Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens

文献类型： 外文期刊

第一作者： Loconsole, Giuliana

作者： Loconsole, Giuliana;Savino, Vito;Liao, Hui-Hong;Jiang, Bo;Saponari, Maria;Yokomi, Raymond K.

作者机构：

关键词： Citrus pathogens;Detection;Real time-PCR multiplex assay

期刊名称：JOURNAL OF VIROLOGICAL METHODS （ 影响因子：2.014； 五年影响因子：2.001 ）

ISSN： 0166-0934

年卷期： 2013 年 193 卷 2 期

页码：

收录情况： SCI

摘要： A number of important citrus pathogens are spread by graft propagation, arthropod vector transmission and inadvertent import and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs require pathogen detection systems which are economical and sensitive to maintain a healthy industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed allowing high-throughput and simultaneous detection of some major invasive citrus pathogens. Automated high-throughput extraction comparing several bead-based commercial extraction kits were tested and compared with tissue print and manual extraction to obtain nucleic acids from healthy and pathogen-infected citrus trees from greenhouse in planta collections and field. Total nucleic acids were used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including a virus, two viroids, a bacterium associated with huanglongbing and a citrus RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were developed and tested with target templates to determine sensitivity and detection efficiency. The first assay included primers and probes for 'Candidatus Liberibacter asiaticus' (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control. Primers and TaqMan probes for the viroids were designed in this work; whereas those for the other pathogens were from reports of others. Based on quantitation cycle values, automated high-throughput extraction of samples proved to be as suitable as manual extraction. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series as singleplex assays and yielded similar quantitation cycle values. Taken together, high throughput extraction and multiplex RT-qPCR assays reported in this study provided a rapid and standardized method for routine and simultaneous diagnosis of different RNA and DNA citrus pathogens. Published by Elsevier B.V.

分类号：

  • 相关文献

[1]Establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (CPA-LFD) for rapid and specific detection of red-spotted grouper nervous necrosis virus. Su, Zi Dan,Shi, Cheng Yin,Huang, Jie,Shen, Gui Ming,Li, Jin,Wang, Sheng Qiang,Fan, Chao,Shi, Cheng Yin,Huang, Jie,Su, Zi Dan,Shen, Gui Ming,Wang, Sheng Qiang,Fan, Chao. 2015

[2]Detection and quantification of hepatopancreatic parvovirus in penaeid shrimp by real-time PCR assay. Liu, Tianqi,Yang, Bing,Song, Xiaoling,Wang, Xiuhua,Yuan, Yanyan,Liu, Li,Huang, Jie,Liu, Tianqi,Yuan, Yanyan. 2013

[3]Development of a real-time reverse transcription loop-mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus. Yu, Xuewu,Shi, Lin,Lv, Xiaoping,Zheng, Shimin,Yu, Xuewu,Shi, Lin,Yao, Wei,Cao, Minghui,Yu, Hanxun,Wang, Xiurong,Wang, Xiurong. 2015

[4]An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene. Xie Guosi,Zhang Qingli,Han Nana,Shi Chengyin,Wang Xiuhua,Liu Qinghui,Huang Jie,Xie Guosi,Huang Jie. 2012

[5]Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification. Chen, Hao-tai,Zhang, Jie,Yang, Sheng-hai,Ma, Li-na,Ma, Yan-ping,Liu, Xiang-tao,Cai, Xue-peng,Zhang, Yong-guang,Liu, Yong-sheng. 2009

[6]Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification. Chen, Hao-tai,Zhang, Jie,Sun, De-hui,Chu, Yue-feng,Cai, Xue-peng,Liu, Xiang-tao,Luo, Xue-nong,Liu, Yong-sheng,Liu, Qing. 2008

[7]Development and validation of a TaqMan (TM) fluorescent quantitative real-time PCR assay for the rapid detection of Edwardsiella tarda. Xie Guosi,Huang Jie,Zhang Qingli,Han Nana,Shi Chengyin,Wang Xiuhua,Xie Guosi,Huang Jie. 2012

