No abstract

On-site monitoring the plantation of genetically modified (GM) crops is of critical importance in agriculture industry throughout the world. In this paper, a simple, visual and instrument-free method for instant on-site detection of GTS 40-3-2 soybean has been developed. It is based on body-heat recombinase polymerase amplification (RPA) and followed with naked-eye detection via fluorescent DNA dye. Combining with extremely simplified sample preparation, the whole detection process can be accomplished within 10 min and the fluorescent results can be photographed by an accompanied smart phone. Results demonstrated a 100% detection rate for screening of practical GTS 40-3-2 soybean samples by 20 volunteers under different ambient temperatures. This method is not only suitable for on-site detection of GM crops but also demonstrates great potential to be applied in other fields.

Respiratory syncytial virus (RSV) is a leading viral pathogen responsible for lower respiratory tract infections, particularly in children under five years worldwide, often resulting in hospitalization.

Nicking enzyme assisted amplification (NEAA) is an extremely rapid method for molecular diagnosis. However, this technology is not widely applied for real sample analysis because the overproduced non-specific products limit its sensitivity and raise the threshold of detection methods. Here, we have found that the non-specific amplification is mainly caused by the coexistence of Bst polymerase, nicking primers and dNTP. The highly active nicking enzyme directs and accelerates the non-specific amplification in a way which favors nicking. To suppress the non-specific amplification, the nicking enzyme concentration, reaction temperature, and magnesium ion concentration are optimized. The compatibility of Bst polymerase with the concentration of the monovalent cation is also crucial. Besides, the sensitivity could be enhanced by shortening the target sequences and priming the 3' end of the target.

The Zushima tablet (ZT) has been used for decades in the clinical treatment of rheumatoid arthritis (RA) in China. However, its therapeutic mechanism is unclear. In this study, we aimed to explore the distinctive metabolic patterns in collagen-induced arthritis (CIA) rats and evaluate the therapeutic effects of ZT on RA using untargeted serum and fecal metabolomics approaches based on gas chromatography coupled with mass spectrometry. Body weight, hind paw swelling, TNF-α and IL-1β levels, arthritis scores, and histopathological parameters were assessed. In the metabolomics study, 31 altered metabolites in the serum and 30 in the feces were identified by comparing the model with the control group using statistical processing. These altered metabolites revealed that the tricarboxylic acid cycle, glycolysis metabolism, fatty acid metabolism, and purine metabolism were disturbed in CIA rats, and most of these altered metabolites including l-isoleucine, l-aspartic acid, pyruvic acid, cholic acid, and hypoxanthine, were rectified by ZT. Furthermore, short-chain fatty acids in feces were quantitatively determined, and the results showed that ZT could regulate the levels of propionate, butyrate, and valerate in CIA rats. Then, gut microbiota were analyzed by 16S rRNA analysis. Our results showed that Firmicutes and Bacteroidetes were the most abundant bacteria in rats. The levels of 19 types of bacteria at the family level were altered in RA rats, and most of them could be regulated by ZT. This study demonstrated that metabolomics analysis is a powerful tool for providing novel insight into RA and for elucidating the potential mechanism of ZT.

' Candidatus Liberibacter asiaticus' (Las) is the most prevalent bacterium associated with huanglongbing, which is one of the most destructive diseases of citrus. In this paper, an extremely rapid and simple method for field detection of Las from leaf samples, based on recombinase polymerase amplification (RPA), is described. Three RPA primer pairs were designed and evaluated. RPA amplification was optimized so that it could be accomplished within 10 min. In combination with DNA crude extraction by a 50-fold dilution after 1 min of grinding in 0.5 M sodium hydroxide and visual detection via fluorescent DNA dye (positive samples display obvious green fluorescence while negative samples remain colorless), the whole detection process can be accomplished within 15 min. The sensitivity and specificity of this RPA-based method were evaluated and were proven to be equal to those of real-time PCR. The reliability of this method was also verified by analyzing field samples.

ABSTRACT:Gambogic acid (GA) is a promising natural anticancer candidate. Although the anticancer activity of GA has been well demonstrated, information regarding the metabolic fate of GA is limited. Previous studies suggested that GA is mainly excreted into intestinal tract in rats through bile after intravenous administration, whereas only traces appeared in the feces, suggesting that GA is metabolized extensively in the intestine. However, there has been no report about the intestinal metabolism of GA either in animals or humans. In this study, large amounts of two sulfonic acid metabolites of GA were found in the feces samples of rats after intravenous administration, and their structures were identified as 10-␣ sulfonic acid GA and 10-␤ sulfonic acid GA by comparison of the retention times and spectral data with those of synthesized reference substances using liquid chromatography-diode array detector-tandem mass spectrometry. This rare intestinal metabolic pathway mainly involves Michael addition of the sulfite ion to the 9,10 carbon-carbon double bond of ␣,␤-unsaturated ketone. In addition, a more detailed metabolic profile in rats is proposed, according to the results of in vitro and in vivo studies. It was found that GA can be metabolized by a variety of routes, including monooxidation, hydration, glutathionylation, glucuronidation, and glucosidation in the liver of rats. These findings provide information on the major metabolic soft spot of GA in the intestine and liver of rats, which is not only useful in the future human metabolic study of this compound but also of value in the metabolic studies of GA analogs.

Huanglongbing is a devastating citrus disease, and 'Candidatus Liberibacter asiaticus' (Las) is the most prevalent huanglongbing-associated bacterium. Its field detection remains challenging. In this work, a visual, rapid, sensitive, and carryover contamination-free method was developed for field detection of Las. Leaf samples were treated with 500 μL of 0.5 M sodium hydroxide solution for 3 min, and 50-fold dilutions were directly amplified by loop-mediated isothermal amplification. Then, a novel SYTO-9-based visual detection method was used to evaluate amplification results without uncapping operation. Negative samples remained colorless, while positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye with a mini-fluorescent-emission cartridge developed originally. The proposed detection method could be accomplished within 40 min and is about 100 times more sensitive than conventional TaqMan polymerase chain reaction. The reliability of this method was also verified by analyzing practical samples.