Optimized Reaction System Research Articles

Development of a visual detection method of porcine deltacoronavirus using loop-mediated isothermal amplification.

The emergence of porcine deltacoronavirus (PDCoV) presents a significant threat to both human and animal health due to its ability to cause highly contagious enteric diseases. This underscores the crucial need for timely and accurate diagnosis to facilitate effective epidemiological investigation and clinical management. This research aimed to establish a visual detection method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for PDCoV testing. In this study, six pairs of primers were designed according to the conserved sequences of PDCoV ORF1a/b genes. The primer sets and parameters that affect LAMP reaction were optimized. The visual RT-LAMP method was developed by incorporating methyl red into the optimized reaction system, it exclusively detected PDCoV without cross-reactivity with other viruses and the detection limits for PDCoV could reach 10 copies/μL. In comparison with RT-PCR for testing 132 clinical samples, the relative specificity and sensitivity of the visual RT-LAMP were found to be 99.2 and 100%, respectively, with a concordance rate of 99.2% and a kappa value of 0.959, indicating that the visual RT-LAMP is a reliable method for the application of PDCoV detection in clinical samples.

First specific detection and validation of tomato wilt caused by Fusarium brachygibbosum using a PCR assay.

Tomato wilt is a widespread soilborne disease of tomato that has caused significant yield losses in many tomato growing regions of the world. Previously, it was reported that tomato wilt can be caused by many pathogens, such as Fusarium oxysporum, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Fusarium acuminatum, and Plectosphaerella cucumerina. In addition, we have already reported that Fusarium brachygibbosum caused symptomatic disease of tomato wilt for the first time in China. The symptoms of tomato wilt caused by these pathogens are similar, making it difficult to distinguish them in the field. However, F. brachygibbosum specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for Fusarium brachygibbosum to establish a more effective plan to control the disease. In this study, we designed F. brachygibbosum-specific forward primers and reverse primers with a fragment size of 283bp located in the gene encoding carbamoyl phosphate synthase arginine-specific large chain by whole genome sequence comparison analysis of the genomes of eight Fusarium spp.. We then tested different dNTP, Mg2+ concentrations, and annealing temperatures to determine the optimal parameters for the PCR system. We evaluated the specificity, sensitivity and stability of the PCR system based on the optimized reaction system and conditions. The PCR system can specifically identify the target pathogens from different fungal pathogens, and the lower detection limit of the target pathogens is at concentrations of 10 pg/uL. In addition, we can accurately identify F. brachygibbosum in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose F. brachygibbosum.

In situ transesterification of the catolé coconut (Syagrus Cearensis) utilizing experimental design by Taguchi method

This work proposes the optimization of biodiesel synthesis utilizing in situ transesterification, through experimental design by the Taguchi method. In this process, nine experiments were carried out and the influence of reaction parameters as catalyst mass, proportion of methanol in relation to oil content and temperature were statistically evaluated. The mass yield was analyzed as a response. The optimized condition was obtained through the reaction with 2 wt% of catalyst, ratio of methanol to oil 5:1 content and 60ºC. The experiment generated a yield of 66.17 wt%. In the experimental study, the evaluated parameters presented relevant p-value, with the catalyst rate being the factor that most impacted the synthesis yield. The synthesized biodiesel obtained from the confirmation experiment of the Taguchi modelling was characterized through the physical-chemical parameters. The material complies with international standards for biodiesel, highlighting the result of oxidative stability greater than 18 h, the combustion events in thermal analysis ranging from 224 to 258 ºC, and activation energy of 67.3 KJ.mol−1, justified by the profile of mostly saturated fatty acids demonstrated in the chromatography analysis. From nuclear magnetic resonance analysis, the synthesis proved to be effective to convert all the triglycerides extracted from the kernel into biodiesel, without the necessity of additional steps of oil extraction and purification. In this sense, an optimized reaction system allied to biodiesel with expressive quality makes catolé coconut a raw material with high commercial potential. The use of Taguchi design to obtain the best conditions for the reaction allowed for an easy interpretation of the results, providing good comprehension of the impacts of the reaction parameters in the synthesis.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

Engineering of an ene-reductase for producing the key intermediate of antiepileptic drug Brivaracetam.

