scholarly journals Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

2007 ◽  
Vol 35 (16) ◽  
pp. e101-e101 ◽  
Author(s):  
S. Huang ◽  
J. Salituro ◽  
N. Tang ◽  
K.-C. Luk ◽  
J. Hackett ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Probe ◽  
Probe Design ◽  
Linear Dna

scholarly journals Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR

BioTechniques ◽  
2004 ◽  
Vol 36 (2) ◽  
pp. 266-275 ◽  
Author(s):  
Anthony T. Yeung ◽  
Brian P. Holloway ◽  
Pamela Scott Adams ◽  
Gregory L. Shipley
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Probe

Ultrasensitive quantification of mature microRNAs by real-time PCR based on ligation of a ribonucleotide-modified DNA probe

2011 ◽  
Vol 47 (33) ◽  
pp. 9465 ◽  
Author(s):  
Jiangyan Zhang ◽  
Zhengping Li ◽  
Hui Wang ◽  
Yucong Wang ◽  
Hongxia Jia ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Probe ◽  
Modified Dna ◽  
Mature Micrornas

scholarly journals Synergetic effect of non-complementary 5’ AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis

2019 ◽  
Author(s):  
Adriana Larrea-Sarmiento ◽  
Anne M. Alvarez ◽  
James P. Stack ◽  
Mohammad Arif
Keyword(s):  
Real Time ◽  
Abc Transporter ◽  
Real Time Pcr ◽  
High Efficiency ◽  
Synergetic Effect ◽  
The United States ◽  
Comparative Genomic ◽  
Probe Design ◽  
Clavibacter Michiganensis ◽  
Reaction Efficiency

AbstractClavibacter is an agriculturally important genus comprising a single species, Clavibacter michiganensis, and multiple subspecies, including, C. michiganensis subsp. nebraskensis which causes Goss’s wilt/blight of corn and accounts for high yield losses - listed among the five most significant diseases of corn in the United States of America. Our research objective was to develop a robust and rapid multiplex TaqMan real-time PCR (qPCR) to detect C. michiganensis in general and C. michiganensis subsp. nebraskensis with enhanced reliability and accuracy by adding non-complementary AT sequences to the 5’ end of the forward and reverse primers. Comparative genomic analyses were performed to identify unique and conserved gene regions for primer and probe design. The unique genomic regions, ABC transporter ATP-binding protein CDS/ABC-transporter permease and MFS transporter were determined for specific detection of C. michiganensis and C. m. subsp. nebraskensis, respectively. The AT-rich sequences at the 5’ position of the primers enhanced the reaction efficiency and sensitivity of rapid qPCR cycling; the reliability, accuracy and high efficiency of the developed assay was confirmed after testing with 59 strains from inclusivity and exclusivity panels – no false positives or false negatives were detected. The assays were also validated through naturally and artificially infected corn plant samples; all samples were detected for C. michiganensis and C. m. subsp. nebraskensis with 100% accuracy. The assay with 5’ AT-rich sequences detected up to 10- and 100-fg of C. michiganensis and C. michiganensis subsp. nebraskensis genome targets, respectively. No adverse effect was observed when sensitivity assays were spiked with host genomic DNA. Addition of 5’ AT rich sequences enhanced the qPCR reaction efficiency from 0.82 (M = -3.83) and 0.91 (M = -3.54) to 1.04 (with optimum slope value; M = -3.23) for both C. michiganensis and C. michiganensis subsp. nebraskensis, respectively; a increase of 10-fold sensitivity was also obtained with C. michiganensis primer set. The methodology proposed here can be used to optimize the reaction efficiency and to harmonize the diagnostic protocols which have prodigious applications in routine diagnostics, biosecurity and microbial forensics.

scholarly journals Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design

2017 ◽  
Vol 19 (5) ◽  
pp. 742-754 ◽  
Author(s):  
Hou-Sung Jung ◽  
Gregory J. Tsongalis ◽  
Joel A. Lefferts
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Probe Design ◽  
Hydrolysis Probe

scholarly journals DESAIN TAQMAN PROBE SECARA IN SILICO SEBAGAI PENDETEKSI MUTASI PADA KODON 516 GEN rpoB Mycobacterium tuberculosis UNTUK METODE REAL-TIME PCR

2017 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Dek Pueteri Dewi Suryani ◽  
Putu Sanna Yustiantara ◽  
Sagung Chandra Yowani
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Aspartic Acid ◽  
Real Time Pcr ◽  
In Silico ◽  
Rpob Gene ◽  
Taqman Probe ◽  
Probe Design ◽  
Accession Number ◽  
Pcr Method

The aim of this research was to in silico design of TaqMan probe. Design of TaqMan probe were conducted using software Clone Manager Suite 6. As a template, the rpoB gene of M. tuberculosis H37Rv (accession number U12205.1) was used. The results of this research were 8 sequences such as, R516MV-2, R516MV-3, R516MV-4, R516MV-5, R516MV-7, R516MV-8, R516MV-11, R516MV-13. These sequences were met the criteria of TaqMan probe, such as length of nucleotide (23-28 nucleotide), Tm value (72ºC), %GC (50-58%), runs and repeats (?4 bases), dimer structure in accordance to the requirements and does not form hairpin structures. In addition, these sequences were met labeling criteria of TaqMan probe which are including the location of G bases and the number of G-C bases in sequences. Therefore, these sequences could be labeled by FAM (reporter) at 5' end and TAMRA (quencher) at 3' end. The conclusion of this research, the sequences were met the criteria of TaqMan probe. Therefore, it could be targeted to detect mutations at codon 516 with a change of aspartic acid into valine (GAC ? GTC) by using real-time PCR method.

scholarly journals Specific detection and differentiation of classic goose parvovirus and novel goose parvovirus by TaqMan real-time PCR assay, coupled with host specificity

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Chunhe Wan ◽  
Cuiteng Chen ◽  
Longfei Cheng ◽  
Rongchang Liu ◽  
Shaohua Shi ◽  
...  
Keyword(s):  
Real Time ◽  
Host Specificity ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Probe Design ◽  
Muscovy Duck ◽  
Specific Detection ◽  
Goose Parvovirus ◽  
Taqman Qpcr

Abstract Background Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. Results After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/μl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. Conclusions We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.

Feldvalidierung einer real-time PCR zum Nachweis von Mycoplasma hyopneumoniae in Nasentupfermaterial von lebenden Schweinen

2005 ◽  
Vol 147 (9) ◽  
pp. 373-379 ◽  
Author(s):  
F. Zeeh ◽  
P. Kuhnert ◽  
R. Miserez ◽  
M. G. Doherr ◽  
W. Zimmermann
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mycoplasma Hyopneumoniae
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