[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional tou...[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.展开更多
[Objective] Grb-AST7 concatemer including 17 copies of Grb-AST7 was constructed by ＂self template-primer＂ PCR. [Method] Two primers of which were synthesized based on the amino acid sequence of Gryllus bimaculatus’...[Objective] Grb-AST7 concatemer including 17 copies of Grb-AST7 was constructed by ＂self template-primer＂ PCR. [Method] Two primers of which were synthesized based on the amino acid sequence of Gryllus bimaculatus’ Grb-AST7. [Result] Through splicing, a length of 570 bp, containing 17 copies of the Grb-AST7 gene repeats were obtained. Appropriate primer splicing conditions were as follows：splice time 20 min, 25 μl PCR system, containing 2 μl template. [Conclusion] The results laid foundation for the future studies on Grb-AST7 gene expression and bio-activity analysis.展开更多
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...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.展开更多
There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical ...There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.展开更多
The information of single nucleotide polymorphisms （SNPs） is quite unknown in sweetpotato. In this study, two sweetpotato varieties （Xushu 18 and Xu 781） were sequenced by Illumina technology, as well as de novo t...The information of single nucleotide polymorphisms （SNPs） is quite unknown in sweetpotato. In this study, two sweetpotato varieties （Xushu 18 and Xu 781） were sequenced by Illumina technology, as well as de novo transcriptome assembly, functional annotation, and in silico discovery of potential SNP molecular markers. Tetra-primer Amplification Refractory Mutation System PCR （ARMS-PCR） is a simple and sufficient method for detecting different alleles in SNP locus. Total 153 sets of ARMS-PCR primers were designed to validate the putative SNPs from sequences. PCR products from 103 sets of primers were different between Xu 781 and Xushu 18 via agarose gel electrophoresis, and the detection rate was 67.32%. We obtained the expected results from 32 sets of primers between the two genotypes. Furthermore, we ascertained the optimal annealing temperature of 32 sets of primers. These SNPs might be used in genotyping, QTL mapping, or marker-assisted trait selection further in sweetpotato. To our knowledge, this work was the first study to develop SNP markers in sweetpotato by using tetra-primer ARMS-PCR technique. This method was a simple, rapid, and useful techn!que to develop SNP markers, and will provide a potential and preliminary application in discriminating cultivars in sweetpotato.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 order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal ...In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy （GEC）/μL, the intraand inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship （r 2 = 0.997） between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 10 0 to 2.31 × 10 9 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.展开更多
The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed f...The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed for lepidopteran phylogenetics might work in Trichoptera. DNA from 8 caddisfly species （Asynarchus nigriculus （Banks, 1908）, Grammotaulius lorettae Denning, 1941, Hesperophylax occidentalis （Banks, 1908）, Limnephilus externus Hagen, 1861, Limnephilus picturatus McLachlan, 1875, Limnephilus secludens Banks, 1914, Limnephilus sublunatus Provancher, 1877 and Agrypnia deflata （Milne, 1931）） was used to screen for amplification. 107 primer pairs for 45 nuclear and 3 mitochondrial genes were tested. Primers for 1 new gene （40S ribosomalprotein $2 （RPS2）） and 8 genes previously used in Trichopteran phylogenetics were recovered （16S rRNA, 18S rRNA, carbamoyl-phosphate synthetase （CAD）, cytoehrome oxidase I （CO1）, cytochrome oxidase 11 （COIl）, elongation factor-1 alpha （EF-1 alpha）, isoeitrate dehydrogenase （IDH）, and RNA polymerase-II （POL-I1））. New primer pairs extended the genomic region sampled for many genes. Evolution rates among loci varied by 2 orders of magnitude. Differences among evolution rates and modes of inheritance offer flexible tools for resolving phylogenetic questions and examining genome evolution in the Trichoptera. Screening libraries of PCR primers is a useful approach for identifying PCR primers in related taxa with limited molecular genetic resources.展开更多
Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-bas...Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene （18S rDNA） libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18S rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.展开更多
Objective:To establish a suitable method of diagnosis of visceral Leishmania sis(VL)using peripheral blood,spleen or bone marrow aspirates.Methods:Peripheral blood,bone marrow and spleen aspirate samples were collecte...Objective:To establish a suitable method of diagnosis of visceral Leishmania sis(VL)using peripheral blood,spleen or bone marrow aspirates.Methods:Peripheral blood,bone marrow and spleen aspirate samples were collected from clinically suspected VL patients(n=26).A new PCR primer pair(MK1F/R)was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani,and Leishmania infantum,and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions.Test was validated by comparing with several other diagnostic methods.Results:The designed primer set showed 100%specificity and 98%sensitivity in detecting VL using blood samples,when compared with more invasive samples:bone marrow or spleen aspirates.Conclusions:The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample,peripheral blood instead of bone marrow or spleen aspirates.展开更多
文摘[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.
基金Supported by National Science and Technology Support Projects(No.2007BAD48B02)Agriculture Science and Technology Achievement Transformation Project(No.2007GB23260410)Basic Research Operating Expenses in Central Public Research Institutes(2007HZS1J007)~~
文摘[Objective] Grb-AST7 concatemer including 17 copies of Grb-AST7 was constructed by ＂self template-primer＂ PCR. [Method] Two primers of which were synthesized based on the amino acid sequence of Gryllus bimaculatus’ Grb-AST7. [Result] Through splicing, a length of 570 bp, containing 17 copies of the Grb-AST7 gene repeats were obtained. Appropriate primer splicing conditions were as follows：splice time 20 min, 25 μl PCR system, containing 2 μl template. [Conclusion] The results laid foundation for the future studies on Grb-AST7 gene expression and bio-activity analysis.
