Consumers are becoming increasingly interested in the quality of their food and the effect it has on their health. In conjunction with recent food scandals, this customer interest drives food producers to offer increased transparency in their food products. One major topic is the source of raw materials.
This matter refers to both the inclusion of materials originating from genetically modified organisms, and the use of the appropriate materials as shown on the ingredient listing.
Technologies based on Polymerase Chain Reaction (PCR) are highly effective in examining the origins of food materials and the genetic backgrounds of materials in processed food, as they offer great sensitivity. A specific quality and quantity of DNA are needed to make use of the PCR-based processes to analyse food.
Certain sample materials (including plant samples and processed food) hold substantial levels of inhibitors, which obstruct PCR-based downstream applications and thus impede analysis.
In the study outlined here, DNA was extracted from northern shrimp, hazelnut and two distinct varieties of cheese. DNA extraction was carried out with the innuPREP DNA Kit-IPC16 and innuPREP Food DNA Kit-IPC16. Extraction was accomplished with InnuPure® C16/C16 touch.
Following optional homogenization and lysis, lysed samples were placed in the Reagent Plastics of the kits. The Reagent Plastics of the kits come ready filled with all necessary buffers and magnetic particles for DNA extraction.
The Reagent Plastics were placed in the InnuPure® C16/C16 touch, before a fully automated extraction process was implemented. After extraction, DNA quality was examined using gel electrophoresis, spectrophotometry and quantitative real-time PCR (qRT-PCR) for species analysis.
Thanks to the high pre-processing state of the kits (pre-filled, sealed buffer reservoirs) and automated purification, the amount of manual effort required of the customer is kept to a minimum. Aside from the advantage of saved time, pre-processing also decreases the risk of human error and loss of valuable samples.
Analysis of extracted DNA by agarose gel electrophoresis, spectrophotometry and qRT-PCR analysis indicates that DNA extracted with devices and kits from Analytik Jena can be readily used for related downstream applications.
Materials and Methods
Frozen northern shrimps (50 mg) underwent homogenization in 200 µl H2O for 15 seconds, using SpeedMill PLUS and Lysis Tubes P (Analytik Jena). Homogenates were blended with 200 µl Lysis Solution CBV and 20 µl Proteinase K and incubated at 50 °C, shaking with 600 rpm for one hour.
400 µl of the lysate was transferred to the Reagent Plastic of the innuPREP DNA KitIPC16 ahead of extraction with InnuPure® C16.
100 mg and 200 mg ground hazelnut was re-suspended in 800 µl Lysis Solution CBV and 20 µl Proteinase K. The samples were lysed for one hour at 65 °C. Following lysis, 400 µl supernatant was used for extraction with the innuPREP Food DNA KitIPC16 and InnuPure® C16.
DNA was extracted from two types of commercially obtainable cheese. Cheeses were stated as being derived from goat and cow milk, respectively. Extraction was carried out in quadruplicates using InnuPure® C16 touch and innuPREP Food DNA KitIPC16. Each replicate used 200 mg of cheese. Elution volume was set to 100 µl.
After extraction, extracts were examined by photometry using ScanDrop® 250 and qRT-PCR. For qRT-PCR qTOWER3 and innuDETECT, Cheese Assay were used. The analysis was carried out using Endpoint method provided by qPCRsoft.
Samples and Reagents
- innuPREP DNA Kit-IPC16
- innuPREP Food DNA Kit-IPC16
- Lysis Tubes P
- innuDETECT Cheese Assay
- Commercially available kit for hazel analysis
- InnuPure® C16
- InnuPure® C16 touch
- SpeedMill PLUS
- BioShake iQ
- ScanDrop® 250
- Standard equipment for agarose gel electrophoresis and gel documentation
- qTOWER3 G
Results and Discussion
Following the extraction of DNA from frozen shrimp samples, the quality and integrity of the DNA was evaluated by spectrophotometrical analysis with ScanDrop® 250 and agarose gel electrophoresis.
Table 1. Spectrophotometrical analysis of DNA extracted from shrimp samples.
Figure 1. Agarose gel electrophoresis of DNA extracted from frozen shrimp samples. Lane 1: DNA ladder; lanes 2–5: Samples 1–4.
The innuPREP DNA Kit-IPC16, in combination with the InnuPure® C16, permits the automated DNA extraction from shrimps with low manual input. Extraction produces pure DNA with A260/A280 ratios ranging from 1.87 to 2.32. Mean yields of 22.1 ng/µl are adequate for PCR-based downstream applications.
Agarose gel analysis indicates that a portion of the DNA is degraded, appearing as a smear in lanes 2–5 of Figure 1. DNA degradation is a natural development which begins shortly after the animals’ death and halts upon freezing. Aside from degraded DNA, quite fairly high molecular weight DNA can be detected as a distinct band, with a size over 1,500 bp.
