scholarly journals Simplified Algorithms for Determining Cycle Shift between qPCR Fluorescence Curves

2015 ◽  
Vol 2015 ◽  
pp. 1-6
Author(s):  
Michael E. Jones ◽  
George C. Mayne ◽  
Tingting Wang ◽  
David I. Watson ◽  
Damian J. Hussey
Keyword(s):  
Fold Increase ◽  
Delayed Fluorescence ◽  
Cyclic Process ◽  
Central Component ◽  
Simple Computation ◽  
Chain Reaction ◽  
Two Samples ◽  
Polymerase Chain ◽  
Template Dna ◽  
Current Guidelines

The polymerase chain reaction is a central component of current molecular biology. It is a cyclic process, in each early cycle of which the template DNA approximately doubles. An indicator which fluoresces when bound to DNA quantifies the DNA present at the end of each cycle, giving rise to a fluorescence curve which is characteristically sigmoid in shape. The fluorescence curve quantifies the amount of DNA initially present; the more the initial DNA, the earlier the rise in the fluorescence. Accordingly the amount of DNA initially present in two samples can be compared: the sample with the less DNA gives rise to a relatively delayed fluorescence curve and the ratio of the DNAs can be deduced from the separation of the curves. There is, however, a second determinant of this separation, the fold increase in DNA per cycle: ideally a twofold increase but frequently less. Current guidelines recommend that this be determined experimentally by carrying out PCR on a series of dilutions. If the value of the fold increase is known, then the algorithm for determining the separation can be reduced to a relatively simple computation, rather than employing a multidimensional nonlinear optimization such as the Marquardt-Levenberg as currently employed.

scholarly journals A method for mapping germ line sequences in Tetrahymena thermophila using the polymerase chain reaction.

Genetics ◽  
1994 ◽  
Vol 137 (1) ◽  
pp. 95-106 ◽  
Author(s):  
D Cassidy-Hanley ◽  
M C Yao ◽  
P J Bruns
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Repetitive Sequences ◽  
Mapping Technique ◽  
Ciliated Protozoan ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Template Dna

Abstract A method for mapping DNA sequences to specific germinal chromosomes in the ciliated protozoan Tetrahymena thermophila has been developed. This mapping technique (PCR mapping) utilizes the polymerase chain reaction and template DNA derived from nullisomic strains to directly assign micronuclear DNA sequences to specific micronuclear chromosomes. Using this technique, a number of unique sequences and short repetitive sequences flanked by unique sequences have been mapped to four of the five germinal chromosomes.

scholarly journals Potentially a new subtype of the cytoplasmic male sterility S- type in maize

Genetika ◽  
2013 ◽  
Vol 45 (1) ◽  
pp. 145-151
Author(s):  
Jelena Vancetovic ◽  
Dragana Ignjatovic-Micic ◽  
Ana Nikolic ◽  
Sofija Bozinovic ◽  
Ksenija Markovic ◽  
...  
Keyword(s):  
Cytoplasmic Male Sterility ◽  
Male Sterility ◽  
Multiplex Polymerase Chain Reaction ◽  
Laboratory Experiments ◽  
Gene Bank ◽  
Chain Reaction ◽  
Additional Band ◽  
Two Samples ◽  
Polymerase Chain ◽  
Maize Research Institute

In gene-bank maize collection of Maize Research Institute Zemun Polje (MRI) two samples with untypical mtDNA profile for cytoplasmic male sterility (cms) were identified. These two samples showed typical multiplex polymerase chain reaction (PCR) band for cms-S, but also an additional band of unknown nature. It is assumed that the additional band is the result of a rearrangement of the two mitochondrial episomes characteristic for the cms-S in maize or a duplication of the part of cms-S mitochondrial genome. Additional field and laboratory experiments are necessary in the further lightening of this phenomenon.

scholarly journals Occurrence of zoonotic Enterocytozoon bieneusi in cats in Brazil

2019 ◽  
Vol 28 (1) ◽  
pp. 80-90 ◽  
Author(s):  
Jamille Batista Faria Prado ◽  
Carlos Alberto do Nascimento Ramos ◽  
Vagner Ricardo da Silva Fiuza ◽  
Veronica Jorge Babo Terra
Keyword(s):  
Enterocytozoon Bieneusi ◽  
Domestic Cat ◽  
Domestic Cats ◽  
Chain Reaction ◽  
Mato Grosso ◽  
Source Of Infection ◽  
Two Samples ◽  
Intestinal Pathogen ◽  
Polymerase Chain ◽  
Mato Grosso Do Sul

Abstract Enterocytozoon bieneusi is an opportunistic intestinal pathogen that infects humans and a wide variety of animals worldwide. Our aim in this study was to investigate the occurrence of E. bieneusi in a domestic cat population in Campo Grande, Mato Grosso do Sul, Brazil. Sixty fecal samples from diarrheic cats were subjected to polymerase chain reaction (PCR) and the amplicons were sequenced for identification. E. bieneusi was detected in two samples (3.3%), both identified as genotype D. This genotype has already been reported in animals and humans and is considered a zoonotic genotype. Our findings represent the first report of E. bieneusi in domestic cats in Brazil, reinforcing the importance of identifying this agent as a source of infection in animals and humans.