(R)-4-Propyldihydrofuran-2(3H)-one (R-PDFO) is the key chiral intermediate for the antiepileptic drug Brivaracetam. Lacking a simple and economical method to approaching R-PDFO, the production of R-PDFO also remains environmentally unfriendly. Here, we developed a straightforward bioreduction way from easily synthesized 4-propylfuran-2(5H)-one (PFO) using ene-reductases. After screened with 27 ene-reductases, E116 stood out with 25.7% yield and 97% ee (R) as the starting enzyme. To improve the catalytic efficiency of E116, several rounds of directed evolution were first carried out. Through rational design, alanine scanning and random mutagenesis, engineered ene-reductase E116-M3 was obtained, with a 2.63-fold improvement in yields over WT, a 12.6-fold improvement in kcat/Km over WT, and stereoselectivity increased to 99% (R). To further improve the yield of R-PDFO, the reaction conditions were then optimized. The catalytic activity of the optimized reaction system was increased again by 2.3 times and the turnover number (TON) of E116-M3 reached 705. Subsequently, whole cells harboring E116-M3 were also shown to have similar capabilities of synthesizing R-PDFO. Finally, E116-M3 was employed in the 50-mL-scale synthesis of R-PDFO under 20mM of PFO loading to achieve 81% isolated yield and 99% ee. In conclusion, this new approach of engineered ene-reductase catalyzing the asymmetric reduction of PFO could be a green alternative for the efficient synthesis of R-PDFO. KEY POINTS: • An ene-reductase library was first used to screen the bioreduction of PFO. • Rational design contributed to the enhanced R-stereoselectivity of PFO reduction. • E116-M3 was obtained with high activity and stereoselectivity for R-PDFO.

Conversion of Dioscorea zingiberensis saponins to prosapogenin A by enzymatic hydrolysis

Prosapogenin A is a secondary saponin in Dioscorea zingiberensis, and it showed remarkable pharmacological effects. Due to very low content and lack of well-developed biotransformation, its preparation was not efficient and clean. This study aims to establish an eco-friendly strategy for preparation of Prosapogenin A from plant material. Physical separation was employed to recycle starch and cellulose, and then D101 resin and polyamide packed-bed column was incorporated for purification of total steroidal saponins (TSS). After these pretreatments, purity of TSS was largely increased to 83.2% with recovery at 87.6%, which was subjected to enzymatic hydrolysis. Optimized reaction system was constructed in 0.20 M HAc-NaAc buffer (pH4.2) containing cellulase/TSS (3:1, w/w), and the hydrolysis was performed at 53 °C for 6 h. Consequently, TSS was almost completely hydrolyzed to Prosapogenin A, while the highest yield reached 5.62%. The newly proposed approach is promising for efficient preparation of Prosapogenin A in industrial applications.

Rapid visualization and detection of Staphylococcus aureus based on loop-mediated isothermal amplification.

Staphylococcus aureus is a common clinical bacterial pathogen that can cause a diverse range of infections. The establishment of a rapid and reliable assay for the early diagnosis and detection of S. aureus is of great significance. In this study, we developed a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus using the colorimetric indicator hydroxy naphthol blue (HNB). The LAMP reaction was optimized by adjusting the amplification temperature, the concentrations of Mg2+, dNTP, and HNB, and the incubation time. In the optimized reaction system, the specificity of LAMP for S. aureus was 100%. The results established that this method accurately identified S. aureus, with no cross-reactivity with 14 non-S. aureus strains. The limit of detection (LOD) of LAMP was 8 copies/reaction of purified plasmid DNA or 400 colony-forming units/reaction of S. aureus. Compared with conventional PCR, LAMP lowered the LOD by tenfold. Finally, 220 clinically isolated strains of S. aureus and 149 non-S. aureus strains were used to evaluate the diagnostic efficacy of LAMP (test accuracy, 99.46%). The findings indicated that LAMP is a reliable test for S. aureus and could be a promising tool for the rapid diagnosis of S. aureus infections.

Establishment of rapid detection method for multiple real-time fluorescent PCR of toxin-producing fungi

To establish a multiplex real-time fluorescent PCR rapid detection method for simultaneous detection of aflatoxin-producing fungi and three latent toxin-producing fungi in a single system. Primers and probes were designed based on the protein activation genes AflR, ITS sequence, β-tubulin coding region and LS rRNA of aflatoxin-producing fungi, Aspergillusochraceus, Penicillium and Fusarium, respectively. A real-time fluorescent PCR method for simultaneous detection of commontoxin-producing fungi was performed, and the sensitivity and specificity of the method were analyzed by optimizing the reaction components and reaction conditions. The detection limits of the optimized reaction system for aflatoxin-producing fungi, Aspergillusochraceus, Penicillium and Fusarium were 3. 37×10~4, 1. 91×10~4, 1. 53×10~4, 3. 95×10~4 copies/reaction, respectively. The multiplex real-time fluorescent PCR assay established in this study is highly specific and sensitive. It can detect toxin-producing fungi within 2 hours and provide technical support for fungal monitoring in food.

