Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Dro...Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.展开更多
[ Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. [ Method] From aspects of preparing procedure, prepare time and the quality of DNA, ad...[ Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. [ Method] From aspects of preparing procedure, prepare time and the quality of DNA, advantages and disadvantages of three sample preparation methods were compared, include two Direet-PCR extraction methods and one universal genomic DNA extraction kit method. In addition, PCR amplification effect on specific primers for 16S rDNA of ＂Candidatua Libefibacter asiaticus＂ （CLas） had also been evaluated. [ Result] The results showed that RT-qPCR detected CLas by using DNA obtained from one of Direct-PCR extraction method as templates. Under improved Direct-PCR extraction method, 16S rDNA of CLas could also be amplified by routine PCR. [ Conclusion] A simple and effective DNA extraction method of citrus Huanglongbing pathogen have been established, which provides technical supports for prepa- ration of large number of samples for detection of CLas.展开更多
Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that ca...Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that causes high mortality rates during outbreaks. The identification of pathogenic species using a fast and reliable method of diagnosis is essential for successful epidemiological studies and disease control. The present study evaluated the use of direct colony PCR in combination with 16S rRNA gene sequencing to diagnose fish bacterial diseases, with the goal of reducing the costs and time necessary for bacterial identification. The method was successful for all 178 isolates tested and produced bands with the same intensity as the standard PCR performed using pure DNA. In conclusion, the genetics methods allowed detecting the most common and important pathogens in Aquaculture, including 12 species of occurrence in Brazilian fish farms. The results of the present study constitute an advance in the available diagnostic methods for bacterial pathogens in fish farms.展开更多
文摘Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.
基金Supported by Most-USDA Cooperation Program in Agriculture Science and Technology(10-8100-1452-CA)National Natural Science Foundation of China(31071712)
文摘[ Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. [ Method] From aspects of preparing procedure, prepare time and the quality of DNA, advantages and disadvantages of three sample preparation methods were compared, include two Direet-PCR extraction methods and one universal genomic DNA extraction kit method. In addition, PCR amplification effect on specific primers for 16S rDNA of ＂Candidatua Libefibacter asiaticus＂ （CLas） had also been evaluated. [ Result] The results showed that RT-qPCR detected CLas by using DNA obtained from one of Direct-PCR extraction method as templates. Under improved Direct-PCR extraction method, 16S rDNA of CLas could also be amplified by routine PCR. [ Conclusion] A simple and effective DNA extraction method of citrus Huanglongbing pathogen have been established, which provides technical supports for prepa- ration of large number of samples for detection of CLas.
基金thank the State of Sao Paulo Research Foundation(FAPESP-Process 2011/07951-5)for the financial support.
文摘Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that causes high mortality rates during outbreaks. The identification of pathogenic species using a fast and reliable method of diagnosis is essential for successful epidemiological studies and disease control. The present study evaluated the use of direct colony PCR in combination with 16S rRNA gene sequencing to diagnose fish bacterial diseases, with the goal of reducing the costs and time necessary for bacterial identification. The method was successful for all 178 isolates tested and produced bands with the same intensity as the standard PCR performed using pure DNA. In conclusion, the genetics methods allowed detecting the most common and important pathogens in Aquaculture, including 12 species of occurrence in Brazilian fish farms. The results of the present study constitute an advance in the available diagnostic methods for bacterial pathogens in fish farms.