文摘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.
基金supported by the Shandong Seed Projectthe National Natural Science Foundation of China(No.31372524)Science and Technology Development Plan of Shandong Province,China(No.2014GHY 115002)
文摘There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.
基金supported by the China Agriculture Research System (CARS-11)the National High-Tech R&D Program of China (2012AA101204)the Jiangsu Independent Inno vation Funds of Agriculture,China (CX(13)2032)
文摘The information of single nucleotide polymorphisms （SNPs） is quite unknown in sweetpotato. In this study, two sweetpotato varieties （Xushu 18 and Xu 781） were sequenced by Illumina technology, as well as de novo transcriptome assembly, functional annotation, and in silico discovery of potential SNP molecular markers. Tetra-primer Amplification Refractory Mutation System PCR （ARMS-PCR） is a simple and sufficient method for detecting different alleles in SNP locus. Total 153 sets of ARMS-PCR primers were designed to validate the putative SNPs from sequences. PCR products from 103 sets of primers were different between Xu 781 and Xushu 18 via agarose gel electrophoresis, and the detection rate was 67.32%. We obtained the expected results from 32 sets of primers between the two genotypes. Furthermore, we ascertained the optimal annealing temperature of 32 sets of primers. These SNPs might be used in genotyping, QTL mapping, or marker-assisted trait selection further in sweetpotato. To our knowledge, this work was the first study to develop SNP markers in sweetpotato by using tetra-primer ARMS-PCR technique. This method was a simple, rapid, and useful techn!que to develop SNP markers, and will provide a potential and preliminary application in discriminating cultivars in sweetpotato.
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 the National Natural Sci-ence Foundation of China (No. 50908185)the National Program of Water Pollution Control (No. 2008ZX07317-004)+2 种基金the Basic Research Foundation of Xi’an University of Architecture and Technology (No. JC0910)the Research Foundation for Talented Scholars of Xi’an University of Architecture and Technology (No. RC0824)the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0853)
文摘In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy （GEC）/μL, the intraand inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship （r 2 = 0.997） between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 10 0 to 2.31 × 10 9 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.
基金provided by a University of Manitoba Graduate Fellowshipthe University of Manitoba Research Grants Program+3 种基金the Field Work Support Program of the Faculty of Science at the University of Manitobaa Rocky Mountain Biological Laboratory Research Fellowshipan NSERC Discovery Grant RGPIN386337-2011a Canada Foundation for Innovation Award
文摘The molecular phylogenetics of the Lepidoptera （butterflies and moths） is well studied, but that of Trichoptera （caddisflies）, the sister clade of Lepidoptera, is less studied. The PCR primer libraries developed for lepidopteran phylogenetics might work in Trichoptera. DNA from 8 caddisfly species （Asynarchus nigriculus （Banks, 1908）, Grammotaulius lorettae Denning, 1941, Hesperophylax occidentalis （Banks, 1908）, Limnephilus externus Hagen, 1861, Limnephilus picturatus McLachlan, 1875, Limnephilus secludens Banks, 1914, Limnephilus sublunatus Provancher, 1877 and Agrypnia deflata （Milne, 1931）） was used to screen for amplification. 107 primer pairs for 45 nuclear and 3 mitochondrial genes were tested. Primers for 1 new gene （40S ribosomalprotein $2 （RPS2）） and 8 genes previously used in Trichopteran phylogenetics were recovered （16S rRNA, 18S rRNA, carbamoyl-phosphate synthetase （CAD）, cytoehrome oxidase I （CO1）, cytochrome oxidase 11 （COIl）, elongation factor-1 alpha （EF-1 alpha）, isoeitrate dehydrogenase （IDH）, and RNA polymerase-II （POL-I1））. New primer pairs extended the genomic region sampled for many genes. Evolution rates among loci varied by 2 orders of magnitude. Differences among evolution rates and modes of inheritance offer flexible tools for resolving phylogenetic questions and examining genome evolution in the Trichoptera. Screening libraries of PCR primers is a useful approach for identifying PCR primers in related taxa with limited molecular genetic resources.
基金Supported by the National Natural Science Foundation of China(No.41076085)
文摘Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene （18S rDNA） libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18S rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.
基金supported by a grant from Bangladesh Medical Research Council(BMRC)(Reference number BMRC/HPNSDP/Research Fund/2012–2013/3449344(34),Dated,20 March 2013)
文摘Objective:To establish a suitable method of diagnosis of visceral Leishmania sis(VL)using peripheral blood,spleen or bone marrow aspirates.Methods:Peripheral blood,bone marrow and spleen aspirate samples were collected from clinically suspected VL patients(n=26).A new PCR primer pair(MK1F/R)was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani,and Leishmania infantum,and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions.Test was validated by comparing with several other diagnostic methods.Results:The designed primer set showed 100%specificity and 98%sensitivity in detecting VL using blood samples,when compared with more invasive samples:bone marrow or spleen aspirates.Conclusions:The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample,peripheral blood instead of bone marrow or spleen aspirates.
