An in-situ self-etching enabled high-power electrode for aqueous zinc-ion batteries

Sluggish storage kinetics is considered as the main bottleneck of cathode materials for fast-charging aqueous zinc-ion batteries(AZIBs).In this report,we propose a novel in-situ self-etching strategy to unlock the Palm tree-like vanadium oxide/carbon nanofiber membrane(P-VO/C)as a robust freestanding electrode.Comprehensive investigations including the finite element simulation,in-situ X-ray diffraction,and in-situ electrochemical impedance spectroscopy disclosed it an electrochemically induced phase transformation mechanism from VO to layered Zn_(x)V_(2)O_5·nH_(2)O,as well as superior storage kinetics with ultrahigh pseudocapacitive contribution.As demonstrated,such electrode can remain a specific capacity of 285 mA h g^(-1)after 100 cycles at 1 A g^(-1),144.4 mA h g^(-1)after 1500 cycles at 30 A g^(-1),and even 97 mA h g^(-1)after 3000 cycles at 60 A g^(-1),respectively.Unexpectedly,an impressive power density of 78.9 kW kg^(-1)at the super-high current density of 100 A g^(-1)also can be achieved.Such design concept of in-situ self-etching free-standing electrode can provide a brand-new insight into extending the pseudocapacitive storage limit,so as to promote the development of high-power energy storage devices including but not limited to AZIBs.

Effect of addition of chitosan to self-etching primer:antibacterial activity and push-out bond strength to radicular dentin

The purpose of this study was to evaluate the antibacterial activity of a modified self-etching primer incorporating chitosan and whether this modification affected the bond strength to radicular dentin.A modified self-etching primer was prepared by adding chitosan solutions at 0.03%,0.06%,0.12% and 0.25%（W/W） to RealSeal selfetching primer.RealSeal primer without chitosan was used as the control.The antibacterial activity of the modified self-etching primer was evaluated using the direct contact test against Enterococcus faecalis.The bonding ability of the RealSeal system to radicular dentin was evaluated using the push-out bond strength test.The modes of failure were examined under a stereomicroscope.Data were analyzed using analysis of variance（ANOVA） and Tukey’s test,with a P-value 〈 0.05 indicating statistical significance.The results showed that the antibacterial properties of the freshly prepared and aged modified self-etching primer incorporating chitosan exhibited potent antibacterial effect against Enterococcus faecalis compared with the unmodified primer.The RealSeal system with the aged modified self-etching primer incorporating chitosan showed no significant differences in the bond strength as compared with the control（P = 0.99）.The findings suggest that modified self-etching primer incorporating chitosan is a promising antibacterial primer which does not adversely affect the bond strength of the RealSeal system to radicular dentin.

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Screening of SSR Core Primers for Purity Identification of Pepper(Capsicum) Hybrids

[Objective] This study aimed to screen a set of SSR core primers suitable for purity identification of pepper （Capsicum） hybrids. [Method] DNA fingerprint of 100 pepper hybrids was analyzed using 17 SSR primers. [Result] According to the polymorphism and heterozygosity, Hpms1-214, Es395 and Hpmsl-5 were determined as three preferred core primers for purity identification of pepper hybrids. By using these three preferred core primers, 97 pepper hybrids （accounting for 97%） had heterozygous band pattern with at least one primer. Es330, Es363, Epms923, Es120 and Es64 were determined as candidate core primers for purity identification of pepper hybrids. Specific primers of 14 varieties were obtained, which could be used to further screen parent-complementary primers of each pepper hybrid. [Con- clusion] This study laid the foundation for constructing standard DNA fingerprints for purity identification of pepper hybrids.

3'-terminus shifted bases degeneracy primers increasing sensitivity of polymerase chain reaction

目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。

Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers

In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.

Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Development of Species-Specific PCR Primers and Sensitive Detection of the Tylenchulus semipenetrans in China

Tylenchulus semipenetrans is the most economically important and widespread nematode pest of citrus in China.rDNA-ITS of 14 populations of T.semipenetrans which were collected from different citrus groves or Chinese fir（Cunninghamia lanceolata） plantations in China were amplified and sequenced.The species-specific primers were designed for the first time to diagnosis T.semipenetrans based on the sequences of rDNA-ITS regions of geographic population above.The primers were sensitive to amplify the expected band size（297 bp） from DNA template of a single second-stage juvenile（J2） or different life stages of T.semipenetrans.No specific band was amplified from 15 non-target nematode species which were commonly found in citrus groves.Specificity and reliability of the primers were validated by further PCR amplification of 16 extra populations of T.semipenetrans collected from 4 provinces of China.The primers successfully detected a single J2 of T.semipenetrans within a whole nematode community comprising a large numbers of non-target nematode.The developed diagnostic technique can be used for accurate identification of T.semipenetrans and also as a decision tool for nematode management for citrus or Chinese fir in China.

An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens

Objective To provide a feasible and cost-effective next-generation sequencing （NGS） method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure： reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide （random hexamer or non-ribosomal hexanucleotide） primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease （HFMD） and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Species-specific COI primers for rapid identification of a globally significant invasive pest, the cassava mealybug Phenacoccus manihoti Matile-Ferrero

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava,and its recent introduction into Asia has raised considerable alarm.To slow or prevent further invasion,an accurate,simple,and developmental-stage-independent detection method for P.manihoti is required.In the present study,a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I(SS-COI)marker was developed for rapid identification of P.manihoti.One pair of SS-COI primers(PMSSZW-1F and PMSSZW-1R)was designed based on sequence variations in the COI gene among P.manihoti and related mealybug species.Specificity of the primer pair was validated on 21 closely related species.Sensitivity tests were performed on four immature developmental stages and female adults.Efficacy tests demonstrated that at the relatively low concentration of(135.2±14.7)pgresuspended DNA,the specific fragment was detected in all replicates.Furthermore,the SS-COI primer pair was assayed on three populations of P.manihoti from major exporting countries of cassava.The PCR assay was proved to be a rapid,simple,and reliable molecular measure for the identification of P.manihoti.This tool will be useful for quarantine,monitoring,and management of this invasive pest.

Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment

AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD).METHODS: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specifi c to rtL180M and rtM204V were selected for YVDD detection. Primer specif ic to rtM204I with an additional 3’-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.RESULTS: We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 108-109 copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations. CONCLUSION: We developed a highly sensitive and specifi c assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.

Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene （rDNA） fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

Studies on the Primers Screening for AFLP Fingerprints of Rice Cultivars

AFLP(amplified fragment length polymorphism) is a very powerful fingerprinting technology. The key of making variety fingerprints is to select specific powerful primers for each crop. A quick and effective procedure for selecting AFLP primers for rice variety fingerprinting was established as the following: (1) Choose3 or more group materials that have close genetic relations. (2) Select potential polymorphic primers from primer pairs that are 2 + 2 primer crosses and same at two ends. (3) Recombine the selected potential polymorphic primers and choosing more polymorphic primers. (4) Add one selecting base at one end to become 2 + 3 or 3 + 2 primers and further selecting more polymorphic primers. Some primers were selected with this procedure, such as M21 P87 and M73P17, with which the fingerprints had more polymorphism and high quality.

Effect of Multiple Coatings of One-step Self-etching Adhesive on Microtensile Bond Strength to Primary Dentin

Objective To investigate the effect of multiple coatings of the one-step self-etching adhesive on immediate microtensile bond strength to primary dentin.Methods Twelve caries-free human primary molars were randomly divided into 2 groups with 6 teeth each.In group 1,each tooth was hemisected into two halves.One half was assigned to control subgroup 1,which was bonded with a single-step self-etching adhesive according to the manufacturer's instructions;the other half was assigned to experimental subgroup 1 in which the adhesive was applied three times before light curing.In group 2,the teeth were also hemisected into two halves.One half was assigned to control subgroup 2,which was bonded with the single-step self-etching adhesive according to the manufacturer's instructions;the other half was assigned to experimental subgroup 2 in which three layers of adhesive were applied with light curing each successive layer.Microtensile bond strength was immediately tested after specimen preparation.Results When the adhesive was applied three times before light curing,the bond strength of the experimental subgroup 1(n=33,57.49±11.61 MPa) was higher than that of the control subgroup 1(n=31,49.71±11.43 MPa,P<0.05).When using the technique of applying multiple layers of adhesive with light curing each successive layer,no difference of immediate bond strength was observed between the control subgroup 2 and the experimental subgroup 2(P>0.05).Conclusion Multiple coatings of one-step self-etching adhesive can increase the immediate bond strength to primary dentin when using the technique of light-curing after applying three layers of adhesive.

Effect of Saliva Contamination on Shear Bond Strength of Orthodontic Brackets When Using a Self-Etch Primer

The effect of saliva contamination on the shear bond strength of orthodontic brackets, at various stages of the bonding procedure using a new self-etch primer was studied. The samples were divided into 4 groups according to 4 different enamel surface conditions: Group A: dry; Group B: saliva contamination before priming; Group C: saliva contamination after priming, and Group D: saliva contamination before and after priming. Stainless steel brackets were bonded in each test group with a light-cured composite resin (TransbondXT 3M). The shear bond strength was determined in the first 30 min after bonding. The analysis of variance indicated that the shear bond strengths of the 4 groups were significantly different (F=11.89, P<0.05). Tukey HSD tests indicated that contamination both before and after the application of the acid-etch primer resulted in a significantly lower (=4.6±1.7 MPa) shear bond strength than either the control group (=8.8±1.9 MPa) or the groups where contamination occurred either before (=7.9±2.0 MPa) or after (=6.9±1.5 MPa) the application of the primer. It was concluded that the new acid-etch primer could maintain adequate shear bond strength if contamination occurred either before or after the application of the primer. On the other hand, contamination both before and after the application of the primer could significantly reduce the shear bond strength of orthodontic brackets.

Preliminary Study on Applicability of Microsatellite DNA Primers from Parasite Protozoa Trypanosoma cruzi in Free-living Protozoa

In this paper, we took the lead in studying on specificity of the microsatellite DNA loci and applicability of mi crosatellite DNA primers in protozoa. In order to study characters of microsatellites in free living protozoa, eight microsatellite loci primers developed from Trypanosoma cruzi (MCLE01, SCLE10, MCLE08, SCLE11, MCLF10, MCLG10, MCL03, MCL05) were employed to amplify microsatellite in four free living protozoa, including Bodo designis, Euglena gracilis FACHB848, Paramecium bruzise and Tetrahymena thermophila BF1. In the amplification systems of P. bruzise, four loci (SCLE10, SCLE11, MCLF10, MCL03) were amplified successfully, and four amplification fragments were in proper size. In genome of E. gracilis FACHB848, five of eight primers brought five clear amplification bands. In B. designis, three (No.4, 5 and 7) of eight loci produced clear and sharp products without stutter bands, whereas no bands appeared in T. thermophila BF1. Further, eight 300-500 bp amplification fragments were cloned and sequenced. Nevertheless, all sequenced products did not contain corresponding microsatellite sequence, although Bodo is in the same order and has the nearest phylogenetic relation with Trypanosoma among these four species. Thus, the microsatellite DNA primers can not be applied among order or more far taxa, and the specificity of microsatellite DNA is very high in protozoa. The results of this study will contribute to our understanding of microsatellite DNA in protozoa.

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS)region of peritrich ciliates in environmental samples

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.

Heavy metal free primers: Polymorphism of gadolinium and titanium in the context of GSR glass phase

The possibility of identifying gunshot residue (GSR) particles produced by non-toxic primers containing only titanium and zinc is a very difficult task using SEM/EDX analysis employed in the analysis of GSR originating from primers containing lead, barium and antimony. However, Bauer et al. demonstrated that non-toxic (TieZn) primers form a TiZn2O4 spinel crystalline structure using SEM/EDX with EBSD (Electron Back Scatter Diffraction) and TKD (Transmission Kikuchi Diffraction), whereas GSR originating from gadolinium-doped TieZn primers form a non-crystalline glass phase. Here, a possible explanation of these different phenomena is hypothesized.

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing was developed by a synthesis method （DP-SBS） for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number （n） of degenerate nucleotides at the 3＇-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides （n＋1） on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.