Following DNA extraction from 100 mg and 200 mg ground hazelnut with the innuPREP Food DNA Kit-IPC16, extracts were used for qRT-PCR-based detection of hazelnut DNA. So as to identify inhibitory impacts of substances held in the extracted DNA, aliquots of the extract were diluted at a ratio of 1:10 ahead of amplification of 1 µl undiluted and 1:10 diluted extract.
Figure 2. DNA extracted from 100 mg ground hazelnut, amplified and detected with qRT-PCR in duplicates (blue curves undiluted, green curves 1:10 dilution).
Figure 3. DNA extracted from 200 mg ground hazelnut, amplified and detected with qRT-PCR in duplicates (blue curves undiluted, green curves 1:10 dilution).
Figures 2 and 3 indicate that DNA extraction is achievable from both 100 mg and 200 mg of ground hazelnut, and yields DNA which can readily be used for qRT-PCR. Sample materials with high contents of lipids are often difficult in regards to extraction, and also face the risk of contamination of the extracted DNA with PCR inhibitors.
For the extraction of DNA from hazelnut, PCR inhibition can be omitted, since ten-fold dilution of the sample is carried out. This leads to a shift of the Ct value of around 3.3 cycles which is the case for extracts from 100 mg and 200 mg, respectively. DNA was extracted from 200 mg cheese as outlined above.
Following extraction, extracts were studied through photometry using ScanDrop® 250 and qRT-PCR. qTOWER3 and innuDETECT Cheese Assay were used for qRT-PCR. Analysis was carried out with Endpoint method provided by qPCRsoft.
Table 2. Photometric analysis of nucleic acid extracted from cheese samples.
Figure 4. Detection of sheep-specific target gene.
Figure 5. Detection of goat-specific target gene.
Figure 6. Detection of cow-specific target gene.
Findings indicate that automated extraction, using InnuPure® C16 touch in combination with the innuPREP Food DNA Kit-IPC16, can be employed to yield DNA from cheese. The achieved quality and quantity are adequate for ensuing qRT-PCR application, as represented in Figure 4-6.
Analysis of 200 mg starting material leads to eluates with concentrations ranging from 9-16 ng/µl. Furthermore, proteins are entirely eliminated using the innuPREP Food DNA Kit-IPC16, as shown by ratios A260/A280 in Table 2. The samples originating from milk with known origins underwent qRT-PCR analysis of sheep-, goat- and cow-specific genes.
In each case, internal positive control (light red) was identified. Nevertheless, goat-specific genes were only identified in cheese stated to be derived from goat milk while cow-specific genes were only identified in cheese stated to be derived from cow milk. None of the samples tested positive for sheep-specific genes.
In conclusion, the findings indicate that a combination of InnuPure® C16 touch and DNA extraction kits for this device can be employed for DNA extraction from a variety of samples linked to the food industry.
DNA extracted in this manner boasts top quality. Most significantly, DNA extracted with InnuPure C16® touch and the related kits can readily be employed for qRT-PCR.
About Analytik Jena US
Analytik Jena is a provider of instruments and products in the areas of analytical measuring technology and life science. Its portfolio includes the most modern analytical technology and complete systems for bioanalytical applications in the life science area.
Comprehensive laboratory software management and information systems (LIMS), service offerings, as well as device-specific consumables and disposables, such as reagents or plastic articles, complete the Group’s extensive range of products.
About Life Science
The Life Science product area demonstrates the biotechnological competence of Analytik Jena AG. We provide a wide product spectrum for automated total, as well as individual solutions for molecular diagnostics. Our products are focused to offer you a quality and the reproducibility of your laboratory results.
This will surely ease your daily work and speed up your work processes in a certain way. All together we support you through the complete process of the lab work. Besides we offer customized solutions and are able to adapt our products to your needs. Automated high-throughput screening systems for the pharmaceutical sector are also part of this segment’s extensive portfolio.
About Analytical Instrumentation
Analytik Jena has a long tradition in developing high-performance precision analytical systems which dates back to the inventions made by Ernst Abbe and Carl Zeiss. We have grown to become one of the most innovative manufacturers of analytical measuring technology worldwide.
Our business unit Analytical Instrumentation offers excellent competencies in the fields of optical spectroscopy, sum parameters and elemental analysis. Being proud of our core competency we grant all our customers a long-term warranty of 10 years for our high-performance optics.
About Lab Automation
With more than 25 years of market experience, Analytik Jena with its CyBio® Product Line is a leading provider for high quality liquid handling and automation technologies. In the pharmaceutical and life science industries, our products enjoy the highest reputation for precision, reliability, robustness and simplicity.
Moreover, the Automation Team designs, produces and installs fully automated systems tailored to our clients' application, throughput and capacity requirements. From stand-alone CyBio® Well up to fully customized robotic systems we handle your compounds, biomolecules and cells with great care.
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