[8]Quantitative detection of the rice false smut pathogen Ustilaginoidea virens by real-time PCR. Li, H.,Ni, D. H.,Li, J.,Li, H.,Ni, D. H.,Duan, Y. B.,Li, J.,Song, F. S.,Li, L.,Wei, P. C.,Yang, J. B.,Li, H.,Wei, P. C.,Duan, Y. B.,Song, F. S.,Chen, Y.. 2013

[9]Development of cross-priming amplification coupled with vertical flow visualization for rapid detection of infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish, Siniperca chuatsi. Liu, Wenzhi,Zhou, Yong,Fan, Yuding,Jiang, Nan,Zeng, Lingbing,Cain, Kenneth,Cain, Kenneth. 2018

[10]Rapid typing of foot-and-mouth disease serotype Asia 1 by reverse transcription loop-mediated isothermal amplification. Liu, Yong-sheng,Liu, Xiang-tao. 2011

[11]The Applications of Gold Nanoparticle-Initialed Chemiluminescence in Biomedical Detection. Liu, Zezhong,Zhao, Furong,Gao, Shandian,Shao, Junjun,Chang, Huiyun. 2016

[12]A Miniature Handheld Automatic Detected Device for Register. Zhang Tiefeng,Wang Jun,Sun Zhihui,Zhao Bo,Lu Guotao. 2011

[13]DEVELOPMENT OF A SIMPLE AND EFFECTIVE METHOD FOR SPECIFIC DETECTION OF PEPPER MILD MOTTLE VIRUS. Liu, Y.,Wang, X.,Zhou, G.,Li, X.. 2009

[14]Aptamer-Based Fluorescence Assay for Hg2+ Determination. Lei Zhao-Jing,Hu Qiu-Hui,Lei Zhao-Jing,Zhang Cun-Zheng,Liu Yuan,Zhang Qiang,Liu Xian-Jin. 2012

[15]Detection and quantification of Rhizoctonia cerealis in soil using real-time PCR. Guo, Yingpeng,Li, Wei,Sun, Haiyan,Wang, Ning,Chen, Huaigu,Guo, Yingpeng,Yu, Hanshou. 2012

[16]A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda. Xie Guosi,Huang Jie,Zhang Qingli,Han Nana,Shi Chengyin,Wang Xiuhua,Liu Qinghui,Xie Guosi,Huang Jie. 2012

[17]Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of ostreid herpesvirus 1 DNA. Ren, Weicheng,Cai, Yuyong,Wang, Chongming,Renault, Tristan,Cai, Yuyong. 2010

[18]Fluorescence polarization assay for the simultaneous determination of bisphenol A, bisphenol F and their diglycidyl ethers in canned tuna. Guan, Tianzhu,Li, Tiezhu,Li, Zhuolin,Wang, Yongzhi,Zhang, Jie,Wang, Yongjun,Zhang, Tiehua,Yu, Hansong,Ruan, Ping. 2017

[19]Rapid detection of Haemophilus parasuis using cross-priming amplification and vertical flow visualization. Li, Chunling. 2018

[20]Development of a loop-mediated isothermal amplification method for rapid detection of porcine boca-like virus. Li, Bin,Ma, Jun-jie,Zhang, Xue-han,Wen, Li-bin,Mao, Li,Ni, Yan-xiu,Guo, Rong-li,Zhou, Jun-ming,Lv, Li-xin,He, Kong-wang,Li, Bin,Ma, Jun-jie,Zhang, Xue-han,Wen, Li-bin,Mao, Li,Ni, Yan-xiu,Guo, Rong-li,Zhou, Jun-ming,Lv, Li-xin,He, Kong-wang,Li, Bin,Ma, Jun-jie,Zhang, Xue-han,Wen, Li-bin,Mao, Li,Ni, Yan-xiu,Guo, Rong-li,Zhou, Jun-ming,Lv, Li-xin,He, Kong-wang,Xiao, Shao-bo. 2012

作者其他论文 更多>>

微信小程序