Development and Application of a Multiplex Reverse-Transcription Polymerase Chain Reaction Assay for Screening a Global Collection of Citrus tristeza virus Isolates

Abstract

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

Simultaneous detection and differentiation of three genotypes of Brassica yellows virus by multiplex reverse transcription-polymerase chain reaction

“…Multiplex RT-PCR assay has been developed successfully to detect and differentiate closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus [7], each of the major Potato virus Y strains and strain mixtures [43–46]. CMV subgroups and tobamoviruses infecting tomato [17], genotypes A and B of Beet necrotic yellow vein virus [11], five genotypes of Citrus tristeza virus from 29 citrus-growing countries [29]. There are three BrYV genotypes (BrYV -A, -B, and -C) based on sequence comparisons and phylogenetic analysis [1, 4], however, the pathogenicity, distribution and host range of the three BrYV genotypes remained to be determined.…”

Citrus tristeza virus: evolution of complex and varied genotypic groups

“…Complicating matters is a lack of consistency in choosing a region or regions to analyze, with CP, ORF1a/b, and sundry 3′ genes all being targeted in different assays (Hilf et al, 2005; Nolasco et al, 2009; Roy et al, 2010). Diagnosis with the coat protein alone is a historical legacy, as it is the most highly conserved and least variable of the 3′ genes with 93.4% nucleotide identity and 96.3% amino acid identity between isolates examined in this study.…”

Past and future of a century old Citrus tristeza virus collection: a California citrus germplasm tale

“…The MMM genotype analysis of CTV was performed using reverse transcription polymerase chain reaction (RT-PCR) with specific primers for the genotypes T30, VT, T36, T3, and B165/T68, as previously described (Hilf et al, 2005; Roy and Brlansky, 2010; Roy et al, 2010) (Table 2). Total RNA from ~0.2 g of bark tissue was extracted using the Spectrum Plant Total RNA kit (Sigma, Saint Louis, Missouri, USA) according to the manufacturer's instructions and eluted in 30 μl of RNase-free water.…”

“…aTwo MMM methodologies were developed independently by Hilf et al (2005)(-H) and Roy and Brlansky (2010)(-R) and Roy et al (2010). CP-U: Coat protein universal primer was developed in this study and was used as positive internal control for CTV detection.…”

“…In this study, genotypes of 48 CTV isolates, representing approximately 90% of the CCPP CTV collection, were characterized using multiple molecular markers (MMM) assays (Hilf et al, 2005; Yokomi and Deborde, 2005; Moreno et al, 2008; Folimonova et al, 2010; Roy et al, 2010; Roy and Brlansky, 2010). The MMM genotype characterization was complimented by CTV phylogenetic analysis of the major coat protein (CP) gene.…”