
Author(s):  
B. T. Chia ◽  
S.-A. Yang ◽  
M.-Y. Cheng ◽  
C.-W. Lin ◽  
Y.-J. Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Fluid Transport ◽  
Point Of Care ◽  
Reaction System ◽  
Thermal Characteristics ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Rapid Temperature ◽  
Small Footprint ◽  
Polymerase Chain

ABSTRACTIn this paper, the development of a portable polymerase chain reaction (PCR) device is presented. Integrating electromagnetic mini-actuators for bi-directional fluid transport, the proposed device, whose dimension is 67mm × 66mm × 25mm, can be fully operated with a 5V DC voltage. The device consists of four major parts: A disposable channel chip in which PCR mixture is manipulated and reacted, a heater chip which generates different temperature zones for PCR reaction, a linear actuator array for pumping PCR mixture, and a circuit module for controlling and driving the system. The advantages of the device include the rapid temperature responses associated with continuous-flow-type PCR devices, as well as the programmable thermal cycling associated with chamber-type PCR devices. The thermal characteristics are measured and discussed. PCR amplification is successfully performed for the 122 bp segment of MCF-7/adr cell line. Due to its small footprint, this self-contained system potentially can be employed for point-of-care (POC) applications.

scholarly journals Suitability of different tissue fixatives for subsequent PCR analysis of Cysticercus ovis

2012 ◽  
Vol 49 (2) ◽  
pp. 67-70 ◽  
Author(s):  
M. Kolesárová ◽  
R. Herich ◽  
M. Levkut ◽  
J. Čurlík ◽  
M. Levkut
Keyword(s):  
Retrospective Studies ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Fresh Tissue ◽  
Carnoy’S Solution ◽  
Negative Results ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Cysticercus Ovis

AbstractPCR amplification of specific DNA regions is a powerful tool for retrospective studies, but not all preservation or fixation methods render DNA that is suitable for subsequent amplification. Several factors affect sensitivity of polymerase chain reaction (PCR) amplification. There were reported the effects of commonly used fixation solutions — 10 % neutral buffered formalin, 20 % neutral buffered formalin and Carnoy’s solution and the efficiency of PCR amplification in fresh tissue and paraffin (or wax) embedded samples of Cysticercus ovis. DNA from samples was isolated and PCR product of 1300 bp was amplified. Results indicated that the samples fixed in Carnoy’s solution produced reliable amplification of desired fragments. The samples that were fixed in 10 % and 20 % neutral buffered formalin brought negative results.

Sign in / Sign up

Export Citation Format

Share Document