A multiplex RT-PCR for rapid and simultaneous detection of viruses and viroids in chrysanthemum

Abstract

Significance and Impact of the Study: The multiplex RT-PCR assay described in this study is the first report of simultaneous detection of virus and viroid in chrysanthemum, which provides a fast, convenient, cost-saving way to detect the virus and viroid mixed infections in plants. AbstractChrysanthemum plants are subject to serious virus diseases, so detection and identification of virus pathogens is important to prevent the virus spread. A reliable one-step multiplex RT-PCR was developed to simultaneously detect two viruses and two viriods: chrysanthemum virus B, tomato Aspermy virus, chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid. In addition, we investigated the detection limit and the efficiency of single and multiplex RT-PCR assays. The results showed that the multiplex RT-PCR assay proved to be as sensitive as the single one. In conclusion, this technique is potentially useful in routine diagnosis of chrysanthemum viruses and viroids.

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

“…Particularly, this technique has been used successfully for simultaneous detection and differentiation of the five seedborne legume viruses [35], three ilarviruses affecting stone fruit trees [27], five potato viruses and a viroid [8], six citrus viroids and apple stem grooving virus from citrus plants [36], four viruses and Pseudomonas savastanoi pv. savastanoi in olive trees [10], five tospoviruses in ornamental crops [28], eight stone fruit viruses [37], nine grapevine viruses [12], three beet poleroviruses in sugarbeet and aphids [38, 39], eight wheat viruses [13], nine crinivirus infecting vegetable and small fruit crops [15], four viruses infecting cassava [18], five tobacco viruses [19], three cucurbit-infecting poleroviruses [30], seven main tomato-infecting viruses [20], three rice viruses [21], three lily-infecting viruses [22], four cherry viruses [23], four viruses in peach [24], three viruses in pear plants [40], five viruses and two viriods infecting chrysanthemum [41], three viruses infecting papaya [42], three orchid viruses [25], five fabaviruses [31] and so on.…”

RT‐PCR tests for sensitive detection of the major Ranunculus‐infecting viruses: field and in vitro applications

“…A new primer pair for RWMV was designed (Table ) and the 5StRNAarg primer pair (Table ) was added in the multiplex procedure to serve as an internal control, amplifying a specific fragment only if the RNA extraction and following cDNA synthesis were correctly performed. The reaction mixture and protocol were optimized following guidelines in published methods (Henegariu et al ., ; Gambino & Gribaudo, ; Song et al ., ). The reaction mixture had a final volume of 25 μL and was as follows: 1.6× PCR buffer (without Mg 2+ ), 3.7 m m MgCl 2 , 0.4 m m dNTPs, 1.5 U Platinum Taq DNA polymerase, 0.16 μ m each TSWV primer, 0.4 μ m each RWMV primer, 0.1 μ m each RanMMV primer, 0.16 μ m each universal primer.…”

Diagnostic techniques for viroids

“…Simultaneous detection lessens costs, labour and time but, at the same time, the amount of amplified DNA is lower than with single RT‐PCR (Ito et al ., ; Narayanasamy, ). Nevertheless, multiplex RT‐PCR reactions, such as fluorescent mRT‐PCR (Di Serio et al ., ), mRT‐PCR‐ELISA (Shamloul et al ., ) and regular mRT‐PCR (Nie & Singh, ; Ito et al ., ; Ragozzino et al ., ; Hataya, ; Wang et al ., ; Matsushita et al ., ; Hajizadeh et al ., ; Lin et al ., ; Lu et al ., ; Song et al ., ; Gambino et al ., ; Kumar et al ., ; Olivier et al ., ) are increasingly being used.…”