The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among...The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I （COI） of five common Crassostrea species in China （including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas） and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers （Oyster- COI-1）, we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.展开更多
Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin,psilocin and ibotenic acid,etc.The mushrooms containing hallucinogenic components are various,widely d...Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin,psilocin and ibotenic acid,etc.The mushrooms containing hallucinogenic components are various,widely distributed and lack of standard to define,which made a great challenge to identification.Traditional identification methods,such as morphology and toxicology analysis,showed shortcomings in old or processed samples,while the DNA-based identification of hallucinogenic mushrooms would allow to identify these samples due to the stability of DNA.In this paper,four primer sets are designed to target Psilocybe cubensis DNA for increasing resolution of present identification method,and the target markers include largest subunit of RNA polymerase II(marked as PC-R1),psilocybin-related phosphotransferase gene(marked as PC-PT),glyceraldehyde 3-phosphate dehydrogenase(marked as PC-3)and translation EF1α(marked as PC-EF).Real-time PCR with high-resolution melting(HRM)assay were used for the differentiation of the fragments amplified by these primer sets,which were tested for specificity,reproducibility,sensitivity,mixture analysis and multiplex PCR.It was shown that the melting temperatures of PC-R1,PC-PT,PC-3 and PC-EF of P.cubensis were(87.93±0.12)℃,(82.21±0.14)℃,(79.72±0.12)℃ and(80.11±0.19)℃ in our kinds of independent experiments.Significant HRM characteristic can be shown with a low concentration of 62.5pg/µL DNA sample,and P.cubensis could be detected in mixtures with Homo sapiens or Cannabis sativa.In summary,the method of HRM analysis can quickly and specifically distinguish P.cubensis from other species,which could be utilized for forensic science,medical diagnosis and drug trafficking cases.展开更多
Targeting Induced Local Lesions IN Genomes （TILLING） is a reverse genetics strategy for the high-throughput screening of induced mutations.γ, radiation, which often induces both insertion/deletion （Indel） and poi...Targeting Induced Local Lesions IN Genomes （TILLING） is a reverse genetics strategy for the high-throughput screening of induced mutations.γ, radiation, which often induces both insertion/deletion （Indel） and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting （HRM） analysis. Pooled rice （Oryza sativa） samples mixed at a 1：7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ, ray-induced mutants in rice. For demonstration, a γ, ray-mutagenized M2 rice population （n=4560） was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution （G→A） and one single nucleotide insertion （A） were identified in OsLCT1, and one tdnucleotide （TTC） deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ, ray mutagenesis to the breeding of rice and other seed crops.展开更多
Objective:Second-generation antipsychotics are widely used in mental illness,but the treatment effects and side effects are affected by single nucleotide polymorphisms(SNPs)of related genes.Quetiapine and aripiprazole...Objective:Second-generation antipsychotics are widely used in mental illness,but the treatment effects and side effects are affected by single nucleotide polymorphisms(SNPs)of related genes.Quetiapine and aripiprazole are two frequently used secondgeneration antipsychotic drugs.The aim of this study was to develop two different SNP detection methods for four SNP alleles associated with the pharmacokinetics of quetiapine and aripiprazole,based on high-resolution melting(HRM)and multicolor melting curve assay(MMCA)respectively.Methods:Whole genome DNA samples were obtained from 240 healthy people(107 females and 133 males)without genetic diseases.HRM methods were established using four kinds of specific primers and a saturated fluorescent dye.Each SNP allele with their own primers was detected in a single reaction.In the MMCA method,a multiplex polymerase chain reaction with 4 different-colored fluorescent probes was established to detect four SNP alleles in a single reaction.All experimental protocols were approved by the Ethics Committee of the Shanghai Children’s Medical Center,China(SCMC-201015)on November 22,2010.Results:Two detection methods for the pharmacogenomics of quetiapine and aripiprazole,based on HRM and MMCA respectively,were established in this study.The single-target HRM method can be completed in 96 minutes,whereas the quadruplex MMCA method takes 133 minutes.It was found that the results of HRM and MMCA for the four SNP alleles had 100%coincidence with Sanger sequencing in the 240 samples.Conclusion:This study developed two methods for the detection of four pharmacogenomic SNP alleles that correlated with quetiapine and aripiprazole.Both methods are rapid,cost-saving,highly accurate and potentially facilitate rational use of second-generation antipsychotics for clinical medication.展开更多
基金Supported by the National Basic Research Program of China(973 Program)(No.2010CB126402)the National Natural Science Foundation of China(Nos.40730845,41206149)+4 种基金the Shandong Provincial Natural Science Foundation(No.ZR2010DQ024)the National High Technology Research and Development Program of China(863 Program)(No.2012AA10A405)the Earmarked Fund for Modern Agro-Industry Technology Research System(No.CARS-48)the Taishan Scholar Program of Shandong Provincethe Taishan Scholar Climbing Program of Shandong Province
文摘The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I （COI） of five common Crassostrea species in China （including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas） and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers （Oyster- COI-1）, we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.
