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
BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for com...BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.展开更多
Klinefelter syndrome （KS） is the set of symptoms that result from the presence of an extra X chromosome in males. Postnatal population-based KS screening will enable timely diagnosis of this common chromosomal disea...Klinefelter syndrome （KS） is the set of symptoms that result from the presence of an extra X chromosome in males. Postnatal population-based KS screening will enable timely diagnosis of this common chromosomal disease, providing the opportunity for early intervention and therapy at the time point when they are most effective and may prevent later symptoms or complications. Therefore, through this study, we introduced a simple high-resolution melting （HRM） assay for KS screening and evaluated its clinical sensitivity and specificity in three medical centers using 1373 clinical blood samples. The HRM assay utilized a single primer pair to simultaneously amplify specific regions in zinc finger protein, X-linked （ZFX） and zinc finger protein, Y-linked （ZFY）. In cases of KS, the ratios of ZFX/ZFYare altered compared to those in normal males. As a result, the specific melting profiles differ and can be differentiated during data analysis. This HRM assay displayed high analytical specificity over a wide range of template DNA amounts （5 ng-50 ng） and reproducibility, high resolution for detecting KS mosaicism, and high clinical sensitivity （100%） and specificity （98.1%）. Moreover, the HRM assay was rapid （2 h per run）, inexpensive （0.2 USD per sample）, easy to perform and automatic, and compatible with both whole blood samples and dried blood spots. Therefore, this HRM assay is an ideal postnatal population-based KS screening tool that can be used for different age groups.展开更多
Discrimination among grapevine varieties based on quantitative traits,such as flowering,veraison and ripening dates is crucial for variety selection in the context of climate change and in breeding programs.These trai...Discrimination among grapevine varieties based on quantitative traits,such as flowering,veraison and ripening dates is crucial for variety selection in the context of climate change and in breeding programs.These traits are under complex genetic control for which 6 linked SSR loci(VVS2,VVIn16,VMC7G3,VrZAG29,VMC5G7,and VVIB23)have been identified.Using these markers in HRM-PCR analysis,we assessed genetic diversity among a large collection of 192 grapevine varieties.The grapevine germplasm used encompasses the majority of Greek vineyard with 181 varieties,3 prominent foreign varieties and 11 varieties of Palestinian origin.The SSR markers used were highly polymorphic,displaying unique melting curves for unusually higher number of samples than generally observed in SSR analysis.This prompted us to examine sequence composition for selected samples and found that variation present as SNPs in the flanking sequences of SSR motifs was responsible for the observed polymorphism.Hence,HRM-PCR proved to be a tool of higher analytical power to distinguish genotypes surpassing the discrimination power of conventional gel-based SSR analysis.The study provides a better understanding of genetic variation of SSR marker loci associated to phenological traits in grapevine varieties,signifying an analytical methodology that may be of higher discrimination power in detection of polymorphism for utilization in breeding programs.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BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.
文摘Klinefelter syndrome （KS） is the set of symptoms that result from the presence of an extra X chromosome in males. Postnatal population-based KS screening will enable timely diagnosis of this common chromosomal disease, providing the opportunity for early intervention and therapy at the time point when they are most effective and may prevent later symptoms or complications. Therefore, through this study, we introduced a simple high-resolution melting （HRM） assay for KS screening and evaluated its clinical sensitivity and specificity in three medical centers using 1373 clinical blood samples. The HRM assay utilized a single primer pair to simultaneously amplify specific regions in zinc finger protein, X-linked （ZFX） and zinc finger protein, Y-linked （ZFY）. In cases of KS, the ratios of ZFX/ZFYare altered compared to those in normal males. As a result, the specific melting profiles differ and can be differentiated during data analysis. This HRM assay displayed high analytical specificity over a wide range of template DNA amounts （5 ng-50 ng） and reproducibility, high resolution for detecting KS mosaicism, and high clinical sensitivity （100%） and specificity （98.1%）. Moreover, the HRM assay was rapid （2 h per run）, inexpensive （0.2 USD per sample）, easy to perform and automatic, and compatible with both whole blood samples and dried blood spots. Therefore, this HRM assay is an ideal postnatal population-based KS screening tool that can be used for different age groups.
文摘Discrimination among grapevine varieties based on quantitative traits,such as flowering,veraison and ripening dates is crucial for variety selection in the context of climate change and in breeding programs.These traits are under complex genetic control for which 6 linked SSR loci(VVS2,VVIn16,VMC7G3,VrZAG29,VMC5G7,and VVIB23)have been identified.Using these markers in HRM-PCR analysis,we assessed genetic diversity among a large collection of 192 grapevine varieties.The grapevine germplasm used encompasses the majority of Greek vineyard with 181 varieties,3 prominent foreign varieties and 11 varieties of Palestinian origin.The SSR markers used were highly polymorphic,displaying unique melting curves for unusually higher number of samples than generally observed in SSR analysis.This prompted us to examine sequence composition for selected samples and found that variation present as SNPs in the flanking sequences of SSR motifs was responsible for the observed polymorphism.Hence,HRM-PCR proved to be a tool of higher analytical power to distinguish genotypes surpassing the discrimination power of conventional gel-based SSR analysis.The study provides a better understanding of genetic variation of SSR marker loci associated to phenological traits in grapevine varieties,signifying an analytical methodology that may be of higher discrimination power in detection of polymorphism for utilization in breeding programs.
