Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.
Chronic kidney disease (CKD) is a global health concern, and recent clinical evidence suggests the potential of traditional Chinese medicine (TCM) to slow CKD progression. This offers alternative strategies for CKD patients, mitigating risks related to polypharmacy and adverse drug reactions. Our self-controlled, prospective study aims to assess the impact of Eefooton (EFT), a TCM-based regimen, on kidney health in stage 3-5 CKD patients. Additionally, we conduct a cell culture study to explore the potential mechanisms of EFT in protecting renal function.
Routine cell number determination for specific Lactobacillus strain by cultivation requires at least 4-7 days. Thus rapid and specific cell number determine methods such as strain-specific quantitative PCR (qPCR) are valuable. However, qPCR method is vulnerable to difficult PCR target such as dimer/secondary structure forming sequence.In this study, a two-component, "Ct contrast" approach was applied to strain-specific qPCR system following the development of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) strain-specific PCR with random amplification of polymorphic DNA (RAPD)-derived strain-specific sequences.The quantitative range of the NTU 101 strain-specific qPCR system was 3.0 × 101 to 3.0 × 105 copies for pure cultures, and 3.0 × 102 to 3.0 × 105 copies for multi-strain or unknown food samples. The results of spike in test and real sample testing suggested that non-specific weak background signals did not compromise test specificity, and demonstrated the potential of the NTU 101 strain-specific qPCR system in food samples.The two-component, "Ct contrast" approach is useful for qPCR discrimination when no ideal PCR target is available or the variance of the target site is unpredictable. The Ct contrast approach might provide a simple and robust solution for other challenging qPCR targets.
Red mold rice (RMR) is a traditional Chinese medicine prepared using Monascus fermentation. M. ruber (pilosus) and M. purpureus have a long history of use as food and medicine. As an economically important starter culture, the relationship between the taxonomy of Monascus and production capabilities of secondary metabolites is crucial for the Monascus food industry. In this study, monacolin K, monascin, ankaflavin, and citrinin production by M. purpureus and M. ruber were genomically and chemically investigated. Our findings suggest that M. purpureus can produce monascin and ankaflavin in a correlated manner, whereas M. ruber produces monascin with minimum ankaflavin. M. purpureus is capable of producing citrinin; however, it is unlikely able to produce monacolin K. In contrast, M. ruber produces monacolin K, but not citrinin. We suggest that the current monacolin K content-related regulation of Monascus food should be revised, and labeling of Monascus species should be considered.
Genetically modified (GM) crops are a major product of the global food industry. From 1996 to 2014, 357 GM crops were approved and the global value of the GM crop market reached 35% of the global commercial seed market in 2014. However, the rapid growth of the GM crop-based industry has also created controversies in many regions, including the European Union, Egypt, and Taiwan. The effective detection and regulation of GM crops/foods are necessary to reduce the impact of these controversies. In this review, the status of GM crops and the technology for their detection are discussed. As the primary gap in GM crop regulation exists in the application of detection technology to field regulation, efforts should be made to develop an integrated, standardized, and high-throughput GM crop detection system. We propose the development of an integrated GM crop detection system, to be used in combination with a standardized international database, a decision support system, high-throughput DNA analysis, and automated sample processing. By integrating these technologies, we hope that the proposed GM crop detection system will provide a method to facilitate comprehensive GM crop regulation.
The effects of a genetically modified cucumber mosaic virus (CMV)-resistant tomato on soil microbial communities were evaluated in this study. Soil position and environmental factors played more dominant roles than the tomato genotype in the variation of soil microbial communities.
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