Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

Currently, several diagnostic methods have been developed for the detection of PRSV, PLDMV, or PapMV, such as enzyme-linked immunosorbent assay (ELISA), Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, and reverse transcription loop-mediated isothermal amplification (RT-LAMP) [17][18][19][20][21][22][23][24][25][26].However, these techniques only can detected individual papaya viruses in each reaction and are time-consuming, laborious, and costly, especially when large numbers of samples need to be tested for mixed infections.These limitations can be overcome by employing a multiplex reverse transcription polymerase chain reaction method (multiplex RT-PCR), which can amplify simultaneously more than one target sequences of different viruses by several primer pairs in one signal reaction, offering a significant time and cost-saving advantage [27,28].To date, multiplex RT-PCR has been used widely to diagnose infection in garlic, cassava, pear, cherry, apple, and potato plants by related plant viruses [21][22][23][29][30][31][32][33][34][35].However, this method has not been applied to the simultaneous detection of PRSV, PLDMV, and PapMV.In this study, the development and evaluation of a multiplex RT-PCR assay for the simultaneous detection and differentiation of PRSV, PLDMV, and PapMV from papaya is described.

Viruses
PRSV (Accession No. HQ424465), PLDMV (Accession No. JX974555), and PapMV (Accession No. JX524226) strains isolated from Hainan were used as the reference strains in this study [5,14,15].These three virus isolates propagated persistently in papaya were used as positive controls for the optimization of all RT-PCR assays.

Nucleic Acid Extraction
The papaya leaves (50-100 mg) were homogenized with liquid nitrogen.Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to manufacturer's protocol.The yield and the quality of RNA were analyzed using a NanoVue TM Plus Spectrophotometer (GE Healthcare, Freiburg, Germany).

The Uniplex PCR
Uniplex PCR for PRSV, PLDMV, or PapMV was carried out in a 25 μL mixture containing 12.5 μL 2 × Eco Taq PCR SuperMix (+dye) (TransGen Biotech, Beijing, China), 0.5 μL of specific primers (20 μM) (Table 1), 2 μL cDNA template, and 10.5 or 9.5 μL sterile deionized H2O.In a negative control reaction, nuclease free water or cDNA from healthy (virus-free) plant was used as the template.To determine the best annealing temperature for a correct amplification preventing non-specific products, a gradient PCR was performed in a TGradient Thermocycler (Biometra, Goettingen, Germany) using the following parameters: one cycle at 94 °C for 3 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 50-65 °C (with an increase of 2 °C per tested sample) for 30 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 5 min.PCR products were examined by electrophoresing 10 μL aliquots on 1.5% agarose gels in TAE buffer.Each amplified viral target fragments was cloned into the pMD18-T vector (Takara), and the resulting plasmid construct pPRSV-953, pPRSV-613, pPRSV-515, pPLDMV-355, pPapMV-476, and pPapMV-205, was confirmed by sequencing (Life Technologies, Guangzhou, China) and BLASTN at NCBI [41].

Optimization of the Multiplex PCR Conditions
To simultaneously amplify all three viral targets in one reaction using three sets of virus-specific primers, six primer combinations (PRSV953- ), ten primer combinations for concentration (0.4 μM, 0.2 μM, or 0.1 μM of each primer pair), and multiplex PCR cycling parameters were optimized.For multiplex RT-PCR, artificial mixture of total plant RNA was prepared from the different papaya samples known to be infected with PRSV, PapMV or PLDMV.The multiplex PCR was performed using a PCR reaction mixture containing 12.5 μL 2 × Eco Taq PCR SuperMix (+dye) (TransGen Biotech), 0.4 μM, 0.2 μM, or 0.1 μM of each primers, 2 μL cDNA template and sterile deionized H2O was added to a final volume of 25 μL.The PCR program was an initial denaturation at 94 °C for 3 min; 35 cycles of at 94 °C for 30 s, annealing at 50-65 °C (with an increase of 2 °C per tested sample) for 30 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 5 min.The amplicons of 10 μL aliquots were determined by electrophoresis through 1.5% agarose-TAE gel.

