Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify ...Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.展开更多
In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM ric...In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.展开更多
Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specifi...Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.展开更多
Real Time PCR,即实时监测PCR扩增产物并进行解析的方法,目前已广泛应用于分子生物学研究的各个领域。Real Time PCR技术秉承及发展了普通PCR的快速、高灵敏度检出等优点,同时克服了普通PCR不能准确定量、容易污染等缺点,无需在反应结...Real Time PCR,即实时监测PCR扩增产物并进行解析的方法,目前已广泛应用于分子生物学研究的各个领域。Real Time PCR技术秉承及发展了普通PCR的快速、高灵敏度检出等优点,同时克服了普通PCR不能准确定量、容易污染等缺点,无需在反应结束后通过电泳操作确认扩增产物。目前,Real Time PCR可设计多对引物在同一反应体系中同时对多个靶基因进行扩增,实现多重实时定量检测。Real Time PCR使PCR技术发生了质的飞跃,扩展了PCR技术的应用范畴,是一种具有划时代意义的技术。本文主要介绍Real Time PCR的主要原理、解析方法、技术发展趋势及其在海洋病原生物检测方面的应用。展开更多
Ticks harbor multiple pathogens, most of which can be transmitted to humans. The ensuing zoonoses display non-specific symptoms that make definitive diagnosis difficult. We report here the development and evaluation o...Ticks harbor multiple pathogens, most of which can be transmitted to humans. The ensuing zoonoses display non-specific symptoms that make definitive diagnosis difficult. We report here the development and evaluation of multiplex real time polymerase chain reaction (qPCR) assays for eight tick-borne zoonoses (TBZ). The assays were organized in duo formats of 4-plex each. Format 1 was optimized for Anaplasma phagocytophilum, Coxiella burnetii, Borrelia burgdoferi and Ehrlichia chaffeensis. Format 2 was optimized for Rickettsia species (spp.), Bartonella spp., Borrelia spp. other than B. burgdoferi and Babesia spp. Synthetic plasmids were used to show that the assays can specifically detect all target sequences in the same reaction tube. Assays were assayed eight times to determine assay performance and the limit of detection was determined as the lowest plasmid concentration that was amplified for all the targets. Standard curves of threshold cycle (Ct) versus copy numbers were generated and used to determine linearity and efficiency of the assays. Pairwise comparison of singleplex and multiplex assays was done using Bland-Altman plots. Prevalence was calculated as overall percentage of positive patients to each TBZ tested Assay 1 had a limit of detection of 2 copy numbers for all targets. Assay 2 was less sensitive and on average had a limit of detection of 18 gene copies. In replicate tests, both assays had intra-assay variation of less than two cycles. Multiplex assays performance was comparable to respective singleplex assays. Evaluation of 512 clinical samples collected between 2008 and 2016 from acute febrile illness patients attending hospitals in different counties in Kenya revealed a 20% prevalence of tick-borne pathogens comprising B. burgdorferi (6%), non B. burgdorferi Borrelia spp. (3%), C. burnetii (5%), A. phagocytophilum (5%), Rickettsia spp. (2%), E. chaffeensis (0.8%), Bartonella spp. (0.8%), and Babesia spp. (0.4%). The high analytical sensitivity suggests potential for the duo 4-plex qPCR for detection of common TBZ.展开更多
基金supported by the Center of Excellence in Clinical Virology.Chulalongkorn University,CU Centenary Academic Development ProjectKing Chulalongkorn Memorial Hospital,the National Research University Project of CHEthe Ratchadaphiseksonphot Endowment Fund(HR1155A)
文摘Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.
基金Supported by Key Special Project for Breeding and Cultivation of GMO Varieties(2011ZX08001-001,2014ZX0800101B)Special Fund from the Department of Finance of Hubei Province(2011-2015)Collaborative Breeding Project for Rice(2013-2017)
文摘In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.
文摘Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.
文摘Real Time PCR,即实时监测PCR扩增产物并进行解析的方法,目前已广泛应用于分子生物学研究的各个领域。Real Time PCR技术秉承及发展了普通PCR的快速、高灵敏度检出等优点,同时克服了普通PCR不能准确定量、容易污染等缺点,无需在反应结束后通过电泳操作确认扩增产物。目前,Real Time PCR可设计多对引物在同一反应体系中同时对多个靶基因进行扩增,实现多重实时定量检测。Real Time PCR使PCR技术发生了质的飞跃,扩展了PCR技术的应用范畴,是一种具有划时代意义的技术。本文主要介绍Real Time PCR的主要原理、解析方法、技术发展趋势及其在海洋病原生物检测方面的应用。
文摘Ticks harbor multiple pathogens, most of which can be transmitted to humans. The ensuing zoonoses display non-specific symptoms that make definitive diagnosis difficult. We report here the development and evaluation of multiplex real time polymerase chain reaction (qPCR) assays for eight tick-borne zoonoses (TBZ). The assays were organized in duo formats of 4-plex each. Format 1 was optimized for Anaplasma phagocytophilum, Coxiella burnetii, Borrelia burgdoferi and Ehrlichia chaffeensis. Format 2 was optimized for Rickettsia species (spp.), Bartonella spp., Borrelia spp. other than B. burgdoferi and Babesia spp. Synthetic plasmids were used to show that the assays can specifically detect all target sequences in the same reaction tube. Assays were assayed eight times to determine assay performance and the limit of detection was determined as the lowest plasmid concentration that was amplified for all the targets. Standard curves of threshold cycle (Ct) versus copy numbers were generated and used to determine linearity and efficiency of the assays. Pairwise comparison of singleplex and multiplex assays was done using Bland-Altman plots. Prevalence was calculated as overall percentage of positive patients to each TBZ tested Assay 1 had a limit of detection of 2 copy numbers for all targets. Assay 2 was less sensitive and on average had a limit of detection of 18 gene copies. In replicate tests, both assays had intra-assay variation of less than two cycles. Multiplex assays performance was comparable to respective singleplex assays. Evaluation of 512 clinical samples collected between 2008 and 2016 from acute febrile illness patients attending hospitals in different counties in Kenya revealed a 20% prevalence of tick-borne pathogens comprising B. burgdorferi (6%), non B. burgdorferi Borrelia spp. (3%), C. burnetii (5%), A. phagocytophilum (5%), Rickettsia spp. (2%), E. chaffeensis (0.8%), Bartonella spp. (0.8%), and Babesia spp. (0.4%). The high analytical sensitivity suggests potential for the duo 4-plex qPCR for detection of common TBZ.