scholarly journals Presence of Arcobacter species in pet cats and dogs in the Czech Republic

2017 ◽  
Vol 61 (No. 8) ◽  
pp. 449-455
Author(s):  
M. Pejchalova ◽  
S. Zabcikova ◽  
L. Silhova ◽  
D. Silha ◽  
I. Brozkova ◽  
...  
Keyword(s):  
Czech Republic ◽  
Gel Electrophoresis ◽  
Dna Isolation ◽  
Human Infection ◽  
Chain Reaction ◽  
The Czech Republic ◽  
Two Samples ◽  
Arcobacter Butzleri ◽  
Polymerase Chain

This study was conducted to evaluate the occurrence of the genus Arcobacter in cats and dogs in the Czech Republic. These animals may be carriers of the bacteria and potential sources of human infection. Oral smears were collected from animals using smear swabs and brushes. Based on previous studies, commercially available DNA kits were used for DNA isolation. Samples were analysed using polymerase chain reaction (PCR) and evaluated using gel electrophoresis. Overall, 178 oral smears were tested, of which 108 were from dogs and 70 were from cats. Out of all smears, five were positive, of which four samples were from dogs and one from a cat. In all five positive cases, PCR confirmed the presence of Arcobacter butzleri. In follow-up sampling, the presence of Arcobacter butzleri was demonstrated in two samples from a dog.

scholarly journals Evidence for insect transmission of rabbit haemorrhagic disease virus

2002 ◽  
Vol 129 (3) ◽  
pp. 655-663 ◽  
Author(s):  
K. A. McCOLL ◽  
J. C. MERCHANT ◽  
J. HARDY ◽  
B. D. COOKE ◽  
A. ROBINSON ◽  
...  
Keyword(s):  
Laboratory Experiments ◽  
Infectious Virus ◽  
Fold Increase ◽  
Field Trials ◽  
Rabbit Haemorrhagic Disease Virus ◽  
Chain Reaction ◽  
Rabbit Haemorrhagic Disease ◽  
Insect Populations ◽  
Polymerase Chain ◽  
Haemorrhagic Disease

The spread of rabbit haemorrhagic disease (RHD) virus from quarantine on Wardang Island to mainland Australia in 1995 suggested that insects could be potential vectors. Field observations and laboratory experiments were conducted to address aspects of this hypothesis. Firstly, the variation in insect populations on the island during the field trials was examined. There was approximately a 1000-fold increase in the number of bushflies, Musca vetustissima, shortly before the spread of the virus. Secondly, M. vetustissima were tested in the laboratory as potential vectors of RHD virus, and it was demonstrated that disease could be transmitted between rabbits by flies. Finally, 13 of 16 insect samples, collected from Wardang Island and from several sites on the mainland following the spread of virus off the island, were positive for the presence of RHD virus by a specific polymerase chain reaction (PCR). Only one sample contained sufficient infectious virus to kill a susceptible rabbit. These data, combined with previously published information on fly biology, suggested that flies, particularly bushflies, may be involved in the transmission of RHD virus. Other possible routes of spread were not assessed in this study.

scholarly journals PCR detection of Bartonella spp. in the dog

2014 ◽  
Vol 83 (2) ◽  
pp. 79-82 ◽  
Author(s):  
Jarmila Konvalinová ◽  
Vlasta Svobodová ◽  
Dobromila Molinková ◽  
Miroslav Svoboda
Keyword(s):  
Polymerase Chain Reaction ◽  
Czech Republic ◽  
Clinical Symptoms ◽  
Bartonella Henselae ◽  
Chain Reaction ◽  
The Czech Republic ◽  
Two Samples ◽  
Healthy Dogs ◽  
Polymerase Chain ◽  
First Time

Our study aimed at using PCR to identify the incidence ofBartonellaspp. in blood of dogs. Altogether 286 dogs of 92 breeds aged 3 month to 17 years were tested from October 2008 to December 2009. Healthy dogs as well as dogs with various clinical symptoms of disease were included in the group. Samples were tested by polymerase chain reaction (PCR) specific for the presence ofBartonellaspp. Following the DNA examination in 286 dogs by PCR and subsequent sequencing, two samples were identified asBartonella henselae(0.7%). Other species ofBartonellawere not found. It was the first time in the Czech Republic when incidence ofBartonellaspp. was determined in dogs.

