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
In this paper,minimum-fuel rendezvous is investigated for the case in which the reference orbit is highly elliptic.To this end,the well-known Tschauner-Hempel equations are used to describe the relative motions betwee...In this paper,minimum-fuel rendezvous is investigated for the case in which the reference orbit is highly elliptic.To this end,the well-known Tschauner-Hempel equations are used to describe the relative motions between rendezvous spacecraft and the target.Lawden’s primer vector theory is then applied on this linear but time-varying system.The analytical solution of the required primer vector for this problem is then derived by using a recently developed method.For the existing non-optimal solutions which don’t satisfy the conditions,the methods are further designed to improve the performance by shifting impulses or adding a new one.Finally,two algorithms are developed for free-impulse time-fixed rendezvous problems.The first algorithm can determine the globally optimal trajectory with the optimal number of impulses.The second one enables for fast trajectory planning.The proposed algorithms have been successfully applied to coplanar and three-dimensional rendezvous problems in which the target is flying on highly elliptical orbits.展开更多
[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 su...[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.展开更多
[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. [Re...[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.展开更多
[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
[Objective] The aim was to explore the reasons of false positives in Different Display Reverse Transcription（DDRT）analysis.[Method] Soybean varieties ＂Jilin 30＂ and ＂Tongnong 13＂ were used as materials to carry ...[Objective] The aim was to explore the reasons of false positives in Different Display Reverse Transcription（DDRT）analysis.[Method] Soybean varieties ＂Jilin 30＂ and ＂Tongnong 13＂ were used as materials to carry out analysis on false positives in DDRT analysis.[Result] An important origin of false positives appeared in DDRT analysis was the non-specific amplification caused by the combination of single primer and cDNA.The parallel PCR test of single primer should be set so as to verify whether the obtained fragments were the false positives or the PCR productions combined with single primer.[Conclusion] This study had provided basis for improving the success rate of DDRT experiment.展开更多
[Objective] This research aimed to develop walnut EST-SSR markers and design corresponding primers.[Method] 5213 EST sequences of walnut（Juglans regia Linn.） public online in NCBI were used for character analysis wi...[Objective] This research aimed to develop walnut EST-SSR markers and design corresponding primers.[Method] 5213 EST sequences of walnut（Juglans regia Linn.） public online in NCBI were used for character analysis with bioinformatics methods,and primers were designed for the selected EST sequences by using Primer 3.0 software.[Result] 207 SSRs were obtained from the EST sequences,including 188 non-redundant sequences,the detection rate was 3.97% with an average distribution distance of 21.12 kb.Totally 92 types of repeat motifs were involved,which were mainly composed of dinucleotide and trinucleotide,accounting for 31.40% and 35.27% of the total number of repeat motifs,respectively.30 pairs of primers were initially selected from the 50 randomly-selected SSR primers by PCR amplification.[Conclusion] This research would lay foundations for the development of EST-SSR molecular markers in walnut and design of the targeted EST-SSR primers by mining and analyzing the SSR sites in walnut EST sequences.展开更多
At present,there are unharmonious factors existing in Chinese family education,which particular manifest in the means of family education.The Three Character Primer,as an epitome of the essence of Chinese traditional ...At present,there are unharmonious factors existing in Chinese family education,which particular manifest in the means of family education.The Three Character Primer,as an epitome of the essence of Chinese traditional culture,can provide enlightenment on building a democratic and harmonious family education means.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
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 ...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.展开更多
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 extract...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.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
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
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.
基金supported by National Natural Science Foundation of China(No.12172288)National Key Basic Research Program of China:Gravitational Wave Detection Project(Nos.2021YFC2202601 and 2021YFC2202603)General Program of Natural Science Foundation of Higher Education of Jiangsu Province(No.21KJB590001)。
文摘In this paper,minimum-fuel rendezvous is investigated for the case in which the reference orbit is highly elliptic.To this end,the well-known Tschauner-Hempel equations are used to describe the relative motions between rendezvous spacecraft and the target.Lawden’s primer vector theory is then applied on this linear but time-varying system.The analytical solution of the required primer vector for this problem is then derived by using a recently developed method.For the existing non-optimal solutions which don’t satisfy the conditions,the methods are further designed to improve the performance by shifting impulses or adding a new one.Finally,two algorithms are developed for free-impulse time-fixed rendezvous problems.The first algorithm can determine the globally optimal trajectory with the optimal number of impulses.The second one enables for fast trajectory planning.The proposed algorithms have been successfully applied to coplanar and three-dimensional rendezvous problems in which the target is flying on highly elliptical orbits.
基金Supported by Important Project of Jinlin Provincial Science and Technology Department(20065020)~~
文摘[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.
基金Supported by Excellent Team Training Program of Yunnan Academy of Agriculture Sciences(YAAS2014YY002)~~
文摘[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.
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[Objective] The aim was to explore the reasons of false positives in Different Display Reverse Transcription（DDRT）analysis.[Method] Soybean varieties ＂Jilin 30＂ and ＂Tongnong 13＂ were used as materials to carry out analysis on false positives in DDRT analysis.[Result] An important origin of false positives appeared in DDRT analysis was the non-specific amplification caused by the combination of single primer and cDNA.The parallel PCR test of single primer should be set so as to verify whether the obtained fragments were the false positives or the PCR productions combined with single primer.[Conclusion] This study had provided basis for improving the success rate of DDRT experiment.
基金Supported by Natural Science Foundation of Zhejiang Province(Y307469)Talent Start-up Fund Project of Zhejiang Agriculture and Forestry University~~
文摘[Objective] This research aimed to develop walnut EST-SSR markers and design corresponding primers.[Method] 5213 EST sequences of walnut（Juglans regia Linn.） public online in NCBI were used for character analysis with bioinformatics methods,and primers were designed for the selected EST sequences by using Primer 3.0 software.[Result] 207 SSRs were obtained from the EST sequences,including 188 non-redundant sequences,the detection rate was 3.97% with an average distribution distance of 21.12 kb.Totally 92 types of repeat motifs were involved,which were mainly composed of dinucleotide and trinucleotide,accounting for 31.40% and 35.27% of the total number of repeat motifs,respectively.30 pairs of primers were initially selected from the 50 randomly-selected SSR primers by PCR amplification.[Conclusion] This research would lay foundations for the development of EST-SSR molecular markers in walnut and design of the targeted EST-SSR primers by mining and analyzing the SSR sites in walnut EST sequences.
文摘At present,there are unharmonious factors existing in Chinese family education,which particular manifest in the means of family education.The Three Character Primer,as an epitome of the essence of Chinese traditional culture,can provide enlightenment on building a democratic and harmonious family education means.
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(in part)Grants UH2CA140233 from the Human Microbiome Project of the NIH Roadmap Initiative and National Cancer InstituteR01AI063477 from the National Institute of Allergy and Infectious Diseases+1 种基金DE-11443 from the National Institute of Dental and Craniofacial ResearchU19DE018385 from the National Institute of Dental & Craniofacial Research
文摘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.
基金This work was funded by the Special Scientific Foundation of Guangdong Province (No. A305030301)was partly supported by a grant from KLFEE, Ministry of Agriculture (2003-04).
文摘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.
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 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.
