scholarly journals Development of Multiplex PCR Primer Sets for the Identification of Rice Varieties

2005 ◽  
Vol 7 (2) ◽  
pp. 87-94 ◽  
Author(s):  
Kazunori Shinmura ◽  
Hiroshi Kanagawa ◽  
Takashi Mikami ◽  
Takeshi Fukumori
Keyword(s):  
Multiplex Pcr ◽  
Rice Varieties ◽  
Primer Sets ◽  
Pcr Primer

scholarly journals Designing Highly Multiplex PCR Primer Sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)

2021 ◽  
Author(s):  
David Zhang ◽  
Nina Xie ◽  
Michael Wang ◽  
Ping Song ◽  
Yifan Wang ◽  
...  
Keyword(s):  
Simulated Annealing ◽  
Multiplex Pcr ◽  
Dna Sequences ◽  
Simulated Annealing Algorithm ◽  
Likelihood Estimation ◽  
Stochastic Algorithm ◽  
Gene Fusions ◽  
Dimer Formation ◽  
Primer Sets ◽  
Pcr Primer

Abstract The design of highly multiplex PCR primers to amplify and enrich many different DNA sequences is increasing in biomedical importance as new mutations and pathogens are identified. One major challenge in the design of highly multiplex PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of highly multiplex PCR primer sets that minimize primer dimer formation. Our approach uses a rapidly computable Loss function to approximate the degree of primer dimer formation within a primer set, and randomly swaps primers in the set with alternative candidates using a simulated annealing algorithm. In a 96-plex PCR primer set (192 primers), we show that we can reduce the fraction of primer dimers from 90.7% in a naively designed PCR primer set to 4.9% in our optimized primer set. Running the optimized 96-plex primer set on FFPE DNA samples from cancer patients, we likewise observe a low fraction of primer dimer reads. Even when scaling to 384-plex (768 primers), the PCR primer set designed by our algorithm maintains low primer dimer fraction. In addition to NGS, we also show that our SADDLE-designed multiplex primer sets can be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.

scholarly journals Development of multiplex PCR primer sets for identification of major strawberry (Fragaria×ananassa Duchesne) cultivars in Japan

2008 ◽  
Vol 10 (3) ◽  
pp. 111-115
Author(s):  
Kimihisa Tasaki ◽  
Yuuki Kashiwaya ◽  
Shun-ichi Kobayashi ◽  
Masayuki Amagai
Keyword(s):  
Multiplex Pcr ◽  
Primer Sets ◽  
Pcr Primer

scholarly journals Minimum Multicolored Subgraph Problem in Multiplex PCR Primer Set Selection and Population Haplotyping

2006 ◽  
pp. 758-766 ◽  
Author(s):  
M. T. Hajiaghayi ◽  
K. Jain ◽  
L. C. Lau ◽  
I. I. Măndoiu ◽  
A. Russell ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Pcr Primer

scholarly journals A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing

2009 ◽  
Vol 58 (8) ◽  
pp. 1045-1057 ◽  
Author(s):  
Lin Cai ◽  
Fanrong Kong ◽  
Qinning Wang ◽  
Huiping Wang ◽  
Meng Xiao ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Blot Hybridization ◽  
Rapid Identification ◽  
Reverse Line Blot ◽  
Discriminatory Power ◽  
Hybridization Assay ◽  
Coagulase Negative Staphylococci ◽  
Primer Sets ◽  
Mrsa Strains

The aim of this study was to develop a new discriminatory method for MRSA SCCmec typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCCmec types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared in silico to 14 whole-genome sequences and 18 partial SCCmec gene sequences of Staphylococcus aureus and complete genome and partial SCCmec genes of seven non-MRSA strains, including meticillin-susceptible S. aureus and meticillin-resistant coagulase-negative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCCmec types and 26 subtypes, including established and new subtypes of SCCmec III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCCmec typing was similar to that of MLST and spa typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCCmec typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCCmec typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals.

scholarly journals Deep Sequencing of a Dimethylsulfoniopropionate-Degrading Gene (dmdA) by Using PCR Primer Pairs Designed on the Basis of Marine Metagenomic Data

2009 ◽  
Vol 76 (2) ◽  
pp. 609-617 ◽  
Author(s):  
Vanessa A. Varaljay ◽  
Erinn C. Howard ◽  
Shulei Sun ◽  
Mary Ann Moran
Keyword(s):  
Sequence Data ◽  
Gene Clusters ◽  
Amino Acid Identity ◽  
Metagenomic Data ◽  
Data Sets ◽  
Free Living ◽  
Metagenomic Sequence ◽  
Primer Sets ◽  
Design And Testing ◽  
Pcr Primer

ABSTRACT In silico design and testing of environmental primer pairs with metagenomic data are beneficial for capturing a greater proportion of the natural sequence heterogeneity in microbial functional genes, as well as for understanding limitations of existing primer sets that were designed from more restricted sequence data. PCR primer pairs targeting 10 environmental clades and subclades of the dimethylsulfoniopropionate (DMSP) demethylase protein, DmdA, were designed using an iterative bioinformatic approach that took advantage of thousands of dmdA sequences captured in marine metagenomic data sets. Using the bioinformatically optimized primers, dmdA genes were amplified from composite free-living coastal bacterioplankton DNA (from 38 samples over 5 years and two locations) and sequenced using 454 technology. An average of 6,400 amplicons per primer pair represented more than 700 clusters of environmental dmdA sequences across all primers, with clusters defined conservatively at >90% nucleotide sequence identity (∼95% amino acid identity). Degenerate and inosine-based primers did not perform better than specific primer pairs in determining dmdA richness and sometimes captured a lower degree of richness of sequences from the same DNA sample. A comparison of dmdA sequences in free-living versus particle-associated bacteria in southeastern U.S. coastal waters showed that sequence richness in some dmdA subgroups differed significantly between size fractions, though most gene clusters were shared (52 to 91%) and most sequences were affiliated with the shared clusters (∼90%). The availability of metagenomic sequence data has significantly enhanced the design of quantitative PCR primer pairs for this key functional gene, providing robust access to the capabilities and activities of DMSP demethylating bacteria in situ.

scholarly journals Prevalence of the Helicobacter pylori babA2 Gene in Children Mainly Depends on the PCR Primer Set Used

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Anja Šterbenc ◽  
Maja M. Lunar ◽  
Matjaž Homan ◽  
Boštjan Luzar ◽  
Nina Zidar ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Helicobacter Pylori ◽  
Diagnostic Tool ◽  
Clinical Relevance ◽  
Detection Methods ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Primer Sets ◽  
H Pylori ◽  
Pcr Primer

Various polymerase chain reaction- (PCR-) based methods with varying positivity rates were designed to detect the Helicobacter pylori babA2 gene. To compare different primer sets, babA2 prevalence was determined in 279 H. pylori-positive pediatric samples using the 832 bp, 139 bp, and 271 bp PCR primer sets, resulting in 34.0%, 51.3%, and 79.6% prevalence of the babA2 gene, respectively. The babA2 status determined using the 832 bp and 139 bp PCR primer sets significantly correlated with bacterial density and activity of inflammation, whereas no such correlations were found using the 271 bp PCR primer set. The 139 and 832 bp PCR primer sets concordantly detected the babA2 gene in 93 cases; however, in comparison to the 832 bp PCR primer set, the 139 bp PCR primer set detected additional 50 babA2 cases, whereas only two 832 bp positive cases were missed. The 271 bp PCR primer set missed 32 babA2 cases that were 832 bp and/or 139 bp PCR positive, but tested solely positive in 109 cases. Interestingly, cloning of a subset of 271 bp PCR positive samples revealed amplification of the babA/B gene chimera. Hence, in our opinion, the 271 bp PCR protocol is not a reliable diagnostic tool for detecting the babA2 gene in children. Our results reaffirm previous observations that the use of certain babA2 PCR primer sets can significantly impact estimation of the prevalence and clinical relevance of the H. pylori babA2 gene in children, suggesting babA2 detection methods should be carefully selected.

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