First report of a simple method of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without PCR amplification.
Shashikala T, Yogi D, Akshay K, Nagesh SN, Manamohan M
Summary
PubMedResearchers developed a CRISPR/Cas13a-based diagnostic test that rapidly detects groundnut bud necrosis virus without PCR amplification, offering a simple, field-deployable method for early virus detection in crops. The test is sensitive enough to detect virus at levels as low as 0.01 ng and works with crude plant extracts in the field.
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Key Findings
CRISPR/Cas13a assay successfully detects GBNV without requiring PCR amplification, enabling faster diagnosis
Achieves high sensitivity with detection limit as low as 0.01 ng of viral RNA from both synthetic and crude field samples
Field-deployable using simple alkaline PEG buffer extraction from infected leaves, eliminating need for specialized laboratory equipment
Original Abstract
Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identifying at the earliest stage and further to initiate disease management. This study presents the first report of a CRISPR/Cas13a-based diagnostic assay for GBNV detection without amplification. GBNV was maintained in cowpea cv. C152 through mechanical inoculation, further viral RNA was isolated to clone the two target genes viz. nucleocapsid (NP) and movement protein (MP) genes. These genes were ligated to pTZ57R/T vector and sequenced. Similarly, the LshCas13a gene was cloned from pUC19 into pET28a, expressed in E. coli BL21, and purified using Ni-NTA affinity chromatography. Guide RNAs targeting conserved regions of NP and MP genes were synthesized by in vitro transcription and mixed with Cas13a protein to form ribonucleoprotein (RNP) complex. Target RNA, obtained either by in vitro-transcription or crude extract of infected tomato was used to detect GBNV, using a fluorescence-based reporter assay. This method found to be highly sensitive that could detect GBNV at as low as 0.01 ng. From the field perspective, GBNV could be detected from the crude extract of the GBNV infected tomato leaves using an alkaline PEG buffer. Thus CRISPR/Cas13a-based assay provides a rapid, amplification-free, and field-deployable diagnostic platform for GBNV. This lays the groundwork for a field adoptable CRISPR diagnostics for other plant RNA viruses also.
This connects to 12 other discoveries — 3 species, 4 topics, 5 related articles
Species Mentioned
The cowpea is an annual herbaceous legume from the genus Vigna. It can be erect, semierect (trailing), or climbing. A high level of morphological diversity is found within the species with large variations in the size, shape, and structure of the plant. Four subspecies are recognised, three of wh...
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