Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the ...Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the history of only short febrile illness were considered.Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome(AES)were selected.Results:Out of 157 samples,42 and 74 were detected as dengue and chikungunya,respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method.Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by EL1SA method.Moreover,this method is capable to detect the viral RNA from patients'sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.展开更多
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
基金supported by the Department of Science and Technology,Goverment of West Bengal.India[grant No.705(Sanc.)ST/P/S&T/9G-27/2007]
文摘Objective:To develop a rapid,cost effective RT-PCR method for the mass scale diagnosis of such diseases at the vireraia stage to find out the actual disease burden in that area.Methods:For this purpose,cases with the history of only short febrile illness were considered.Thus 157 samples with the history of dengue/chikungunya like illness and only 58 samples with a history of acute encephalitis syndrome(AES)were selected.Results:Out of 157 samples,42 and 74 were detected as dengue and chikungunya,respectively and out of 58 AES cases only 23 could be detected as Japanese encephalitis by this RT-PCR method.Conclusions:This cost effective RT-PCR method can detect the total positive cases that remain undetected by EL1SA method.Moreover,this method is capable to detect the viral RNA from patients'sera even after the appearance of IgM antibody at one fifth costs as compared with the other commercially available kits.
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.