Novel fructooligosaccharide conversion from sugarcane syrup using a specialised enzymatic pH-stat bioreactor

Fructooligosaccharides (FOS) have gained significant attention for their prebiotic properties. Given that sugarcane syrup (SS) is sucrose-rich but with other nutritional benefits, its direct transformation into FOS may add value to this product. Therefore, the aims of this study were to develop FOS conversion from SS and to define the kinetic behaviour of FOS synthesized in a 1-L specialized pH-stat bioreactor (SPSB). The SS was composed of sucrose (58.93%) with considerable antioxidant capacities and Ƴ-aminobutyric acid. The developed SPSB process consisted of three stages: evaporation of sugarcane juice into syrup (68–75 °Brix) (stage 1), optimization of the Viscozyme L and SS mixture at different reaction temperatures (47–55 °C) (stage 2), and upscaling of the optimized reaction system under defined conditions in a 1 L-SPSB system (stage 3). In the 1 L-SPSB system, the enzymatic reaction yielded 32.22% of FOS from SS after a 6 h reaction, which is comparable with a pure system containing an equivalent concentration of 10% of sucrose as initial substrate with 39.55% yield. This result demonstrated the efficient conversion of SS into FOS, supporting the utilization of sugarcane juice for its health benefits.

Simulation and optimization of Novozym 40086 kinetic resolution of α‐cyclopentylphenylacetic acid enantiomers

AbstractBACKGROUNDEnzymatic resolution of enantiomers by hydrolysis is a popular method, yet most enantiomers have low solubility in the aqueous phase, resulting in low conversion. Notwithstanding this, the simulation and optimization of the reaction process by modelling has been reported only rarely. Thus, PEG was added to the aqueous phase and a mathematical model was established to obtain optimal conditions.RESULTSThe effects of various parameters were investigated to build an optimized reaction system. The results indicated that temperature and pH had little effect on enantiomeric excess, but they had significant effect on conversion. Conversion was enhanced by 85% with the addition of PEG 400. Kinetic studies showed that pentanol competitively inhibited enzyme activity. A mathematical model was developed, and forward and reverse reactions were taken into consideration. Through simulation and optimization of model, the optimum conditions were obtained as follows: 10 mg mL−1 Novozym 40086, 10 mmol L−1 substrate, 30% PEG 400, temperature 45 °C, pH 6.5 and reaction time 36 h.CONCLUSIONChiral resolution of α‐cyclopentylphenylacetic acid enantiomer (CPPA) was achieved by Novozym 40086 enantioselective hydrolysis of the CPPA ester. Under optimum conditions, eep and cs were 95.83% and 88.52%, respectively. The relative errors between predicted values and experimental values were <5%, confirming that the model could accurately predict the enzymatic resolution reaction. © 2019 Society of Chemical Industry

Optimization for ISSR-PCR system of traditional Chinese medicine Lysimachia christinae by orthogonal design

In order to establish the stable andreliable ISSR-PCR System of Lysimachia christinae, L16 (4(5)) orthogonal design, which based on 7 levels of single factor experiment, were used in this study. The variance analysis was carried out by SPSS 19.0, and 5 main factors affecting the reaction system were optimized in 4 levels. The best annealing temperature was selected by the optimized reaction system. And the stability and reliability of this system was tested by 23 samples from different origins. The results showed that the five factors (DNA template, primer, dNTP, Mg2+ and Taq enzyme) were the most impacts on the amplified results of ISSR-PCR of L. christinae. The order of the influence was: primer > Taq enzyme > DNA template > Mg2+ > dNTP. The optimal system, which was determined by multiple comparison on different levels of each factor, was total volume of 25 microL, including DNA template 60 ng, primer 0.3 micromol x L(-1), dNTP 0.2 mmol x L(-1), Mg2+ 1.8 mmol x L(-1), Taq enzyme 1.25 U. The optimal system was stable and reliable tested by 23 samples from different origins. This study lays the foundation for genetic diversity analysis, fine varieties selection and molecular identification of L. christinae, and provides reference for optimization on ISSR-PCR system of other speciesin future.

Study on optimization of SRAP-PCR reaction System for Pinellia ternata in Suzhou

To investigate the optimization system of SRAP-PCR molecular marker technology in the analysis on Pinellia ternata. SRAP-PCR reaction system for P. ternata was optimized by L16 (5(4)) orthogonal design with five elements (dNTPs, Mg2+, the template DNA, primers, Taq enzyme) and four standards. The most suitable forward primer for SRAP for Pinellia ternata was 5'-TGAGTCCAAACCGGAAG-3', while the reverse primer was 5'-GACTGCGTACGAATTACG-3'. The optimized reaction system contained 70 ng DNA template, 0.9 micromol x L(-1) primer, 0.20 mmol x L(-1) dNTP s, 1.5 - 2.0 mmol x L(-1) Mg2+, and 2 U Taq enzyme. SRAP-PCR system for P. ternata is established to lay a foundation for future construction of SRAP genetic map of P. ternata.

