Rapid on-site nucleic acid testing: On-chip sample preparation, amplification, and detection, and their integration into all-in-one systems.

As nucleic acid testing is playing a vital role in increasingly many research fields, the need for rapid on-site testing methods is also increasing. The test procedure often consists of three steps: Sample preparation, amplification, and detection. This review covers recent advances in on-chip methods for each of these three steps and explains the principles underlying related methods. The sample preparation process is further divided into cell lysis and nucleic acid purification, and methods for the integration of these two steps on a single chip are discussed. Under amplification, on-chip studies based on PCR and isothermal amplification are covered. Three isothermal amplification methods reported to have good resistance to PCR inhibitors are selected for discussion due to their potential for use in direct amplification. Chip designs and novel strategies employed to achieve rapid extraction/amplification with satisfactory efficiency are discussed. Four detection methods providing rapid responses (fluorescent, optical, and electrochemical detection methods, plus lateral flow assay) are evaluated for their potential in rapid on-site detection. In the final section, we discuss strategies to improve the speed of the entire procedure and to integrate all three steps onto a single chip; we also comment on recent advances, and on obstacles to reducing the cost of chip manufacture and achieving mass production. We conclude that future trends will focus on effective nucleic acid extraction via combined methods and direct amplification via isothermal methods.

Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick.

The worrying emergence of multiple resistance genes to last-resort antibiotics in food animals and human populations throughout the food chain and relevant environments has been increasingly reported worldwide. Enterobacteriaceae pathogens are considered the most common reservoirs of such antibiotic resistance genes (ARGs). Thus, a rapid, efficient and accurate detection method to simultaneously screen and monitor such ARGs in Enterobacteriaceae pathogens has become an urgent need. Our study developed a recombinase polymerase amplification (RPA) assay combined with a lateral flow dipstick (LFD) for simultaneously detecting predominant resistance genes to last-resort antibiotics of Enterobacteriaceae pathogens, including mcr-1, blaNDM-1 and tet(X4). It is allowed to complete the entire process, including crude DNA extraction, amplification as well as reading, within 40 min at 37°C, and the detection limit is 101 copies/µl for mcr-1, blaNDM-1 and tet(X4). Sensitivity analysis showed obvious association of color signals with the template concentrations of mcr-1, blaNDM-1 and tet(X4) genes in Enterobacteriaceae pathogens using a test strip reader (R 2 = 0.9881, R 2 = 0.9745, and R 2 = 0.9807, respectively), allowing for quantitative detection using multiplex RPA-LFD assays. Therefore, the RPA-LFD assay can suitably help to detect multiple resistance genes to last-resort antibiotics in foodborne pathogens and has potential applications in the field.

Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral.

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

Melting Curve Analysis of Aptachains: Adenosine Detection with Internal Calibration.

Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak.

High-Resolution Melting Analysis of COI Sequences Distinguishes Pufferfish Species (Takifugu spp.) in China.

Pufferfish is a traditional, delicious dish in Asia. However, eating wild or improperly processed pufferfish causes serious poisoning. This study aimed to exploit the high-resolution melting (HRM) method for authenticating four species of Takifugu pufferfish (Takifugu xanthopterus, T. fasciatus, T. flavidus, and T. rubripes). Candidate DNA barcodes, including the cytochrome c oxidase subunit I (COI), cytochrome oxidase b (Cytb), and the control region (D-loop), were analyzed, with COI selected as the optimal DNA barcode. An HRM method was developed to identify 57 commercial fish samples in China, including 33 commercial pufferfish products and 24 unlabeled fish products. The findings revealed that the pufferfish products were T. rubripes or T. fasciatus, and four T. xanthopterus samples were detected in unlabeled fish products. These results showed that DNA barcode coupled with HRM analysis was a rapid and efficient tool to identify pufferfish, which might aid in the prevention of consumer fraud or mislabeling of fish products.

Efficient whole-cell oxidation of α,ß-unsaturated alcohols to α,ß-unsaturated aldehydes through the cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin.

BACKGROUND: α,ß-Unsaturated aldehydes are widely used in the organic synthesis of fine chemicals for application in products such as flavoring agents, fragrances and pharmaceuticals. In the selective oxidation of α,ß-unsaturated alcohols to the corresponding α,ß-unsaturated aldehydes, it remains challenging to overcome poor selectivity, overoxidation and a low atom efficiency in chemical routes. RESULTS: An E. coli strain coexpressing the NADP+-specific alcohol dehydrogenase YsADH and the oxygen-dependent NADPH oxidase TkNOX was constructed; these components enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with a yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin protein VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB were generated, which completely converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions for the cascade biocatalysis were optimized, in which supplementation with 0.2 mM FAD and 0.4 mM NADP+ was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,ß-unsaturated aldehydes through the selective oxidation of various α,ß-unsaturated alcohols. CONCLUSIONS: The construction of a strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB achieved efficient NADP+ regeneration and the selective oxidation of various α,ß-unsaturated alcohols to the corresponding α,ß-unsaturated aldehydes. Among the available redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the most recent successful example to improve catalytic performance in comparison with its separate components.

Linear Chain Formation of Split-Aptamer Dimers on Surfaces Triggered by Adenosine.

The detection of small molecules impacts various fields; however, their small size and low concentration are usually the cause of limitations in their detection. Thus, the need for biosensors with appropriate probes and signal amplification strategies is required. Aptamers are appropriate probes selected specifically against small targets such as adenosine. The possibility to split aptamers in parts led to original amplification strategies based on sandwich assays. By combining the self-assembling of oligonucleotide dimers with split-aptamer dangling ends and a surface plasmon resonance imaging technique, we developed an original amplification approach based on linear chain formation in the presence of the adenosine target. In this article, on the basis of sequence engineering, we analyzed its performance and the effect of the probe grafting density on the length of the chains formed at the surface of the biosensor.