Extraction and Purification of DNA from Complex Biological Sample Matrices Using Solid-Phase Microextraction Coupled with Real-Time PCR

Abstract The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

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Determination of Organochlorine Pesticides in Saussurea Costus by Ultrasonic Extraction and Solid-Phase Microextraction Method

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.554-556.1947 ◽

2012 ◽

Vol 554-556 ◽

pp. 1947-1951

Author(s):

Wei Liu ◽

Du Shu Huang ◽

Na Wu ◽

Ju Cheng Zhang ◽

Ping Yi ◽

...

Keyword(s):

Petroleum Ether ◽

Organochlorine Pesticides ◽

Solid Phase Microextraction ◽

Solid Phase ◽

Volume Ratio ◽

Ultrasonic Extraction ◽

Extraction And Purification ◽

Ultrasonic Time ◽

Pre Treatment

Ultrasonic extraction and purification using solid-phase microextraction column for the determination of 7 types of organochlorine pesticides including α, β, γ, δ-BHC, 2,4 '-DDT, 4,4'-DDT and heptachlor from the Saussrrea cotus were investigated in this study. The pretreatment of Saussrrea cotus samples, the effect of different ultrasonic time on the extraction efficiency and the effect of different ratio of dichloromethane: petroleum ether on purification efficiency were studied. Results show that the satisfactory extraction conditions for pre-treatment process are: 30 minutes ultrasonic treatment; the volume ratio of dichloromethane and petroleum ether mixture is 3:7.

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Erratum: Corrigendum: Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing

Nature Protocols ◽

10.1038/nprot0415-644b ◽

2015 ◽

Vol 10 (4) ◽

pp. 644-644 ◽

Cited By ~ 2

Author(s):

Rachel Yuan Nong ◽

Di Wu ◽

Junhong Yan ◽

Maria Hammond ◽

Gucci Jijuan Gu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Solid Phase ◽

Proximity Ligation

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Temporal Resolution of Solid-Phase Microextraction: Measurement of Real-Time Concentrations within a Dynamic System

Analytical Chemistry ◽

10.1021/ac102186u ◽

2010 ◽

Vol 82 (22) ◽

pp. 9492-9499 ◽

Cited By ~ 19

Author(s):

Xu Zhang ◽

Ken D. Oakes ◽

Di Luong ◽

John Z. Wen ◽

Chris D. Metcalfe ◽

...

Keyword(s):

Dynamic System ◽

Real Time ◽

Temporal Resolution ◽

Solid Phase Microextraction ◽

Solid Phase

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Task 1.12 - Solid-Phase Microextraction (SPME) coupled with optical spectroscopy for near-real-time determination of fuel and related pollutants in water samples

10.2172/567463 ◽

1995 ◽

Author(s):

Keyword(s):

Real Time ◽

Optical Spectroscopy ◽

Water Samples ◽

Solid Phase Microextraction ◽

Solid Phase ◽

Time Determination

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Assay Optimization Can Equalize the Sensitivity of Real-Time PCR with ddPCR for Detection of Helicoverpa armigera (Lepidoptera: Noctuidae) in Bulk Samples

Insects ◽

10.3390/insects12100885 ◽

2021 ◽

Vol 12 (10) ◽

pp. 885

Author(s):

Thayssa M. R. Oliveira ◽

Frida A. Zink ◽

Renato C. Menezes ◽

Érico C. Dianese ◽

Karina C. Albernaz-Godinho ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Helicoverpa Armigera ◽

Real Time Pcr ◽

Helicoverpa Zea ◽

Agricultural Pests ◽

Assay Optimization ◽

Extraction And Purification ◽

Assay Performance ◽

Helicoverpa Armígera

Helicoverpa armigera (Hübner) is one of the most important agricultural pests in the world. This historically Old World species was first reported in Brazil in 2013 and has since spread throughout much of South America and into the Caribbean. Throughout North America, H. armigera surveys are ongoing to detect any incursions. Each trap is capable of capturing hundreds of native Helicoverpa zea (Boddie). The two species cannot be separated without genitalic dissection or molecular methods. A ddPCR assay is currently used to screen large trap samples, but this equipment is relatively uncommon and expensive. Here, we optimized a newly designed assay for accurate and repeatable detection of H. armigera in bulk samples across both ddPCR and less costly, and more common, real-time PCR methods. Improvements over previously designed assays were sought through multiple means. Our results suggest bulk real-time PCR assays can be improved through changes in DNA extraction and purification, so that real-time PCR can be substituted for ddPCR in screening projects. While ddPCR remains a more sensitive method for detection of H. armigera in bulk samples, the improvements in assay design, DNA extraction, and purification presented here also enhance assay performance over previous protocols.

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Solid-Phase Capture of Pathogenic Bacteria by Using Gangliosides and Detection with Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.02601-07 ◽

2008 ◽

Vol 74 (7) ◽

pp. 2254-2258 ◽

Cited By ~ 15

Author(s):

Prerak T. Desai ◽

Marie K. Walsh ◽

Bart C. Weimer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pathogenic Bacteria ◽

Solid Phase

ABSTRACT We developed a method for concentrating pathogens from samples without enrichment. Immobilized gangliosides concentrated bacteria for detection with real-time PCR. A sensitivity of ∼4 CFU/ml (3 h) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml. The variance was less than one cycle.

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