Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a-based visual assay

被引：107

作者：

Jiao, Jian ^{[1
]}

Kong, Kangkang ^{[1
]}

Han, Jinmeng ^{[1
]}

Song, Shangwei ^{[1
]}

Bai, Tuanhui ^{[1
]}

Song, Chunhui ^{[1
]}

Wang, Miaomiao ^{[1
]}

Yan, Zhenli ^{[2
]}

Zhang, Hengtao ^{[2
]}

Zhang, Ruiping ^{[2
]}

Feng, Jiancan ^{[1
]}

Zheng, Xianbo ^{[1
]}

机构：

[1] Henan Agr Univ, Coll Hort, Zhengzhou, Peoples R China

[2] Chinese Acad Agr Sci, Zhengzhou Fruit Res Inst, Zhengzhou, Henan, Peoples R China

来源：

PLANT BIOTECHNOLOGY JOURNAL | 2021年 / 19卷 / 02期

关键词：

apple viral diseases; LbCas12a; RT-RPA; visualized detection; in-field application; NUCLEIC-ACID DETECTION; NECROTIC MOSAIC-VIRUS; STEM-GROOVING-VIRUS; REVERSE TRANSCRIPTION; GOLD NANOPARTICLES; RAPID DETECTION; ILARVIRUS; SEQUENCE; TREES;

D O I：

10.1111/pbi.13474

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Co-infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a-based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namelyApple necrotic mosaic virus(ApNMV),Apple stem pitting virus(ASPV),Apple stem grooving virus(ASGV),Apple chlorotic leaf spot virus(ACLSV) andApple scar skin viroid(ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide-conjugated gold nanoparticles. The CRISPR/Cas12a-RT-RPA platform exhibited comparable sensitivity to RT-qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT-PCR detection for 52 samples. This novel Cas12a-based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

引用

页码：394 / 405

页数：12

共 44 条

[1] Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay [J].