Simultaneous detection and differentiation of four viruses in passion fruit plants by a multiplex RT-PCR

A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect passion fruit, citrus-associated rhabdovirus (CiaRV), East Asian Passiflora virus (EAPV), Passiflora latent virus (PLV), and Telosma mosaic virus (TeMV). The optimized parameters included the primer concentration, annealing temperature, extension time, and number of cycles. The established multiplex RT-PCR assay produced the corresponding products with sizes of 597 bp, 529 bp, 320 bp, and 235 bp, which were specific for CiaRV, EAPV, PLV, and TeMV, respectively. The fragments could be distinguished clearly by agarose gel electrophoresis. The detection limit of the assay was 100 pg of total RNA for CiaRV and EAPV, 10 pg of total RNA for PLV, and 1.0 ng of total RNA for TeMV. The multiplex RT-PCR assay was also tested using field samples, and the results showed that the developed assay could detect the viruses in single or multiple infections of passion fruit. The multiplex RT-PCR established here will be quite helpful for the diagnosis of passion fruit infected with various combinations of the four viruses in the field.


Introduction
Passion fruit (Passiflora edulis Sims), originated in South America, has been widely cultivated in tropical and subtropical regions worldwide because of its high pharmacological and edible value (Thokchom and Mandal 2017;Kawakami et al. 2021). In China, passion fruit orchards are located mainly in the southern part of the country, in Guangxi, Fujiang, and Jinagxi provinces. However, the cultivation and production of passion fruit are severely affected by various pathogens, such as viruses, bacteria, and fungi. Among them, viral diseases are extremely serious on passion fruit and a yield-limiting factor for the crop. It has been documented that passion fruit is susceptible to infection by more than 30 different viruses (Baker et al. 2011). These include potyviruses, such as East Asian Passiflora virus (EAPV) (Iwai et al. 2006), Telosma mosaic virus (TeMV) (Chiemsombat et al. 2014), and passion fruit woodiness virus (PWV) (Sokhandan et al. 1997); carlaviruses, such as Passiflora latent virus (PLV) (Spiegel et al. 2007); begomoviruses, as passion fruit severe leaf distortion virus (Ferreira et al. 2010), Euphorbia leaf curl virus (ELCV), papaya leaf curl Guangdong virus (PaL-CuGDV) (Cheng et al. 2014), and papaya leaf curl China virus (PaLCuCNV) (Huang et al. 2019); and tentative cytorhabdoviruses, such as citrus-associated rhabdovirus (CiaRV) (Zhang et al. 2020). Many of the known passion fruit viruses have been detected in China, such as TeMV, EAPV, CiaRV, and PLV. TeMV and EAPV are both considered causal agents of passion fruit woodiness disease, which is characterized by foliar rugosity, mosaic and distorted woody, and severely malformed fruits (Iwai et al. 2006;Chiemsombat et al. 2014), and have been detected in many passion fruit production areas in China (Xie et al. 2017(Xie et al. , 2018Chen et al. 2018). TeMV is especially pervasive in passion fruit plants in that country . CiaRV is a tentative cytorhabdovirus that can infect passion fruit, but the symptoms induced in the host are still not clear. Based on our previous investigation, the Aijun  detection rate of CiaRV is also high, and CiaRV always coinfects with TeMV. PLV causes foliar and fruit mosaic symptoms on Passiflora spp. and has been reported to occur naturally in Germany, Australia, the USA, New Zealand, and Korea (Spiegel et al. 2007;Pares et al. 2007;Tang et al. 2008;Cho et al. 2021). We also detected PLV in local field passion fruit plants. Based on our previous survey, these four viruses were most commonly found in mixed infections on the local field passion fruit plants, especially the TeMV, EAPV, and CiaRV. A highly efficient and reliable detection method would be extremely valuable for these viral diseases' management. Currently, several diagnostic methods have been developed for the detection of TeMV and PLV, such as reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription loop mediated isothermal amplification (RT-LAMP), and serological method (Spiegel et al. 2007;Chiemsombat et al. 2014;Fu et al. 2021). RT-PCR is the only available method for EAPV and CiaRV. However, these methods can only detect one virus in a single reaction. Multiplex RT-PCR, a variant of PCR, is a quick, reliable, and cost-effective method that has been used to successfully detect a variety of viruses simultaneously in a single assay. To date, multiplex RT-PCR has been used widely to diagnose infection in citrus, apples, cherry, peach, and papaya plants by related viruses (Osman et al. 2015;Hao et al. 2016;Noorani et al. 2013;Yu et al. 2013;Tuo et al. 2014).
However, the simultaneous detection of CiaRV, EAPV, PLV, and TeMV from infected passion fruit has not been reported. The main goal of this study was therefore to develop a sensitive and specific multiplex RT-PCR assay for the detection of four economically important viruses. The optimized multiplex RT-PCR assay was evaluated by detecting these viruses in field samples, and proven to be reliable and sensitive.

Plants and viruses
A single passion fruit plant ( Fig. 1) confirmed to be coinfected with all four viruses was used as the virus source to develop the multiplex RT-PCR assay. Total RNA was extracted from the leaves of passion fruit plants using RNAeasy isolation reagent (Vazyme Bio Inc., Nanjing, China) according to the manufacturer's instructions. The integrity of the RNA was confirmed by the standard electrophoresis technique. Additionally, RNA quality and quantity were assessed using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), after which the RNA was immediately stored at − 80 ℃ for further procedures.

