TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics

Autor:	Kitty F. Cardwell, Daniela Sempertegui-Bayas, Francisco M. Ochoa-Corona, Andres S. Espindola, Danny F. Bravo-Padilla, Viviana Freire-Zapata
Rok vydání:	2021
Předmět:	0106 biological sciences 0301 basic medicine Nepovirus Genome Viral Computational biology Biology Microbiology 01 natural sciences Article Plant Viruses HTS diagnostics 03 medical and health sciences microbe finder TRSV Virology Plant virus Complementary DNA tobacco ringspot virus diagnostics RNA Viruses Pathogen MiFi Plant Diseases template switching oligo Computational Biology High-Throughput Nucleotide Sequencing RNA sequencing Reverse Transcription TASPERT Ribosomal RNA sensitivity QR1-502 Titer RNA silencing 030104 developmental biology Infectious Diseases Metagenomics Metagenome RNA Viral HTS Closteroviridae 010606 plant biology & botany
Zdroj:	Viruses Volume 13 Issue 7 Viruses, Vol 13, Iss 1223, p 1223 (2021)
ISSN:	1999-4915
Popis:	High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.
Databáze:	OpenAIRE
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