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This study (open access) builds upon previously published work.  The authors expand the scope of multiplex digitalPCR (dPCR) analysis for undeclared GM soy to simultaneously check for 19 different GM events and the lectin (Le1) endogene, and proved the method’s transferability to a popular commercial dPCR platform.

They used a nanowell plate-based all-in-one dPCR system, the QIAGEN QIAcuity One. The method used four 5-plex assays, taking advantage of the platform’s multiple fluorescence detection channels.  DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method, with RNase-A and proteinase-K for removal of RNA and protein, respectively, as described in Annex A.3 of ISO21571:2005.  Quantification by simplex and multiplex assays was compared by testing CRM solutions containing single GM events.  The authors report that all four 5-plex assays showed results comparable with simplex assays

They report that the methods complied with the minimum performance requirements in terms of specificity, trueness, precision, sensitivity and dynamic range, making them suitable for use in routine detection and quantification of GM crops.

They conclude that approach represents a significant step forward by providing discriminative quantification of a large number of targets. The use of a commercial system prooves that quantitative multiplexing can become time and cost-effective, and they believe that their approach is particularly well suited to regulatory compliance testing. Sample compartmentalization, temperature cycling, and fluorescence detection are all performed automatically on the same machine, greatly simplifying the workflow and minimizing hands-on time. The experimental protocol requires no specialized sample handling, allowing it to be performed by operators familiar with qPCR workflow without additional training.

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In this study (open access) researchers developed and validated new new polymerase chain reaction (PCR) systems for detection of rapeseed in small samples of highly processed vegetable oils oils.

They designed primerss targeting the rapeseed acetyl-CoA carboxylase (ACCase; BnACCg8) gene, and optimised the PCR conditions after genomic DNA extraction from ground seeds and 700 µL aliquots of edible oils. DNA was isolated using two commercial kits, and PCR products were assessed by agarose gel electrophoresis.

They report that Uniplex PCRs demonstrated species specificity, producing 147-bp and 174-bp amplicons only in rapeseed DNA, with no amplification in soybean, sunflower, or maize. PCR bands from oils were weak or absent but implementing a double-PCR approach increased detection sensitivity in oils by approximately fivefold. Strong, expected-size amplicons were obtained from all oil extracts, confirming reliable detection of rapeseed in both cold-pressed and refined varieties, regardless of extraction method.

They conclude that this approach offers a sensitive, rapeseed-specific molecular tool for verifying the botanical origin of edible oils. It is suitable for routine authenticity testing and quality control of vegetable oils.

DNA-based verification that gelatin-containing foods and cosmetics do not contain pork products has always been a challenge due to DNA damage and destruction during gelatin production. 

In this study (open access) the authors report that – by careful optimisation of conditions – they could successfully apply a “traditional” PCR test to the problem.

They describe DNA extraction, post-isolation DNA analysis, annealing temperature and primer concentration optimization, specificity assay, amplification efficiency trial, sensitivity test, repeatability examination, and marketed sample analysis.

They report that the developed method demonstrated good specificity under optimized conditions. It achieved a good amplification efficiency of 101.2% with an R² of 0.994. The real-time PCR technique had a limit of detection of 1,316 pg in the sensitivity examination and a coefficient of variation of 0.81% in the repeatability testing.

They tested 10 retail samples (five facial mask cosmetics, food additive gelatin powder, two marshmallow products, and two gummy candy products), reporting that all of the samples displayed no amplification and were thus considered not to contain porcine DNA, consistent with the manufacturers’ labels.

The authors conclude that their real-time PCR method meets the validation criteria for qualitative analysis, including specificity, amplification efficiency, sensitivity, and repeatability.

The canning process has a degrading effect on DNA, making the species verification of canned tuna more challenging than for raw fish.

In this paper (open access) the authors optimised and compared three PCR approaches: real-time PCR (RT-PCR), mitochondrial control region (CR) mini-barcode, and multiplex PCR.  They tested 24 samples labelled as either albacore, yellowfin, skipjack or light tuna.

