An optimized nucleic acid isolation protocol for virus diagnostics in cassava (Manihot esculenta Crantz.)

Abstract

Our group works on the detection and characterization of cassava viruses, supporting projects that involve large scale pathogen surveillance activities and resistance screening assays in multiple and remote locations. In order to comply with these applications, nucleic acid isolation protocols need to be cost effective, adjusted for samples that will stand long distance transport and harsh storage conditions, while maximizing the yield and quality of the nucleic acid extracts obtained. The method we describe here has been widely used and validated using different downstream tests (including, but not limited to, Rolling Circle Amplification and Illumina and Nanopore sequencing), but is currently unpublished. The protocol begins with milligram amounts of dry leaf samples stored in silica gel, does not require liquid Nitrogen nor phenol extraction and produces an average of 2.11 µg of nucleic acids per mg of dry tissue. DNA purity estimations reveal OD260/280 ratios above 2.0 and OD260/230 ratios above 1.7, even for samples stored in silica gel for several months. The high quality of the extracts is suitable for detection of DNA and RNA viruses, with high efficiency. We suggest this method could be used as part of a gold standard kit for virus detection in cassava.

Susceptibility of Cassava Varieties to Disease Caused by Sri Lankan Cassava Mosaic Virus and Impacts on Yield by Use of Asymptomatic and Virus-Free Planting Material

“…A total of 943 samples (314 from 2019, 574 from 2020, and 55 from clean planting material), corresponding to the topmost, youngest leaves of asymptomatic plants were collected from the harvest areas of all treatment plots. Leaf samples were dried in silica gel and stored at 4 • C before CTAB extraction [24]. PCR diagnostics were carried out with nucleic acid extracts diluted to a working concentration of 60 ng/µL and 1µL used per PCR reaction in a total reaction volume of 20 µL.…”

“…The PCR program was as follows: initial denaturation at 94 • C for 5 min followed by 35 cycles of denaturation at 94 • C × 30 s, annealing at 58 • C × 30 s and extension at 72 • C × 1 min. After a final extension at 72 • C × 5 min PCR products were resolved by agarose gel electrophoresis as previously described [24].…”

Cassava Frogskin Disease: Current Knowledge on a Re-Emerging Disease in the Americas

“…Several years of experience working with these pathogens have validated mechanical isolation in Nicotiana benthamiana plants, only for CsCMV and CsVX, which are not associated with root symptoms of CFSD. We have implemented standardized field data collection and nucleic acid extraction protocols [ 5 , 22 ] for cassava pathogen detection and characterization ( Table 2 ), which complement the chip-bud grafting [ 23 ] and greenhouse experiments described above. The availability of a novel virus vector that can be mechanically transmitted to cassava [ 55 ] could be helpful to study the role of different viruses (via RNA silencing) or viral genes (via transient expression) in the development of CFSD.…”

“…Nowadays, grafting tests using indicator plants could still be used to identify the presence of mixed infections, but it is not recommended for general virus indexing. Additional protocols have been validated to detect these pathogens in cassava without the need for grafting [ 20 , 21 , 22 , 23 , 24 ] ( Table 2 ).…”

Complete genome sequence of a novel secovirid infecting cassava in the Americas

“…The complete genome of CsTLV was obtained from a cassava plant of the commercial variety Melua-31, collected in August 2020 in Yopal, Colombia, and total RNA extraction with CTAB was done as reported previously [ 9 ]. For library preparation, we followed the SQK-DCS109 protocol (Oxford Nanopore Technologies), which uses RNase Cocktail Enzyme Mix (Thermo Fisher) to eliminate ribosomal RNA.…”