Verification of clinical samples, positive in AMPLICOR Neisseria gonorrhoeae polymerase chain reaction, by 16S rRNA and gyrA compared with culture

2005 ◽  
Vol 16 (6) ◽  
pp. 415-419 ◽  
Author(s):  
Åsa Airell ◽  
Emma Lindbäck ◽  
Ferda Ataker ◽  
Kirsti Jalakas Pörnull ◽  
Bengt Wretlind
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Neisseria Gonorrhoeae ◽  
False Negative ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Gyra Gene ◽  
Two Samples ◽  
Polymerase Chain

We compared 956 samples for AMPLICOR Neisseria gonorrhoeae polymerase chain reaction (PCR) (Roche) with species verification using the 16S rRNA gene to verification using gyrA gene. Control was the culture method. The gyrA verification uses pyrosequencing of the quinolone resistance-determining region of gyrA. Of 52 samples with optical density ≥0.2 in PCR, 27 were negative in culture, two samples from pharynx were false negative in culture and four samples from pharynx were false positives in verification with 16S rRNA. Twenty-five samples showed growth of gonococci, 18 of the corresponding PCR samples were verified by both methods; three urine samples were positive only in gyrA ; and one pharynx specimen was positive only in 16S rRNA. Three samples were lost. We conclude that AMPLICOR N. gonorrhoeae PCR with verification in gyrA gene can be considered as a diagnostic tool in populations with low prevalence of gonorrhoea and that pharynx specimens should not be analysed by PCR.

scholarly journals Genotypical characterization of thermotolerant coliforms isolated from food produced by a Solidarity Economic Venture of Bahia (Brazil)

2020 ◽  
Author(s):  
J. N. Silva ◽  
M. D. Baliza ◽  
F. Freitas ◽  
E. S Cruz ◽  
V. M. A. Camilo ◽  
...  
Keyword(s):  
Virulence Genes ◽  
Food Contamination ◽  
Microbiological Analysis ◽  
Chain Reaction ◽  
E Coli ◽  
Family Farmers ◽  
Two Samples ◽  
Thermotolerant Coliforms ◽  
Polymerase Chain

Abstract Many Solidarity Economic Venture (SEV) are family farmers who seek to add value to production through artisanal processing, which can lead to food contamination. Thus, this study aimed to genotypically characterize thermotolerant coliforms (TtC) strains from food produced by local agribusinesses of SEV during January to April 2019. Samples from thirteen production units (PU) from the SEV were submitted to a microbiological analysis of thermotolerant coliforms (AFNOR 3M1/2 – 09/89), using a fast count method in Petrifilm™ dishes. The Polymerase Chain Reaction (PCR) technique was used to verify the following virulence genes (VGs) associated with Escherichia coli: stx, typical from enterohemorrhagic E. coli (EHEC); bfpA typical from entheropathogenic E. coli (EPEC) and elt and slt, typical from entherotoxigenic E. coli (ETEC). The results showed that two samples of queijadinha (typical Brazilian candy made with eggs and coconut) and one sample of cassava cake presented characteristic colonies TtC. This way, three strains were isolated in order to perform the PCR technique. However, the genes used in the reaction were not detected in the isolated strains. Therefore, it is suggested that the isolated strains are from E. coli pathotypes with different virulence genes than the ones analyzed belong other types of TtC, such as Enterobacter and Klebsiella. Although the virulence of genes has not been confirmed, the presence of TtC on food indicates hygiene flaws during production and, therefore, measurements to control and prevent contamination should be taken.

scholarly journals Kultur Jaringan Jeruk Keprok Tejakula (Citrus reticulata var. Tejakula) Menggunakan Tunas Muda dan Biji Serta Deteksi CVPD dengan Teknik Polymerase Chain Reaction (PCR)

2020 ◽  
Vol 10 (1) ◽  
pp. 49
Author(s):  
VLORA VERONICA SIPANGKAR ◽  
I NYOMAN WIJAYA ◽  
MADE SRITAMIN
Keyword(s):  
Polymerase Chain Reaction ◽  
Tissue Culture ◽  
Pcr Analysis ◽  
Citrus Reticulata ◽  
Chain Reaction ◽  
Citrus Plant ◽  
Two Samples ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

 Tissue Culture Citrus (Citrus reticulata var. Tejakula) using Shoot and Seed and the Detection of CVPD with Polymerase Chain Reaction (PCR). The sample was taken in Pecatu Village, Kuta Selatan District, Badung Regency and continued with in vitro culture and PCR analysis at UPT. Genetic Resource and Molecular Biology Laboratory, was start from  December of 2018 until May of  2019. The purpose of this research was to create a CVPD free Citrus reticulata var. Tejakula seedling from tissue culture. The sample was chosen based on visual characteristic. Two samples were taken from  citrus  plant  that  show  CVPD  symptoms  and  citrus  plant  that  did  not  show  CVPD symptoms. The seed  and shoot  were cultured using MS media for 12  week after planting (WAP). From this research showed that both explant from the shoot and seed were able to grow well.  The  explant  from  citrus  sample  that  showed  CVPD  symptoms  was  infected  by Liberibacter asiaticus and the explants from citrus that did not show CVPD symptoms was not invected by Liberibacter asiaticus, according to PCR analysis.

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