کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10536302 | 962067 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for the detection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03 to 0.80Â pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. The system is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presence of unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18S detector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA in the sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 339, Issue 1, 1 April 2005, Pages 73-82
Journal: Analytical Biochemistry - Volume 339, Issue 1, 1 April 2005, Pages 73-82
نویسندگان
MarÃa López-Andreo, Laura Lugo, Amando Garrido-Pertierra, M. Isabel Prieto, Antonio Puyet,