The Detection of Viruses in Field Samples by Uniplex RT-PCR and Multiplex RT-PCR
A total of 341 field papaya leaf samples showing symptoms such as mosaic, ring spots and distortion on leaves, and water soaking streaks on petioles, were collected from different geographical regions of Hainan Island (Table 2) between August 2013 and March 2014.Subsequently, these samples were simultaneously used to test PRSV, PLDMV, and PapMV by uniplex RT-PCR and multiplex RT-PCR.

Specificity and Compatibility of Primer Pairs
In uniplex RT-PCR, the expected sizes of viral target genes for PRSV, PapMV, and PLDMV were amplified specifically for all of six primers (Table 1) designed in this study (Figure 1, Lane 1-6).
The sequences of amplified products were identical to the GenBank sequences (Accession No. HQ424465, JX524226, JX974555), respectively.For multiplex RT-PCR, the primer combination PRSV613-F/R + PLDMV355-F/R + PapMV205-F/R gave more clear and specific bands of target products compared to the rest combinations, and then the primer combination was selected for further optimization in multiplex RT-PCR (Figure 1, Lane 10).

Optimization of Multiplex RT-PCR
A gradient RT-PCR was then performed to determine the optimal annealing temperature for uniplex RT-PCR and multiplex RT-PCR.In uniplex PCR assays, all primers performed well in the tested temperature range (50-64.3°C) except at 64.3 °C for PRSV amplification (lane 8 in Figure 2A-C).Similarly, a faint amplification for PRSV was detected at 64.3 °C in multiplex RT-PCR (lane 8 in Figure 2D).According to amplification efficiency and specificity for the targeted viruses as detected by agarose gel electrophoresis, the optimal annealing temperature was determined to be 57.9 °C.The optimized cycle protocol of the RT-PCR was: 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 1 min at 72 °C; and a final extension at 72 °C for 5 min.For the combinations of different concentrations of three primers, the amplification efficiencies of PRSV and PapMV varied at different combinations of primer concentrations, while that of PLDMV remained consistent and reliable (Figure 3).A balanced amplification, with similar fluorescence intensity for the bands corresponding to the three expected templates, was achieved when the primer concentrations were 0.4 μM for PRSV, 0.1 μM for PLDMV, and 0.2 μM for PapMV in the multiplex RT-PCR (Figure 3, Lane 7).

Evaluation of Multiplex RT-PCR Using Field Samples
The multiplex RT-PCR was validated for its practical application by detecting PRSV, PLDMV, and PapMV using 341 field samples (Table 2 and Figure 5).PRSV, PLDMV, and PapMV were detected in 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), respectively.Mixed infection of PRSV and PLDMV was detected in 59/341 (17.3%) of the samples.All of those field-collected samples were further confirmed using uniplex RT-PCR and the data showed that the results were in agreement with the multiplex RT-PCR (Table S1), indicating that the multiplex RT-PCR assay developed in this study is a rapid and validated method.

