利用ELISA与qRT-PCR技术检测番木瓜PRSV病毒的方法研究  

Detection of Papaya PRSV Virus Using ELISA and qRT-PCR Techniques

作　　者：谢秀菊 夏启玉[1] 霍姗姗[1] 杜晓希 麦贤俊 张雨良[1] 郭安平[1] 李峰 孔祥义 赵辉[1] XIE Xiuju;XIA Qiyu;HUO Shanshan;DU Xiaoxi;MAI Xianjun;ZHANG Yuliang;GUO Anping;LI Feng;KONG Xiangyi;ZHAO Hui(Sanya Research Institute,Chinese Academy of Tropical Agricultural Sciences/Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences/Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions,Sanya,Hainan 572024,China;Sanya Academy of Tropical Agriculture,Sanya,Hainan 572022,China;Bellagen Biotechnology Co.,Ltd.,Jinan,Shandong 250300,China)

机构地区：[1]中国热带农业科学院三亚研究院/中国热带农业科学院热带生物技术研究所/海南省南繁生物安全与分子育种重点实验室,海南三亚572024 [2]三亚市热带农业科学院,海南三亚572022 [3]山东舜丰生物科技有限公司,山东济南250300

出　　处：《热带作物学报》2024年第12期2534-2541,共8页Chinese Journal of Tropical Crops

基　　金：三亚市科技创新专项(No.2022KJCX21);海南省院士创新平台项目。

摘　　要：番木瓜环斑病毒(Papaya ringspot virus,PRSV)是番木瓜生产上最严重的病害之一,其发病率高,传播快,危害重。为及时发现感染PRSV的番木瓜植株,本研究建立了分别利用酶联免疫吸附测定(ELISA)技术和荧光定量逆转录PCR(qRT-PCR)技术检测番木瓜植株感染PRSV的方法,利用这2种方法检测多株转基因番木瓜与非转基因番木瓜的PRSV含量,并对结果进行比较分析。结果表明:利用PRSV多肽抗原作为标准品制备的抗体建立的标准曲线拟合度较好,可用于ELISA法检测PRSV;qRT-PCR法中选择的内参基因Cpa03g018830在番木瓜的不同生长阶段均稳定表达,可作为PRSV含量测定的内参基因;ELISA法和qRT-PCR法对多株转基因和非转基因番木瓜植株中PRSV含量的检测结果基本一致,表明这2种方法均可用于番木瓜植株中PRSV含量的检测。利用这2种检测手段,可及时发现并铲除感染了PRSV的番木瓜植株,有效预防与控制PRSV传播。Papaya ringspot virus(PRSV)is one of the most serious diseases in papaya production,with high incidence rate,rapid transmission and serious harm.To detect papaya plants infected with PRSV in a timely manner,this study established methods for detecting papaya plants infected with PRSV using enzyme-linked immunosorbent assay(ELISA)and fluorescence quantitative reverse transcription PCR(qRT-PCR).The two methods were used to detect the PRSV content of multiple transgenic and non transgenic papaya plants,and the results were compared.The results showed that the standard curve established using PRSV peptide antigen as the standard and antibodies prepared from it had good fitting,and could be used for ELISA detection of PRSV;The reference gene Cpa03g018830 selected in qRT-PCR method was stably expressed at different growth stages of papaya and could be used as a reference gene for PRSV content determination;The detection results of PRSV content in multiple transgenic and non transgenic papaya plants using ELISA and qRT-PCR methods were basically consistent,indicating that both methods can be used for the detection of PRSV content in papaya plants.By using thee two detection methods,papaya plants infected with PRSV can be detected and eradicated in a timely manner,effectively preventing and controlling the spread of PRSV.

关 键 词：番木瓜环斑病毒 酶联免疫吸附 荧光定量逆转录PCR 

分 类 号：S436.67[农业科学—农业昆虫与害虫防治]