The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we esta...The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.展开更多
The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization....The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.展开更多
The objective of this study was to identify rice genes that are in response to the striped stem borer （SSB） （Chilo suppressalis Walker） feeding at the first to second larval stage. Using combined suppression subtr...The objective of this study was to identify rice genes that are in response to the striped stem borer （SSB） （Chilo suppressalis Walker） feeding at the first to second larval stage. Using combined suppression subtractive hybridization （SSH） and dot blot approaches, we analyzed the induced defense genes that took place during the first 72 h of infesting intact rice （Oryza sativa L.） plants in sheath tissues with SSB larvae. By sequencing the whole SSH library, 39 expressed sequence tags involved in disease stress, insect stress or other stress responses were identified to be up-regulated by SSB larvae feeding. Among these genes, rice allene oxide cyclase （AOC）, terpene synthase （TPS） and four proteinase inhibitor （PI） genes were up-regulated by SSB larvae feeding. Real-time quantitative reverse transcription polymerase chain reaction analysis showed that four rice PI genes were already up-regulated at 6 h, and reached peaks between 6 h to 12 h. In addition, the transcription ofgene involving in jasmonate signaling pathway such as allene oxide cyclase （AOC） concerning rice early defense response to SSB feeding was activated after rice feeding by SSB for 2 h. Although the expression office terpene synthase （TPS） gene, involved in the biosynthesis ofmonoterpenes or diterpenes, was already up-regulated at 7 h, a significant increase in the expression was delayed until 12 h and reached its peak at 24 h. The present study identified six SSB-response genes and their expression patterns, which provides evidence and information to understand insect stress-response in plants.展开更多
基金supported by the National Natural Science Foundation of China (Nos. 31470271 and 81730110)Guangzhou Science and Technology Program key projects (No. 201803040006)
文摘The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.
基金Supported by the Key Program of Science and Technology Innovation in Ningbo (No.2019B10009)the National Natural Science Foundation of China (No.41476111)。
文摘The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.
基金Acknowledgments We greatly appreciate the grant support from National Natural Science Foundation of China （No. 30871640, No. 30330410）, China national ＂973＂ Basic Research Program （No. 2007CB 109202）, and Research Foundation of State Key Laboratory for Biology of Plant Diseases and Insect Pests （SKL2007SR01）.
文摘The objective of this study was to identify rice genes that are in response to the striped stem borer （SSB） （Chilo suppressalis Walker） feeding at the first to second larval stage. Using combined suppression subtractive hybridization （SSH） and dot blot approaches, we analyzed the induced defense genes that took place during the first 72 h of infesting intact rice （Oryza sativa L.） plants in sheath tissues with SSB larvae. By sequencing the whole SSH library, 39 expressed sequence tags involved in disease stress, insect stress or other stress responses were identified to be up-regulated by SSB larvae feeding. Among these genes, rice allene oxide cyclase （AOC）, terpene synthase （TPS） and four proteinase inhibitor （PI） genes were up-regulated by SSB larvae feeding. Real-time quantitative reverse transcription polymerase chain reaction analysis showed that four rice PI genes were already up-regulated at 6 h, and reached peaks between 6 h to 12 h. In addition, the transcription ofgene involving in jasmonate signaling pathway such as allene oxide cyclase （AOC） concerning rice early defense response to SSB feeding was activated after rice feeding by SSB for 2 h. Although the expression office terpene synthase （TPS） gene, involved in the biosynthesis ofmonoterpenes or diterpenes, was already up-regulated at 7 h, a significant increase in the expression was delayed until 12 h and reached its peak at 24 h. The present study identified six SSB-response genes and their expression patterns, which provides evidence and information to understand insect stress-response in plants.
