scholarly journals Development of a real-time PCR assay to detect the single nucleotide polymorphism causing Warmblood Fragile Foal Syndrome

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259316
Author(s):  
Sharon Flanagan ◽  
Áine Rowe ◽  
Vivienne Duggan ◽  
Erin Markle ◽  
Maureen O’Brien ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Genetic Mutation ◽  
Wild Type ◽  
High Throughput Analysis ◽  
Single Nucleotide ◽  
Cornell University ◽  
Single Nucleotide Change ◽  
Effective Manner

Warmblood Fragile Foal syndrome (WFFS) is an autosomal recessive condition that affects the maturation of collagen in affected foals. Foals affected with the disease typically die or are euthanised shortly after birth. WFFS is caused by a single nucleotide change at position 2032 of the equine PLOD1 gene, causing an impairment of the wild-type enzyme. A commercial test for the causative genetic mutation is currently available from companies operating under licence from Cornell University but it has limitations. This test requires amplification of a region of the PLOD1 gene encompassing the site of interest, followed by Sanger sequencing of that region and computational analysis. We describe here the development of an alternative, real-time PCR based assay that rapidly and reliably differentiates between the wild-type and WFFS associated nucleotides without the need for sequencing, thus increasing the potential for high throughput analysis of large numbers of samples in a cost-effective manner.

scholarly journals Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms

2006 ◽  
Vol 55 (1) ◽  
pp. 43-51 ◽  
Author(s):  
Alex J. Stephens ◽  
Flavia Huygens ◽  
John Inman-Bamber ◽  
Erin P. Price ◽  
Graeme R. Nimmo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Population Structure ◽  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Resolving Power ◽  
Nucleotide Polymorphisms ◽  
Methicillin Resistant ◽  
Effective Manner ◽  
Mrsa Strains

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleotide polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eburst analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbp/sdrE, and the integrated plasmids pT181, pI258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

scholarly journals In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Manash Jyoti Kalita ◽  
Kalpajit Dutta ◽  
Gautam Hazarika ◽  
Ridip Dutta ◽  
Simanta Kalita ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Sybr Green ◽  
Green Approach ◽  
Novel Approach ◽  
Effective Manner ◽  
Wide Range ◽  
Thermal Cycler

AbstractAs the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains

2017 ◽  
Vol 28 (4) ◽  
pp. 248-252 ◽  
Author(s):  
Sachin S. Pawar ◽  
Chetan D. Meshram ◽  
Niraj K. Singh ◽  
Mohini Saini ◽  
B. P. Mishra ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bovine Herpesvirus ◽  
Wild Type ◽  
Pcr Assay ◽  
Bovine Herpesvirus 1 ◽  
Glycoprotein E ◽  
Herpesvirus 1

scholarly journals Prediction of sofosbuvir response using interleukin-6 serum level and single nucleotide polymorphism of interferon lambda- 4

2020 ◽  
Vol 14 (01) ◽  
pp. 80-88
Author(s):  
Amal E Saafan ◽  
Ashraf Abobaker ◽  
Mohamed S Abbas ◽  
Ahmed El-Gendy
Keyword(s):  
Serum Level ◽  
Real Time ◽  
Real Time Pcr ◽  
Dual Therapy ◽  
Snp Genotyping ◽  
Nucleotide Polymorphism ◽  
Time Intervals ◽  
Genotype 4 ◽  
Single Nucleotide ◽  
Taqman Probes

Introduction: In Egypt, 15% of the populations are suffering from chronic hepatitis C especially genotype 4. Sofosbuvir was approved by FDA in December 2013 for treatment of HCV genotypes 2 and 3 in combination with Ribavirin, and for genotypes 1 and 4 in combination with Peg-IFN. Recently, polymorphism of different genes and plasma levels of IL-6 were utilized for better prediction of HCV clearance. This study aimed at early prediction of the efficacy of HCV treatment with Sofosbuvir (Sovaldi) and comparing the antiviral efficacy of dual and triple Sovaldi combination therapy. Methodology: Blood samples were collected from 100 HCV positive patients and detected by real time PCR at three time intervals. SNP genotyping of INFL-4 gene was estimated by using real-time PCR with predesigned primers and Taqman probes. IL-6 serum level was estimated before, during and after the end of the treatment using ELISA assay based on human IL-6 KIT. Results: SNP genotyping of INFL-4 gene showed that 13.1% of patients carried ∆G/∆G, 30.4% patients had TT/TT and 56.5% patients possessed heterozygote allele ∆G/TT. Clinical data displayed that 13 patients were got relapsed at SVR 12. Serum level of IL-6 was noticed higher in HCV patients than healthy ones. Noteworthy, it was increased during treatment then decreased to a minimal level than begining of treatment. Conclusion: SNP in INFL-4 gene has displayed no effect in response to Sofosbuvir. Dual therapy had the same effect like triple therapy, so interferon could be withdrawn from the treatment regimen.

scholarly journals Cost-effective optimization of real-time PCR-based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases

2015 ◽  
Vol 119 (5) ◽  
pp. 1391-1402 ◽  
Author(s):  
M.S.R. Fachmann ◽  
M.H. Josefsen ◽  
J. Hoorfar ◽  
M.T. Nielsen ◽  
C. Löfström
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Polymerases ◽  
Cost Effective
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