They reported RT-PCR as having the highest identification rate (100%), followed by CR mini-barcoding (33%) and multiplex PCR (29%). They consider that the success of RT-PCR may have been due to the short (<100 bp) DNA fragments targeted. In comparison, multiplex PCR and CR mini-barcoding targeted slightly longer fragments of 127–270 and∼236 base pairs, respectively. Regarding species identification, CR mini-barcoding and multiplex PCR confirmed the presence of albacoreor yellowfin tuna in several samples; however, both methods struggled with the identification of skipjack tuna.

CR mini-barcoding enabled sequencing-based detection of a range of species in the products. The authors conclude that a combination of real-time PCR and CR mini-barcoding is the optimum approach for rapid screening of target species along with sequencing-based confirmation.

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There is a growing market for the use of insect protein in feed.  The cricket species Gryllus assimilis. is approved in the EU for feeding farmed animals whilst the closely-related G. locorojo is only permitted for pets. The two are difficult to distinguish analytically in a highly processed product.

This paper (purchase required) reports a method developed on the basis of the cytochrome oxidase I gene, (COI), which was sequenced with thoroughly characterised G. locorojo and G. assimilis samples. The method is highly sensitive, detecting 0.8 pg G. locorojo-DNA or 0.1% G. locorojo incurred in feed, respectively. Authentic G. assimilis specimens were used to ensure that the G. locorojo method (Gloco-PCR) discriminates this closely related sister taxon, with a comfortable Ct-difference of 10-15. For cross analysis of true G. assimilis, similar primers with another probe were employed (Gassim-PCR) and the annealing temperature was increased from 60 °C to 62 °C.

Under these conditions, authentic G. assimilis crickets were detectable with Ct-values around 20, while G. locorojo samples showed a low detection at cycles around Ct 35. An investigation of ten ‘G. assimilis’ samples collected from Germany and four other European countries revealed that all of them were of the G. locorojo type.

The authors conclude that this small preliminary survey proves the usefulness of the method and supports the assumption that many G. assimilis crickets marketed in the EU indeed belong to the species G. locorojo. Consequently, European legislation, currently based on a white list of allowed insect species, is critically questioned.

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DNA analysis is rarely used for the verification of edible oil species, because of the low amount of intact DNA in the refined oil and the genetic similarities between different oil varieties.  In this study (open access pre-print, not yet peer-reviewed) the authors compared different DNA extraction kits and PCR protocols and new genetic markers to try and resolve the issue.  They reported that DNA extraction kits such as NucleoSpin Food, DNeasy mericon Food, and Olive Oil DNA Isolation as well as modified CTAB method were found to be able to isolate amplifiable genomic DNA from highly processed oils. Novel uniplex, double, and nested PCR systems targeting the sunflower-specific helianthinin gene were developed for efficient identification of sunflower. New sunflower DNA markers were revealed by uniplex PCRs.

They concluded that a combination of modified CTAB and nested PCR gave the best performance, and was demonstrated as a reliable, rapid, and cost-effective technology for detecting traces of sunflower in highly processed oil, including refined and used cooking oil.

In this study (purchase required), 14 widely commercialized GM soybean events, including GTS40-3-2, MON89788, CV127, A2704-12, A5547-127, 305423, 356043, MON87701, MON87705, MON87708, MON87769, SYHT0H2, FG72, and DAS-444Ø6-6 were utilised as targets for event-specific identification. A high-throughput detection method was established by integrating a single universal primer multiplex PCR (SUP-M-PCR) with capillary electrophoresis  The authors report that this method enables the identification of 14 soybean events in a single PCR reaction, addressing the limitations of conventional multiplex PCR such as self-inhibition and amplification variations between different primers. Furthermore, it significantly enhances the sensitivity and accuracy of previous multiple detection methods, achieving a detection sensitivity of 0.05% (w/w). They conclude that this approach improves detection efficiency and holds promising potential for the identification and detection of the 14 targeted GM soybean events and their derivatives.

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