Development and Validation of a Duplex RT-qPCR for Detection of Peach Latent Mosaic Viroid and Comparison of Different Nucleic-Acid-Extraction Protocols

Abstract

Peach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis.

Keywords: PLMVd; TPS; rapid-extraction methods; validated detection test.

Figure 1

Example of an amplification plot of dRT-qPCR for PLMVd. Blue curves: PLMVd-positive sample (ID 2 of Table 1) that tested positive with both sRT-PCRs; pink curves: PLMVd sample (ID 1 of Table 1) only detectable with the test by Serra et al. [4]; green curve: healthy sample.

Figure 2

This graph highlights the efficiency-curve comparison. The horizontal axis reports the logarithm of the dilution factor, while the vertical axis shows the Cq values obtained in the reactions. In blue are the points and the interpolating line of the dRT-qPCR (slope = −3.41; R² = 98%); in green are the points and the interpolating line of the sRT-qPCR by Luigi and Faggioli [8] (slope = −3.10; R² = 99%); and in purple are the points and the interpolating line of the sRT-qPCR by Serra et al. [4] (slope = −3.10; R² = 98%).

Figure 3

ΔCq values of amplifications using three different real-time PCR tests: Luigi and Faggioli [8], Serra et al. [4] and the dRT-qPCR developed in this work. Each test was applied to six different classic extraction protocols: liquid nitrogen (N2) + RNeasy Plant mini-kit (Qiagen)—red; liquid nitrogen (N2) + Quick-RNA Plant Kit (Zymo)—orange; liquid nitrogen (N2) + Sbeadex maxi-plant kit (Sbeadex/KF)—yellow; Tissue Lyser (TL) + RNeasy Plant mini-kit (Qiagen)—green; Tissue Lyser (TL) + Quick-RNA Plant Kit (Zymo)—purple; and Tissue Lyser (TL) + Sbeadex maxi-plant kit (Sbeadex/TL)—blue. Cq values were normalized (ΔCq) using the Tissue Lyser (TL) + Quick-RNA Plant Kit (Zymo) as a benchmark. Values are expressed as boxplots of six PLMVd-infected plants (two technical replicates).

Figure 4

Cq values obtained by testing three PLMVd isolates at relative tenfold dilution (reported as logarithm).

Figure 5

Cq values obtained by testing the repeatability (red) and reproducibility (blue) of the dRT-qPCR. Samples were tested at low (■) and medium (●) concentrations, each in three technical replicates.

Figure 6

Comparison of the mean ΔCq values obtained in analyzing TRNA extracted with the following combinations: liquid nitrogen + RNeasy Plant mini-kit—red; liquid nitrogen + Quick-RNA Plant Kit—orange; liquid nitrogen + Sbeadex maxi-plant kit—yellow; Tissue Lyser + RNeasy Plant mini-kit—green; Tissue Lyser + Sbeadex maxi-plant kit—blue. Statistical significance of differences was determined using Tukey’s HSD post hoc test; different letters indicate statistically different groups (p < 0.001).

Figure 7

Boxplot reporting the ΔCq values obtained in analysis of TRNA from rapid extraction: (a) results obtained in analysis of tissue-printed samples; (b) results obtained in analysis of samples macerated in PBS buffer; and (c) results obtained in analysis of samples macerated in PO₄ buffer. Statistical significance of different ΔCq values was determined using Tukey’s HSD post hoc test (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Figure 8

(a) Boxplot reporting the ΔCq values obtained in analysis of leaf samples vs phloem samples through tissue printing (TP) or maceration in PBS (PBS). (b) Comparison of the ΔCq values of the leaf samples macerated in Bioreba buffer, spotted on nylon or paper membranes and released with Triton or glycine buffer. Statistical significance of differences was determined using Tukey’s HSD post hoc test (* = p < 0.05; *** = p < 0.001).

Figure 9

Graphical representation of the different combinations of rapid-extraction tests used.