Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects...Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.展开更多
Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficie...Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time.The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions.Deoxyadenosine monophosphate(dAMP)was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis.The linear range in DNA quantitation by this method is 1.2-5000 ng/mL.In the validation,the inter-day and intra-day accuracies were within 90%-110%,and the inter-day and intra-day precision were acceptable(RSD<10%).The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model.Compared to the quantitation methods using UV absorbance,the reported method provides an enhanced sensitivity,and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.展开更多
基金The National High Technology Research and Development Program ("863" Program) of China under contract No 2006AA09Z178 the National Natural Science Foundation of China under contract No 40706044
文摘Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.
文摘Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time.The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions.Deoxyadenosine monophosphate(dAMP)was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis.The linear range in DNA quantitation by this method is 1.2-5000 ng/mL.In the validation,the inter-day and intra-day accuracies were within 90%-110%,and the inter-day and intra-day precision were acceptable(RSD<10%).The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model.Compared to the quantitation methods using UV absorbance,the reported method provides an enhanced sensitivity,and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.
