03779naa a2200265 a 450000100080000000500110000800800410001910000190006024501770007926000090025652028210026565000310308665000230311765000180314065000330315870000260319170000170321770000220323470000170325670000190327370000190329270000230331170000170333477301620335111326262022-10-18       2022    bl uuuu     u00u1 u    #d1 aGORAYEB, E. S.  aNucleic acid extraction and multiplex polymerase chain reaction analysis for the detection of maize stunt causal agents in plants and insect tissues.h[electronic resource]  c2022  aPresently, the corn stunt complex (CSC) is one of the most important diseases affecting corn crops. This complex is associated with infections of at least one of the following pathogens: maize bushy stunt phytoplasma (MBSP), corn stunt spiroplasma (CSS), and maize rayado fino virus (MRFV). Currently, the control of CSC is focused on their vector Dalbulus maidis (Delong and Wolcott), which combines monitoring programs with chemical spraying and biological control in the field. Owing to its RNA genome composition, MRFV detection is performed separately from mollicutes, which contributes to the omission of virus diagnosis in CSC monitoring activities and epidemiological studies. In this study, we developed a complete CSC diagnostic method comprising an RNA/DNA extraction protocol and a triplex polymerase chain reaction (PCR) for detecting all CSC pathogens in a single reaction step. Therefore, an MRFV-specific primer pair was designed for use along with the widely used F2n/R2 and CSSF2/CSSR6 primers for mollicute detection. Total nucleic acid extraction was performed using the CTAB-based protocol. MRFV-specific primer was designed using the Primer3 software (v. 0.4.0) and the two complete MRFV sequences (from the USA and Costa Rica) available in the GenBank. In silico tests were conducted to determine the primer specificity and secondary structure formation using BLASTn and IDT oligo Analyzer tools, respectively. The newly MRFV-specific designed primer pair was tested in vitro for its specificity and sensibility and subjected to a temperature-gradient PCR to adjust the best virus detection conditions. The MRFV primer was then combined in a triplex PCR with the mollicute-specific primers using samples from different Brazilian states, retrieved from infected insects and plants. The designed MRFV primer pair was species-specific, sensible, and did not form secondary structures or dimerize either with each other or with the other primers used to detect mollicutes. The primer was used to amplify a 270-bp fragment comprising the 5? end of the polyprotein gene of MRFV-infected samples from different geographical locations and tissues (both plants and insects) in a multiplex reaction. Three positive bands comprising the three pathogens (with sizes of ~270, 500, and 1250 bp, for MRFV, CSS, and MBSP, respectively) were visibly separated in the agarose gel, thereby yielding a clear interpretation of the outcomes. The results revealed that the proposed method was effective, cheaper, faster, and easier to perform relative to the other methods reported in the literature. Therefore, we recommend this tool, which is helpful for basic, applied, and epidemiological analyses and for monitoring programs conducted to evaluate the viral incidence in the corn stunting complex and corn crops.  acorn stunt disease complex  ainsect infectivity  aPCR multiplex  aplant pathogens transmission1 aALBUQUERQUE, M. R. M.1 aFERREIRA, J.1 aNASCIMENTO, S. C.1 aSILVA, T. S.1 aSAVARIS, D. M.1 aRIBEIRO, L. P.1 aSILVA, M. C. C. R.1 aSILVA, F. N.  tIn: WORKSHOP BRASILEIRO DE EPIDEMIOLOGIA DE DOENÇAS DE PLANTAS, 6., 2022, Chapecó. Resumos... Brasília: Sociedade Brasileira de Fitopatologia, 2022. p. 37