Miniaturized continuous-flow polymerase chain reaction microfluidic chip system

A miniaturized continuous-flow polymerase chain reaction (PCR) microfluidic chip system was developed to perform DNA amplification. This system consists of a 20-cycle continuous-flow PCR microfluidic chip, an electrical heating system and a miniature air pressure-vacuum pump. The chip was ablated with excimer laser direct-writing micromachining technique on a polymethyl methacrylate (PMMA) sheet. The ablated microchannel was inverse trapezoidal with a depth of 70 μm, top width of 200 μm and bottom width of 120 μm. Its surface roughness Ra was 1.42 μm after being treated with excimer laser polishing. The substrate sheet ablated with the microchannel was bonded with other cover sheets using hot-press bonding method to form a closed structure. The electrical heating system consisted of three groups of heating membranes, Pt100 sensors, copper blocks and PID temperature digital controllers. It could provide three distinct maintained temperature zones and a uniform temperature distribution in each zone. PCR amplification of a 170 base pair (bp) DNA fragment was carried out to validate the system's feasibility. The PCR temperatures were set as 94℃ for denaturation, 55℃ for primer annealing and 72℃ for extension. The flow rate in the microchannel was 40 nL/s and the total time for the completion of a 20-cycle amplification of 20 μL reagent was 15 min.     

Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans.

We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3'-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo.     

Cooking DNA: the effect of ‘domestic’ cooking methods on detection of GM potato

The ability to detect GM material in otherwise unprocessed foods cooked using domestic methods is important should ‘ready‐to‐eat’ foods require labelling. This study addresses the issue of DNA degradation in foods as a result of cooking. A number of ‘domestic’ cooking methods were shown to affect the length of DNA sequences able to be PCR amplified from potato samples and the degree of degradation was treatment‐specific. However, a. real‐time PCR assay was developed and. GM material was positively identified in all cooked GM potato samples. This confirms that GM material should be able to be detected in otherwise unprocessed food samples cooked using domestic methods, even if the cooking process has partially degraded the DNA. Results indicate, however, that there may be implications of the cooking process on the ability to accurately quantify GM content in some cooked samples.     

Functionalized Carbon Dot-Delivered RNA Nano Fungicides as Superior Tools to Control Phytophthora Pathogens through Plant RdRP1 Mediated Spray-Induced Gene Silencing

Spray-induced gene silencing (SIGS) represents an attractive avenue for plant protection, but limited uptake efficiency of double-stranded RNA (dsRNA) has restricted its application against the notorious oomycetes, Phytophthora. To control Phytophthora, dsRNAs targeting two essential genes in Phytophthora are screened for their ability to partially decrease Phytophthora capsici (P. capsici) infection and fecundity. To further facilitate SIGS for Phytophthora control, functionalized carbon dots (CDs) are complexed with the screened dsRNAs (dsRNA-CDs) through electrostatic interaction. dsRNA-CDs significantly enhanced the control efficacy of dsRNA against Phytophthora infestans, Phytophthora sojae, and both the wild-type and fungicide-resistant P. capsici. The synergism is based on enhancing dsRNA stability and internalization. Dual treatment with dsRNA-CDs and the corresponding fungicide reduced the amount of fungicide required to achieve the same protective effect by 90%. Plant RdRP1 is the main effector in processing various lengths of small RNAs to induce translation inhibition in Phytophthora. Notably, here the first application of a nano-delivery system to improve the effect of SIGS against Phytophthora pathogens is reported. Moreover, the elucidation of how CD facilitates dsRNA internalization in recipient cell and the molecular mechanism of SIGS can benefit the application of dsRNA-CDs in other plant-pathogen pathosystems through improving dsRNA bioactivity and reducing chemical fungicides application.     

Detection of the human specific Bacteroides genetic marker provides evidence of widespread sewage contamination of stormwater in the urban environment

Human sewage contamination of surface waters is a major human health concern. We found urban stormwater systems that collect and convey runoff from impervious surfaces act as a conduit for sewage originating from breeches in sanitary sewer infrastructure. A total of 828 samples at 45 stormwater outfalls were collected over a four-year period and assessed by culture based methods, PCR, and quantitative PCR (qPCR) to test for traditional and alternative indicators of fecal pollution. All outfalls had the HF183 (human) Bacteroides genetic marker detected in at least one sample, suggesting sewage contamination is nearly ubiquitous in the urban environment. However, most outfalls were intermittently positive, ranging from detection in 11%-100% of the samples. Positive results did not correlate with seasonality, rainfall amounts, or days since previous rainfall. Approximately two-thirds of the outfalls had high (>5000 copy number, i.e. CN, per 100 ml) or moderate levels (1000-5000 CN per 100 ml) of the human Bacteroides genetic marker. Escherichia coli (E. coli) and enterococci levels did not correlate to human Bacteroides. A total of 66% of all outfall samples had standard fecal indicator levels above 10,000 CFU per 100 ml. A tiered assessment using this benchmark to identify high priority sites would have failed to flag 35% of the samples that had evidence of sewage contamination. In addition, high fecal indicators would have flagged 33% of samples as priority that had low or no evidence of sewage. Enteric virus levels in one outfall with high levels of the human Bacteroides genetic marker were similar to untreated wastewater, which illustrates stormwater can serve as a pathway for pathogen contamination. The major source of fecal pollution at four of five river sites that receive stormwater discharge appeared to be from sewage sources rather than non-human sources based on the ratios of human Bacteroides to total Bacteroides spp. This study shows the feasibility and benefits of employing molecular methods to test for alternative indicators of fecal pollution to identify sewage sources and potential health risks and for prioritization of remediation efforts.     

玛卡及其制品的PCR方法鉴别研究

本研究通过玛卡的defensin基因序列,设计了玛卡的特异性引物,建立了玛卡及其制品中玛卡源性成分检测的一种新方法。通过物种特异性测试,所设计的引物能够特异性扩增玛卡基因,并且,DNA浓度的检测灵敏度达到0.1 ng/μL以上,重量灵敏度达到0.1%以上。并结合芜菁源性成分基因检测方法对市场随机抽取的21份玛卡及其制品进行检测,21份样品均检出了玛卡源性成分而未检出芜菁源性成分。本研究所建立的PCR检测方法快速,准确,高效,适用于市场上玛卡及其制品真伪的快速鉴别。      

创伤弧菌的优化培养及快速检测

研究了创伤弧菌优化培养的方案,用以提高该菌的检出率。利用Design-Expert软件中的Box-Behnken中心组合实验原理,设计一组3因素3水平实验,通过响应曲面分析,得到优化培养参数为:含盐量3.65%,pH6.75,培养温度37.00℃,在该条件下培养液的OD595nm为0.520。利用创伤弧菌优化培养条件对市售海产样品进行了培养,并通过聚合酶链式反应（Polymerase Chain Reaction,PCR）对样品进行快速检测,且该菌的检出率高达23.3%。结果表明:应用本实验的优化培养方案、PCR法能快速有效检测水产品中存在的创伤弧菌。      

State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2

Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a “bucket brigade.” This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication.