Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challengin...Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.展开更多
云纹石斑鱼(Epinephelus moara)和褐石斑鱼(E.bruneus)为石斑鱼属内近缘种,外形相似常被混淆.本研究改良建立了Nest-tetra-primer specific PCR方法,获得了云纹石斑鱼和褐石斑鱼线粒体DNAND2基因内的3个特异性条带,分别为内参序列NC1(39...云纹石斑鱼(Epinephelus moara)和褐石斑鱼(E.bruneus)为石斑鱼属内近缘种,外形相似常被混淆.本研究改良建立了Nest-tetra-primer specific PCR方法,获得了云纹石斑鱼和褐石斑鱼线粒体DNAND2基因内的3个特异性条带,分别为内参序列NC1(394bp)、特异性条带ND2-M(268bp)和ND2-B(122bp),以及核基因组中核糖体DNAITS1区的5个特异性条带,分别为内参序列NC2(588bp)、NC3(563bp),特异性条带rDNA-M(426bp)、ITS1-M(488bp)和ITS1-B(304bp).研究结果不仅为两种石斑鱼的鉴别提供了稳定、可靠、快捷的特异性分子标记,而且也为鱼类近缘种的DNA鉴别提供了新的途径.展开更多
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 ...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.展开更多
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 acc...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.展开更多
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-aspartat...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.展开更多
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 co...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.展开更多
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 experti...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.展开更多
In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic spe...In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.展开更多
Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biologica...Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.展开更多
Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA...Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.展开更多
<div style="text-align:justify;"> Polymerase Chain Reaction (PCR) is an accurate, simple and fast analytical method. This technique is widely used in the identification of meat adulteration and meat-ba...<div style="text-align:justify;"> Polymerase Chain Reaction (PCR) is an accurate, simple and fast analytical method. This technique is widely used in the identification of meat adulteration and meat-based processed food products. Three Mitochondrial DNA (mt-DNA) primers NADH Dehydrogenase sub unit 5 (<em>ND5</em>), <em>D-Loop</em>, and Cytochrome b (<em>Cyt-b</em>) were tested for their specificity in detecting of pig (<em>Sus scrofa</em>) DNA fragments. DNA genome from 6 meat samples (pork, beef, goat, lamb, and chicken) was amplified by PCR technique using three pairs of primers (<em>ND5, D-Loop</em><em>, </em>and <em>Cyt-b</em>) and sequenced. The results of amplification using the three primers produced specific DNA bands with the lengths of 232 bp, 951 bp, and 404 bp, respectively. Comparison results with<em> ND5, D-Loop,</em> and <em>Cyt-b</em> gene sequences resulted in similarity values of 100%, 97%, and 99%, respectively. These showed that the mt-DNA primers of <em>ND5, D-Loop</em>, and<em> Cyt-b </em>genes can be recommended as specific primers in detecting pig (<em>Sus scrofa</em>) DNA fragments. </div>展开更多
[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit f...[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.展开更多
Conventional gas sensing materials(e.g.,metal oxides)suffer from deficient sensitivity and serve cross-sensitivity issues due to the lack of efficient adsorption sites.Herein,the heteroatom atomically doping strategy ...Conventional gas sensing materials(e.g.,metal oxides)suffer from deficient sensitivity and serve cross-sensitivity issues due to the lack of efficient adsorption sites.Herein,the heteroatom atomically doping strategy is demonstrated to significantly enhance the sensing performance of metal oxides-based gas sensing materials.Specifically,the Sn atoms were incorporated into porous Fe_(2)O_(3)in the form of atomically dispersed sites.As revealed by X-ray absorption spectroscopy and atomic-resolution scanning transmission electron microscopy,these Sn atoms successfully occupy the Fe sites in the Fe_(2)O_(3)lattice,forming the unique Sn-O-Fe sites.Compared to Fe-O-Fe sites(from bare Fe_(2)O_(3))and Sn-O-Sn sites(from SnO_(2)/Fe_(2)O_(3)with high Sn loading),the Sn-O-Fe sites on porous Fe_(2)O_(3)exhibit a superior sensitivity(Rg/Ra=2646.6)to 1 ppm NO_(2),along with dramatically increased selectivity and ultra-low limits of detection(10 ppb).Further theoretical calculations suggest that the strong adsorption of NO_(2)on Sn-O-Fe sites(N atom on Sn site,O atom on Fe site)contributes a more efficient gas response,compared to NO_(2)on Fe-O-Fe sites and other gases on Sn-O-Fe sites.Moreover,the incorporated Sn atoms reduce the bandgap of Fe_(2)O_(3),not only facilitating the electron release but also increasing the NO_(2)adsorption at a low working temperature(150°C).This work introduces an effective strategy to construct effective adsorption sites that show a unique response to specific gas molecules,potentially promoting the rational design of atomically modified gas sensing materials with high sensitivity and high selectivity.展开更多
At present,the polymerase chain reaction(PCR)amplification-based file retrieval method is the mostcommonly used and effective means of DNA file retrieval.The number of orthogonal primers limitsthe number of files that...At present,the polymerase chain reaction(PCR)amplification-based file retrieval method is the mostcommonly used and effective means of DNA file retrieval.The number of orthogonal primers limitsthe number of files that can be accurately accessed,which in turn affects the density in a single oligo poolof digital DNA storage.In this paper,a multi-mode DNA sequence design method based on PCR file retrie-val in a single oligonucleotide pool is proposed for high-capacity DNA data storage.Firstly,by analyzingthe maximum number of orthogonal primers at each predicted primer length,it was found that the rela-tionship between primer length and the maximum available primer number does not increase linearly,and the maximum number of orthogonal primers is on the order of 10^(4).Next,this paper analyzes themaximum address space capacity of DNA sequences with different types of primer binding sites for filemapping.In the case where the capacity of the primer library is R(where R is even),the number ofaddress spaces that can be mapped by the single-primer DNA sequence design scheme proposed in thispaper is four times that of the previous one,and the two-level primer DNA sequence design scheme can reach [R/2·(R/2-1)]^(2)times.Finally,a multi-mode DNA sequence generation method is designed based onthe number of files to be stored in the oligonucleotide pool,in order to meet the requirements of the ran-dom retrieval of target files in an oligonucleotide pool with large-scale file numbers.The performance ofthe primers generated by the orthogonal primer library generator proposed in this paper is verified,andthe average Gibbs free energy of the most stable heterodimer formed between the orthogonal primersproduced is−1 kcal·(mol·L^(−1))^(−1)(1 kcal=4.184 kJ).At the same time,by selectively PCR-amplifying theDNA sequences of the two-level primer binding sites for random access,the target sequence can be accu-rately read with a minimum of 10^(3) reads,when the primer binding site sequences at different positionsare mutually different.This paper provides a pipeline for orthogonal primer library generation and multi-mode mapping schemes between files and primers,which can help achieve precise random access to filesin large-scale DNA oligo pools.展开更多
Device-based measurements are recommended to improve population-based physical activity(PA)surveillance.1,2However,implementation remains challenging due to lack of consensus on analytical methods,and the most widely ...Device-based measurements are recommended to improve population-based physical activity(PA)surveillance.1,2However,implementation remains challenging due to lack of consensus on analytical methods,and the most widely used“generic”(absolute intensity)cut-point approach has limited generalisability to population-level free-living data.Further,current methods generally fail to account for differences in people's physical capacity.展开更多
Excited-state intramolecular proton-transfer(ESIPT)based fluorescence probes are particularly attractive due to their unique properties including environmental sensitivity,a large Stokes shift,and potential for ratiom...Excited-state intramolecular proton-transfer(ESIPT)based fluorescence probes are particularly attractive due to their unique properties including environmental sensitivity,a large Stokes shift,and potential for ratiometric sensing.In general,ESIPT-based fluorophore incorporates an intramolecular hydrogen bonding interaction between a hydrogen bond donor(-OH and NH_(2)are common)and a hydrogen bond acceptor(C=N and C=O).More,protection-deprotection of hydroxyl group as hydrogen bond donor could induce an off-on switch of ESIPT-based emission.Therefore,protection-deprotection of hydroxyl group has been the widely used strategy to design fluorescent probes,where the potential key issue is selecting a protective group that can specifically leave in the presence of the target analyte.In this review,we mainly summarize the specific protecting groups(sites)and deprotection mechanisms for biologically important species(including reactive sulfur species(RSS),reactive oxygen species(ROS),enzymes,etc.),and analyze the advantages and disadvantages of different protection mechanisms from some aspects including probe stability,selectivity,response rate and assay system,etc.Based on the aforementioned,we further point out the current challenges and the potential future direction for developing ESIPT-based probes.展开更多
Nanozymes,characterized by their stability,cost-effectiveness,and tunable catalytic activity,are promising alternatives to natural enzymes.However,specifically mimicking a single natural enzyme's activity presents...Nanozymes,characterized by their stability,cost-effectiveness,and tunable catalytic activity,are promising alternatives to natural enzymes.However,specifically mimicking a single natural enzyme's activity presents a challenge.By exploiting the catalytic selectivity derived from the valence-band hybridization of noble metal nanoalloys,we introduce an alloying strategy to modulate the reaction specificity of metallic nanozymes.Ag Pd nanoalloy exhibits enhanced peroxidase-like activity and eliminated oxidase-like activity by adjusting the Ag content.The introduction of Ag changes the hybrid d band energy of the alloyed metal and inhibits the O_(2)adsorption and decomposition on Pd,while improving the peroxidase mimicry by allowing for the H_(2)O_(2)activation.By exemplifying the construction of a highly sensitive and selective colorimetric glucose detection platform with its practicality validated in serum samples,this strategy pioneers a multi-noble metal nanozyme with tailored peroxidase activity based on the chemical structure engineering and would advance the development of single-catalytic function nanozymes for building exclusively specific biosensors through reducing substrate competition.展开更多
基金supported by Central Public-interest Scientific Institution Basal Research Fund(CATAS-Nos.1630152023007,1630152023011,1630152023012,1630152023013)the National Natural Science Foundation of China(Grant No.32071805).
文摘Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.
文摘云纹石斑鱼(Epinephelus moara)和褐石斑鱼(E.bruneus)为石斑鱼属内近缘种,外形相似常被混淆.本研究改良建立了Nest-tetra-primer specific PCR方法,获得了云纹石斑鱼和褐石斑鱼线粒体DNAND2基因内的3个特异性条带,分别为内参序列NC1(394bp)、特异性条带ND2-M(268bp)和ND2-B(122bp),以及核基因组中核糖体DNAITS1区的5个特异性条带,分别为内参序列NC2(588bp)、NC3(563bp),特异性条带rDNA-M(426bp)、ITS1-M(488bp)和ITS1-B(304bp).研究结果不仅为两种石斑鱼的鉴别提供了稳定、可靠、快捷的特异性分子标记,而且也为鱼类近缘种的DNA鉴别提供了新的途径.
基金supported by the National Natural Science Foundation of China (30700526)the Postdoctoral Science Foundation of China (55920)the Science Foundation of the Fujian Province,China (2009N0013)
文摘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.
基金supported by the National Key R&D Program of China(2017YFC1200600,2016YFC1201200 and 2015BAD08A16)the Science and Technology Innovation Program of CAAS(caascx-2013-2018-IAS)
文摘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.
文摘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.
基金This study was supported by grants from the International Found for Sciences(No.A/3224-1)Cooperative Project of the Key Lab of Freshwater Germplasm and Biotechnology of Chinese Ministry of Agriculture(No.LFB20040503)+1 种基金the National Natural Science Foundation of China(No.40176028)the National Key R&D Program(‘973'Program)of China(No.G1999012005).
文摘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.
基金Supported by the National Natural Science Foundation of China(Nos.31572255,41522604,31301867)the Strategic Priority Research Program of CAS(No.XDA11020702)the Science and Technology Development Program of Yantai(No.2014ZH073)
文摘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.
基金supported by grants from the Significant Special Found of "13115" S&T Innovation Project of Shaanxi Province,China(2007 ZDKG-01)"13115" Technology Innovation Engineering and Engineering Technology Research Center of Shaanxi Province,China(2008 ZDGC-02)the Special Capital for the Construction of Modern Agriculture Technical System of Shaanxi Province,China (NYCYTX-001)
文摘In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.
基金funded by a FPU a grant from the Ministry of Education of Spain
文摘Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.
文摘Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.
文摘<div style="text-align:justify;"> Polymerase Chain Reaction (PCR) is an accurate, simple and fast analytical method. This technique is widely used in the identification of meat adulteration and meat-based processed food products. Three Mitochondrial DNA (mt-DNA) primers NADH Dehydrogenase sub unit 5 (<em>ND5</em>), <em>D-Loop</em>, and Cytochrome b (<em>Cyt-b</em>) were tested for their specificity in detecting of pig (<em>Sus scrofa</em>) DNA fragments. DNA genome from 6 meat samples (pork, beef, goat, lamb, and chicken) was amplified by PCR technique using three pairs of primers (<em>ND5, D-Loop</em><em>, </em>and <em>Cyt-b</em>) and sequenced. The results of amplification using the three primers produced specific DNA bands with the lengths of 232 bp, 951 bp, and 404 bp, respectively. Comparison results with<em> ND5, D-Loop,</em> and <em>Cyt-b</em> gene sequences resulted in similarity values of 100%, 97%, and 99%, respectively. These showed that the mt-DNA primers of <em>ND5, D-Loop</em>, and<em> Cyt-b </em>genes can be recommended as specific primers in detecting pig (<em>Sus scrofa</em>) DNA fragments. </div>
基金Supported by Natural Science Foundation of Fujian Province (2011J01066, 2012JO1061)。
文摘[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.
基金supported by the National Key Research and Development Project of China(Grant No.2022YFB3205500)the National Natural Science Foundation of China(Grant No.12275190,12105201)+2 种基金Jiangsu Funding Program for Excellent Postdoctoral Talent(Grant No.2024ZB723)the Shenzhen Research Funding Program(JCYJ20230807154402004)supported by the Collaborative Innovation Center of Suzhou Nano Science&Technology,the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD),the 111 Project,the Joint International Research Laboratory of Carbon-Based Functional Materials and Devices,and the Suzhou Key Laboratory of Functional Nano&Soft Materials and Soochow University-Western University Centre for Synchrotron Radiation Research.
文摘Conventional gas sensing materials(e.g.,metal oxides)suffer from deficient sensitivity and serve cross-sensitivity issues due to the lack of efficient adsorption sites.Herein,the heteroatom atomically doping strategy is demonstrated to significantly enhance the sensing performance of metal oxides-based gas sensing materials.Specifically,the Sn atoms were incorporated into porous Fe_(2)O_(3)in the form of atomically dispersed sites.As revealed by X-ray absorption spectroscopy and atomic-resolution scanning transmission electron microscopy,these Sn atoms successfully occupy the Fe sites in the Fe_(2)O_(3)lattice,forming the unique Sn-O-Fe sites.Compared to Fe-O-Fe sites(from bare Fe_(2)O_(3))and Sn-O-Sn sites(from SnO_(2)/Fe_(2)O_(3)with high Sn loading),the Sn-O-Fe sites on porous Fe_(2)O_(3)exhibit a superior sensitivity(Rg/Ra=2646.6)to 1 ppm NO_(2),along with dramatically increased selectivity and ultra-low limits of detection(10 ppb).Further theoretical calculations suggest that the strong adsorption of NO_(2)on Sn-O-Fe sites(N atom on Sn site,O atom on Fe site)contributes a more efficient gas response,compared to NO_(2)on Fe-O-Fe sites and other gases on Sn-O-Fe sites.Moreover,the incorporated Sn atoms reduce the bandgap of Fe_(2)O_(3),not only facilitating the electron release but also increasing the NO_(2)adsorption at a low working temperature(150°C).This work introduces an effective strategy to construct effective adsorption sites that show a unique response to specific gas molecules,potentially promoting the rational design of atomically modified gas sensing materials with high sensitivity and high selectivity.
基金supported by the fund from Tianjin Municipal Science and Technology Bureau(22JCYBJC01390).
文摘At present,the polymerase chain reaction(PCR)amplification-based file retrieval method is the mostcommonly used and effective means of DNA file retrieval.The number of orthogonal primers limitsthe number of files that can be accurately accessed,which in turn affects the density in a single oligo poolof digital DNA storage.In this paper,a multi-mode DNA sequence design method based on PCR file retrie-val in a single oligonucleotide pool is proposed for high-capacity DNA data storage.Firstly,by analyzingthe maximum number of orthogonal primers at each predicted primer length,it was found that the rela-tionship between primer length and the maximum available primer number does not increase linearly,and the maximum number of orthogonal primers is on the order of 10^(4).Next,this paper analyzes themaximum address space capacity of DNA sequences with different types of primer binding sites for filemapping.In the case where the capacity of the primer library is R(where R is even),the number ofaddress spaces that can be mapped by the single-primer DNA sequence design scheme proposed in thispaper is four times that of the previous one,and the two-level primer DNA sequence design scheme can reach [R/2·(R/2-1)]^(2)times.Finally,a multi-mode DNA sequence generation method is designed based onthe number of files to be stored in the oligonucleotide pool,in order to meet the requirements of the ran-dom retrieval of target files in an oligonucleotide pool with large-scale file numbers.The performance ofthe primers generated by the orthogonal primer library generator proposed in this paper is verified,andthe average Gibbs free energy of the most stable heterodimer formed between the orthogonal primersproduced is−1 kcal·(mol·L^(−1))^(−1)(1 kcal=4.184 kJ).At the same time,by selectively PCR-amplifying theDNA sequences of the two-level primer binding sites for random access,the target sequence can be accu-rately read with a minimum of 10^(3) reads,when the primer binding site sequences at different positionsare mutually different.This paper provides a pipeline for orthogonal primer library generation and multi-mode mapping schemes between files and primers,which can help achieve precise random access to filesin large-scale DNA oligo pools.
文摘Device-based measurements are recommended to improve population-based physical activity(PA)surveillance.1,2However,implementation remains challenging due to lack of consensus on analytical methods,and the most widely used“generic”(absolute intensity)cut-point approach has limited generalisability to population-level free-living data.Further,current methods generally fail to account for differences in people's physical capacity.
基金National Natural Science Foundation of China(Nos.22277104,22325703,22074084)the Natural Science Foundation of Shanxi Province(No.202203021212184)+3 种基金Research Project supported by Shanxi Scholarship Council of China(No.2022-002)the Basic Research Program of Shanxi Province(Free Exploration)(No.202203021221009)2022 Lvliang City science and technology plan project(Nos.2022SHFZ51,2022GXYF15)Scientific Instrument Center of Shanxi University(No.201512)。
文摘Excited-state intramolecular proton-transfer(ESIPT)based fluorescence probes are particularly attractive due to their unique properties including environmental sensitivity,a large Stokes shift,and potential for ratiometric sensing.In general,ESIPT-based fluorophore incorporates an intramolecular hydrogen bonding interaction between a hydrogen bond donor(-OH and NH_(2)are common)and a hydrogen bond acceptor(C=N and C=O).More,protection-deprotection of hydroxyl group as hydrogen bond donor could induce an off-on switch of ESIPT-based emission.Therefore,protection-deprotection of hydroxyl group has been the widely used strategy to design fluorescent probes,where the potential key issue is selecting a protective group that can specifically leave in the presence of the target analyte.In this review,we mainly summarize the specific protecting groups(sites)and deprotection mechanisms for biologically important species(including reactive sulfur species(RSS),reactive oxygen species(ROS),enzymes,etc.),and analyze the advantages and disadvantages of different protection mechanisms from some aspects including probe stability,selectivity,response rate and assay system,etc.Based on the aforementioned,we further point out the current challenges and the potential future direction for developing ESIPT-based probes.
基金financially supported by the National Natural Science Foundation of China(No.22074038)the Luoyang Institute of Science and Technology Natural Science General Project(No.21010905)+1 种基金the Fundamental Research Funds for the Central Universitiessponsored by the Domestic Visiting Scholar Program of Shandong University of Science and Technology。
文摘Nanozymes,characterized by their stability,cost-effectiveness,and tunable catalytic activity,are promising alternatives to natural enzymes.However,specifically mimicking a single natural enzyme's activity presents a challenge.By exploiting the catalytic selectivity derived from the valence-band hybridization of noble metal nanoalloys,we introduce an alloying strategy to modulate the reaction specificity of metallic nanozymes.Ag Pd nanoalloy exhibits enhanced peroxidase-like activity and eliminated oxidase-like activity by adjusting the Ag content.The introduction of Ag changes the hybrid d band energy of the alloyed metal and inhibits the O_(2)adsorption and decomposition on Pd,while improving the peroxidase mimicry by allowing for the H_(2)O_(2)activation.By exemplifying the construction of a highly sensitive and selective colorimetric glucose detection platform with its practicality validated in serum samples,this strategy pioneers a multi-noble metal nanozyme with tailored peroxidase activity based on the chemical structure engineering and would advance the development of single-catalytic function nanozymes for building exclusively specific biosensors through reducing substrate competition.