Selective hydrosilylation of allyl esters with octahydridosilsesquioxane

Hydrosilylation of allyl esters with octahydridosilsesquioxane (T8H8) in the presence of platinum-based catalysts was studied. The effects of the catalysts and the reaction conditions on the conversion and the selectivity of the products were investigated. Karstedt's catalyst with two equivalents of PPh3 was found to be a superior hydrosilylation catalyst in terms of conversion and selectivity, compared with other Pt catalysts examined. An octasilsesquioxane functionalized with epoxy substituents was synthesized using the optimized reaction system, which was converted to a thermosetting polymer and its properties were investigated.

Establishment of an optimized PCR method using sequence-specific primers for screening multiple gene polymorphisms simultaneously

The present study aimed to explore the factors that affect polymerase chain reaction using sequence-specific primers (PCR-SSP) and to establish an optimized PCR-SSP method for detecting multiple gene polymorphisms simultaneously. The amplification system parameters, including the concentrations of Mg2+, dNTPs, pfu Taq, primers and control primers, were optimized using the designed PCR-SSP reactions. The resulting optimized reaction system was used to determine the melting temperature of the genomic DNA and the cycling parameters. The optimized PCR-SSP method was used to analyze the polymorphisms of the following genes: mutations -308A/G and -238G/A in TNF-α, -174G/C in IL-6 and C/T mutation at exon 188 of CYP2D6 *10B. The PCR-SSP amplification system was optimized; in a 20µl reaction system, the quantities of Mg2+, dNTPs, pfu Taq, primers, control primers and genomic DNA were 3.25µM, 0.5mM, 2.5units, 0.5µM, 0.2µM and 0.15µg, respectively. The cycling system comprised 5 start cycles and took 15min to melt a genomic DNA sample using a touchdown protocol. The optimized PCR-SSP is suitable for polymorphism analysis of polygenic SNPs in large genomic DNA samples and a number of different genes.

Multiplex PCR System Optimization with Potato SSR Markers

Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components (enzyme, MgCl 2 , DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L 9 (3 4 ) was applied in the optimization of four sets of primers (STM0014, Pat, SSI, and UGP) in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL: 2.5 μL 25 mmol • L −1 MgCl 2 , 0.6 μL 10 mmol • L −1 dNTPs, 0.8 U Taq , 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol • L −1 primers was 2: 1: 2: 3, and the annealing temperature was 54.7°C. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting.

ISSR analysis of Fusarium oxysporum Schl. in Hebei province

Abstract An Orthogonal design is applied to the optimization of ISSR-PCR reaction system for Fusarium oxysporum Schl. The effect of the five main factors was tested. A most suitable ISSR-PCR reaction system for Fusarium oxysporum Schl. was established. The optimized reaction system consists of 20 ng template DNA, 1 U Taq DNA polymerase, 0.4 μmol/L primer, 0.2 mmol/L dNTPs, 4.0 mmol/L Mg2+ and 2.5 μL 10× buffer. Optimal annealing temperature was decided by different primers. 2 primers with stable amplification and rich polymorphism for ISSR were selected. Based on the optimizing, 30 isolated strains from Hebei were tested in order to analyse the genetic diversity between of Fusarium oxysporum Schl. The isolated 30 strains were clustered into 2 genetic line-ages at 0. 86 genetic similarity. The genetic lineages of 30 strains showed no obvious relation with their geographical originals.

Loop-mediated isothermal amplification for detection of porcine circovirus type 2

BackgroundPorcine circovirus type 2 (PCV2) is the primary causative agent of the emerging swine disease known as postweaning multisystemic wasting syndrome (PMWS). Nowadays, polymerase chain reaction (PCR) is still the most widespread technique in pathogen detection. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method developed in 2000, will possibly replace PCR in the field of detection. To establish a LAMP method for rapid detection of PCV2, two pairs of primers were designed specially from the open reading frame 2 (ORF2) sequences of PCV2. A LAMP method for rapid detection of PCV2 was established. To compare with PCR, sensitivity and specificity of LAMP were evaluated using the optimized reaction system. The LAMP products could be determined by agarose gel electrophoresis or adding SYBR Green I dye.ResultsThe amplification of LAMP could be obtained at 63°C for 60 min. The detection limit was nearly 1 copy of DNA plasmid, more sensitive than PCR. There was no cross-reaction with porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) under the same conditions.ConclusionsLAMP is an useful rapid detection method with high sensitivity and specificity for PCV2.