基金supported by grants from the Shanghai Areas of Development for Society Planning Projects[grant number 19dz1200600]the National Natural Science Foundation of China[grant numbers 81930056 and 81625013]the National Youth Talent Support Program[grant number WRQB2019].
文摘Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin,psilocin and ibotenic acid,etc.The mushrooms containing hallucinogenic components are various,widely distributed and lack of standard to define,which made a great challenge to identification.Traditional identification methods,such as morphology and toxicology analysis,showed shortcomings in old or processed samples,while the DNA-based identification of hallucinogenic mushrooms would allow to identify these samples due to the stability of DNA.In this paper,four primer sets are designed to target Psilocybe cubensis DNA for increasing resolution of present identification method,and the target markers include largest subunit of RNA polymerase II(marked as PC-R1),psilocybin-related phosphotransferase gene(marked as PC-PT),glyceraldehyde 3-phosphate dehydrogenase(marked as PC-3)and translation EF1α(marked as PC-EF).Real-time PCR with high-resolution melting(HRM)assay were used for the differentiation of the fragments amplified by these primer sets,which were tested for specificity,reproducibility,sensitivity,mixture analysis and multiplex PCR.It was shown that the melting temperatures of PC-R1,PC-PT,PC-3 and PC-EF of P.cubensis were(87.93±0.12)℃,(82.21±0.14)℃,(79.72±0.12)℃ and(80.11±0.19)℃ in our kinds of independent experiments.Significant HRM characteristic can be shown with a low concentration of 62.5pg/µL DNA sample,and P.cubensis could be detected in mixtures with Homo sapiens or Cannabis sativa.In summary,the method of HRM analysis can quickly and specifically distinguish P.cubensis from other species,which could be utilized for forensic science,medical diagnosis and drug trafficking cases.
基金Project supported by the National Key Research and Development Program of China(No.2016YFD0102103)
文摘Targeting Induced Local Lesions IN Genomes （TILLING） is a reverse genetics strategy for the high-throughput screening of induced mutations.γ, radiation, which often induces both insertion/deletion （Indel） and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting （HRM） analysis. Pooled rice （Oryza sativa） samples mixed at a 1：7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ, ray-induced mutants in rice. For demonstration, a γ, ray-mutagenized M2 rice population （n=4560） was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution （G→A） and one single nucleotide insertion （A） were identified in OsLCT1, and one tdnucleotide （TTC） deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ, ray mutagenesis to the breeding of rice and other seed crops.
文摘Objective:Second-generation antipsychotics are widely used in mental illness,but the treatment effects and side effects are affected by single nucleotide polymorphisms(SNPs)of related genes.Quetiapine and aripiprazole are two frequently used secondgeneration antipsychotic drugs.The aim of this study was to develop two different SNP detection methods for four SNP alleles associated with the pharmacokinetics of quetiapine and aripiprazole,based on high-resolution melting(HRM)and multicolor melting curve assay(MMCA)respectively.Methods:Whole genome DNA samples were obtained from 240 healthy people(107 females and 133 males)without genetic diseases.HRM methods were established using four kinds of specific primers and a saturated fluorescent dye.Each SNP allele with their own primers was detected in a single reaction.In the MMCA method,a multiplex polymerase chain reaction with 4 different-colored fluorescent probes was established to detect four SNP alleles in a single reaction.All experimental protocols were approved by the Ethics Committee of the Shanghai Children’s Medical Center,China(SCMC-201015)on November 22,2010.Results:Two detection methods for the pharmacogenomics of quetiapine and aripiprazole,based on HRM and MMCA respectively,were established in this study.The single-target HRM method can be completed in 96 minutes,whereas the quadruplex MMCA method takes 133 minutes.It was found that the results of HRM and MMCA for the four SNP alleles had 100%coincidence with Sanger sequencing in the 240 samples.Conclusion:This study developed two methods for the detection of four pharmacogenomic SNP alleles that correlated with quetiapine and aripiprazole.Both methods are rapid,cost-saving,highly accurate and potentially facilitate rational use of second-generation antipsychotics for clinical medication.
