Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation,affecting both yield and quality.Thus,rapid and accurate detection of P.parasitica plays an important role in disease m...Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation,affecting both yield and quality.Thus,rapid and accurate detection of P.parasitica plays an important role in disease management.The aim of this study was to develop a simple diagnostic method to detect P.parasitica infection by combining recombinase polymerase amplification and lateral flow strips(LF-RPA).To establish the LF-RPA assay of P.parasitica,the primers and probe designed based on the Ypt1 gene were tested for specificity to P.parasitica,which showed no cross-reactivity with DNAs of other related oomycete species.The LF-RPA assay detected the amount of genomic DNA of P.parasitica which was as low as 1 pg.To make the LF-RPA assay useful in low-resource settings,four simplified DNA extraction methods were compared,after which the LF-RPA assay was applied,with no specialized equipment,to analyze a diverse range of citrus tissues by using a simplified PEG-NaOH method for DNA extraction.This method was successful in detecting P.parasitica in infected plant samples within 30 min.Combining the LF-RPA assay and a simplified DNA extraction method could be a potential detection test for P.parasitica,especially in areas with limited resources.展开更多
Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and ...Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.展开更多
基金This work was funded by grants from the Fundamental Research Funds for the Central Universities,China(KYT202001 and JCQY201901)the Special Fund for Agro-scientific Research in the Public Interest,China(201503112).
文摘Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation,affecting both yield and quality.Thus,rapid and accurate detection of P.parasitica plays an important role in disease management.The aim of this study was to develop a simple diagnostic method to detect P.parasitica infection by combining recombinase polymerase amplification and lateral flow strips(LF-RPA).To establish the LF-RPA assay of P.parasitica,the primers and probe designed based on the Ypt1 gene were tested for specificity to P.parasitica,which showed no cross-reactivity with DNAs of other related oomycete species.The LF-RPA assay detected the amount of genomic DNA of P.parasitica which was as low as 1 pg.To make the LF-RPA assay useful in low-resource settings,four simplified DNA extraction methods were compared,after which the LF-RPA assay was applied,with no specialized equipment,to analyze a diverse range of citrus tissues by using a simplified PEG-NaOH method for DNA extraction.This method was successful in detecting P.parasitica in infected plant samples within 30 min.Combining the LF-RPA assay and a simplified DNA extraction method could be a potential detection test for P.parasitica,especially in areas with limited resources.
基金supported by the 973 program,Grant No.2012CB721102
文摘Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.
