Determination of Cry1Ac copy number in transgenic pigeonpeaplants using quantitative real time PCR.

Copy number of Cry1Ac in transgenic pigeonpea plants was determined by quantitative real time PCR using Syber Green as fluorescence indicator. Gene specific primers designed to amplify relatively long amplicons (400 – 600 bp), for Cry1Ac was used to increase specificity and sensitivity of Real time PCR. Estimated copy number in transgenic lines using real-time quantitative PCR and southern hybridization was correlated and found to be same i.e. single copy number. This study shows effectivness of real-time PCR method for estimating the transgene copy number in transgenic pigeonpea plants by a simple, accurate and cost effective manner.Keywords: Copy number, Cry1Ac, Pigeonpea transformation, Real-time PCR, Syber green.

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A novel quantitative real-time PCR method for the detection of mammalian and poultry species based on a shared single-copy nuclear DNA sequence

Food Chemistry ◽

10.1016/j.foodchem.2020.128170 ◽

2021 ◽

Vol 341 ◽

pp. 128170

Author(s):

Wenjun Wang ◽

Ming Fu ◽

Qingde Zhang ◽

Yueran Zhen ◽

Jianjian Liu ◽

...

Keyword(s):

Real Time ◽

Dna Sequence ◽

Real Time Pcr ◽

Nuclear Dna ◽

Single Copy ◽

Quantitative Real Time Pcr ◽

Pcr Method

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Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

Analytical Biochemistry ◽

10.1016/j.ab.2007.11.022 ◽

2008 ◽

Vol 375 (1) ◽

pp. 150-152 ◽

Cited By ~ 27

Author(s):

Cheng Xin Yi ◽

Jun Zhang ◽

Ka Man Chan ◽

Xiao Kun Liu ◽

Yan Hong

Keyword(s):

Gossypium Hirsutum ◽

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Transgene Copy Number

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Quantitative real-time PCR method to detect changes in specific transcript and total RNA amounts

Electronic Journal of Biotechnology ◽

10.2225/vol7-issue1-fulltext-5 ◽

2004 ◽

Vol 7 (1) ◽

Author(s):

Daniel Z. Skinner ◽

Kwang-Hyun Baek

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Total Rna ◽

Specific Transcript ◽

Pcr Method

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Collaborative Validation of an Event-Specific Quantitative Real-Time PCR Method for Genetically Modified Rice Event TT51-1 Detection

Journal of Agricultural and Food Chemistry ◽

10.1021/jf401339k ◽

2013 ◽

Vol 61 (25) ◽

pp. 5953-5960 ◽

Cited By ~ 14

Author(s):

Yuhua Wu ◽

Litao Yang ◽

Yinglong Cao ◽

Guiwen Song ◽

Ping Shen ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genetically Modified ◽

Quantitative Real Time Pcr ◽

Genetically Modified Rice ◽

Pcr Method

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A Quantitative Real-Time PCR Method Using an X-Linked Gene for Sex Typing in Pigs

Molecular Biotechnology ◽

10.1007/s12033-012-9589-5 ◽

2012 ◽

Vol 54 (2) ◽

pp. 493-496 ◽

Cited By ~ 5

Author(s):

Maria Ballester ◽

Anna Castelló ◽

Yuliaxis Ramayo-Caldas ◽

Josep M. Folch

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Sex Typing ◽

Linked Gene ◽

Pcr Method

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The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

Journal of Microbiological Methods ◽

10.1016/j.mimet.2005.09.007 ◽

2006 ◽

Vol 65 (3) ◽

pp. 476-487 ◽

Cited By ~ 44

Author(s):

Miguel A. Providenti ◽

Jason M. O'Brien ◽

Robyn J. Ewing ◽

E. Suzanne Paterson ◽

Myron L. Smith

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Sybr Green ◽

Quantitative Real Time Pcr ◽

Genetic Elements

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Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1373 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1373-1378 ◽

Cited By ~ 30

Author(s):

ANNA-CLARA RÖNNER ◽

HANS LINDMARK

Keyword(s):

Detection Limit ◽

Real Time ◽

Dna Extraction ◽

Campylobacter Jejuni ◽

Real Time Pcr ◽

Close Correlation ◽

Quantitative Detection ◽

Quantitative Real Time Pcr ◽

Direct Plating ◽

Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

BMC Hematology ◽

10.1186/2052-1839-14-4 ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 21

Author(s):

Runa M Grimholt ◽

Petter Urdal ◽

Olav Klingenberg ◽

Armin P Piehler

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Globin Gene ◽

Copy Number Variations ◽

Globin Genes ◽

Quantitative Real Time Pcr ◽

Human Genetic Disease ◽

Large Deletions ◽

Wide Range

Abstract Background Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). Results Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. Conclusions HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results.

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