Discussion
A PCR-based method is routinely used to diagnose the presence of plant pathogens, particularly plant viruses.In this study, the uniplex and multiplex PCR and RT-PCR protocols were standardized for the simultaneous detection of PRSV, PLDMV, and PapMV, both as single and as mixed infections in papaya.Generally, primer pairs are the most important factors affecting the efficiency and specificity of a multiplex RT-PCR.Therefore, three primer pairs were designed according to the conserved region of the CP gene of PLDMV and PapMV.However, a different target P3 gene was used for PRSV.Previous methods to detect PRSV targeted the CP gene, a conserved region of the nuclear inclusion protein (NIb) gene, or the 3'-UTR, which are relatively more highly conserved compared to other regions of the complete genomic sequence of PRSV [17,18,20,24].However, these genes or fragments from different geographical PRSV isolates have been used to develop transgenic cultivars with resistance to PRSV in many countries, such as China, America, Brazil, Jamaica, Venezuela, Thailand, Australia, and some countries of East Africa [43][44][45].Indeed, two transgenic papaya lines have been planted commercially in Hawaii, United States, since 1998, and in Guangdong province, China, in 2006 [43,45,46].Therefore, the primers previous designed could not be used to distinguish between transgenic papaya with PRSV CP or NIb gene for the resistance against PRSV and PRSV-infected papaya.In this study, a highly conserved region (2774-4207 nt) within the HC-Pro and P3 gene was chosen as a suitable target to design three primer pairs specific for PRSV through aligning 26 isolates of the complete genomic sequence of PRSV by software.Of the six primers screened in uniplex and multiplex RT-PCR, the primer combination PRSV613-F/R + PLDMV355-F/R + PapMV205-F/R was selected for further optimization in multiplex RT-PCR.The three amplicons (613 bp for PRSV, 355 bp for PLDMV, and 205 bp for PapMV) were easily differentiated on agarose gel electrophoresis since their size difference was 150 bp or more.
In this work, 2 × Eco Taq PCR SuperMix (+dye) (TransGen Biotech) including optimized PCR buffer, Taq DNA polymerase, dNTP, and loading dye was used to optimize the reaction systems and conditions.Currently, premixed master Taq DNA polymerase is supplied by most biotechnology companies worldwide, and successfully amplifies low-titer templates even in the presence of inhibitors.This is among the most popular applications for routine PCR because of several advantages: (i) minimized PCR optimization steps with advanced buffer systems; (ii) enhanced enzyme stability for increasing yield and specificity of PCR amplification; (iii) using convenient master mix formats can save time and reduce the risk of sample contamination during assay setup; (iv) no need to add gel loading dye can save time on agarose gel electrophoresis.Therefore, by the absence of a need to optimize the concentrations of Taq DNA polymerase, dNTP, and Mg 2+ in multiplex RT-PCR, the time, labor, and reagent costs in the detection of these three viruses was reduced, especially for a large number of samples.
A frequent problem in multiplex RT-PCR is the unbalanced amplification of certain viruses due to the presence of multiple targets in one reaction and to primers with different compatibility to their targets, which may result in competition for enzymes and nucleotides in the reactions [23,47].In this study, the simultaneous amplification of PRSV, PLDMV, and PapMV by using mixtures of equal amounts of primer pairs specific for those three viruses resulted in strong differences in band intensity on the agarose gel.Further, the strong amplification of the PLDMV band of 355 bp was produced in all multiplex RT-PCR assays (Figure 3, Lane 1-10).To obtain balanced amplification efficiency of all three viruses, plasmids containing the target genes of those three viruses were used for the optimization of primer concentrations, which ensured that similar band intensities resulted from almost equal amounts of templates.Thus, the optimum combinations of the primer concentrations were 0.4 μM for PRSV, 0.1 μM for PLDMV, and 0.2 μM for PapMV in the multiplex RT-PCR (Figure 3, Lane 7).In addition, the assay sensitivity of multiplex RT-PCR compared to uniplex RT-PCR was approximately 10-100-fold lower for detection of PRSV, PLDMV, and PapMV.Similar findings were reported in other studies [23,32,34,48], which owe to some factor affecting a multiplex RT-PCR efficiency, such as high oligonucleotide and primer concentrations.
Validation of this approach in 341 field samples collected from 18 counties covering Hainan Island identified infection by PRSV, PLDMV, and PapMV in 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), respectively.Furthermore, 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island.These results have shown that PRSV has a high prevalence and is a major papaya pathogen on Hainan Island, China.Additionally, PLDMV was detected in 18 counties, representing nearly the entire island, but it was reported for the first time in Dongfang in 2012 [14], which suggested that PLDMV might be nearing outbreak.Furthermore, the mixed infection with PRSV and PLDMV has been detected by this multiplex RT-PCR.In contrast, PapMV infection remains infrequent.Thus, viral diseases in papaya become more and more complex and papaya production likely faces important new challenges concerning the transmission of PRSV and PLDMV in China.
In conclusion, this is the first report of multiplex RT-PCR for the diagnosis of PRSV, PLDMV, and PapMV.A reliable, specific, sensitive, and cost-effective multiplex RT-PCR technique was developed for detection and differentiation of these three viral infections in samples of papaya.This technique is useful for routine molecular diagnosis and epidemiological studies of papaya.The results of field application of this multiplex RT-PCR method revealed that the major limiting factor for papaya production in Hainan Island comes from separate or combined infections of PRSV and PLDMV.

Table 1 .
List of primers used in the multiplex detection for PRSV, PLDMV, and PapMV.

Table 2 .
Detection of Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) in papaya plants from different geographical regions of Hainan Island.