Design of virus-specific primers
The primer PLVReh-RT-F/R was selected from published articles for simplex and multiplex RT-PCR (Jover-Gil et al. 2018;Spiegel et al. 2007). The respective virus-specific primers for CiaRV, EAPV, and TeMV RT-PCR assays were designed using Oligo 7.0 software. They were based on the conserved region of each virus's coat protein (CP) or nucleocapsid protein (N) gene obtained by comparing the available genomic sequences of each virus in the NCBI GenBank (Table S1) to determine the RNA region with the least amount of sequence variability. The primer names, oligonucleotide sequences, expected size of amplicon, and references are given in Table 1. The specificity of the primers was verified using BLASTn from NCBI (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi). All primers were synthesized by GenScript Biotech Corporation (Nanjing, China). The multiplex RT-PCR products of the expected size were cloned into the pMD19-T cloning vector (TaKaRa), sequenced, and subjected to BLASTn search.

Simplex RT-PCR assays
The simplex RT-PCR reaction was conducted using a one-step RT-PCR kit (Vazyme Bio Inc., Nanjing, China) in a 20-μL reaction mixture containing 10 μL of 2 × onestep mix, 0.4 μL of each primer (10 pM), 1 μL of one-step Enzyme mix, 1 μL of template RNA, and 8.2 μL of RNasefree ddH 2 O. The amplification was carried out using a GeneAmp PCR System (Applied Biosystems, CA, USA) with the following parameters: a 30-min incubation step at 50 °C, followed by one cycle at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 20 s, annealing at 56 °C for 20 s, and extension at 72 °C for 50 s, followed by a final extension 1 3 step of 72 °C for 7 min. The amplicons were visualized by 1.5% agarose gel electrophoresis. Each amplified viral target fragment was purified using a gel extraction kit (TaKaRa Bio Inc., Japan), cloned into a pMD-19 T vector (TaKaRa Bio Inc., Japan), and sequenced (Genewiz, China). The obtained nucleotide sequences were verified by BLASTn from NCBI.

Optimization of multiplex RT-PCR
The multiplex RT-PCR was optimized by sequentially testing the primer concentration, annealing temperature, extension time, and number of cycles using the plant RNA prepared from the virus source plant. To optimize the multiplex RT-PCR system, combinations of different concentrations of four primer pairs were tested. The concentrations of the four primers were tested from 0.15 to 0.30 pM. The annealing temperature was increased from 52 to 59 °C in increments of 1 °C. The number of cycles was set as 30, 35, and 40. The extension times tested were 45 s, 60 s, and 75 s. The multiplex RT-PCR amplifications were performed in a 20-μL reaction volume comprising 10 μL of 2 × one step mix, 0.15-0.30 pM EAPV-F/R primers, 0.15 pM of CiaRV-F/R, PLVReh-RT-F/R, or TeMV-F/R primers, 1 μL of one step enzyme mix, 1 μL template RNA, and RNase-free ddH 2 O to make up the volume to 20 μL. Multiplex RT-PCR amplification parameters were as follows: a 30-min incubation step at 50 °C, followed by one cycle at 94 °C for 3 min, (30-40) cycles of denaturation at 94 °C for 20 s, annealing at (52-59) °C for 20 s, and extension at 72 °C for (45-75) s, followed by a final extension step of 72 °C for 7 min. The amplicons were visualized by 1.5% agarose gel electrophoresis.

Detection limits of multiplex RT-PCR
The sensitivity of the optimized multiplex RT-PCR for all the four viruses was compared to those of the simplex RT-PCRs for CiaRV, EAPV, PLV, and TeMV, using tenfold plant total RNA prepared from the virus source plant (100 ng/ μL, 10 ng/μL, 1.0 ng/μL, 100 pg/μL, 10 pg/μL, and 1.0 pg/ μL). Both RT-PCR amplifications were performed under the optimized multiplex RT-PCR assay conditions using the corresponding primers of the target fragments to be assayed.

Multiplex RT-PCR assays of field samples
During 2018-2020, a total of 59 mature passion fruit leaf samples with symptoms such as yellowing, mosaic, or with no visible symptoms were collected from orchards of Xunwu, Ruijin, and Yudu counties in Jiangxi Province, China. The sampled passion fruit plants were all cultivar of purple passion fruit and planted in the same year. Total RNA was extracted from the 59 field samples as described in "Plants and viruses". The RNA from an identified virusnegative field passion fruit sample was used as a negative control, and RNA from the virus source passion fruit plant was used as a positive control. The multiplex assay results of the field samples were verified using simplex RT-PCR assays. All the above experiments were carried out three times independently to ensure reproducibility and eliminate experimental differences.

Specificity and compatibility of primers
In simplex RT-PCR, the expected sizes of viral target genes for CiaRV (597 bp), EAPV (529 bp), PLV (320 bp), and TeMV (235 bp) were amplified specifically for all four primer pairs (Fig. 2, Lane 2-5). For multiplex RT-PCR, the primer combinations also gave clear and specific bands corresponding to target products. The amplified PCR products were sequenced and submitted to GenBank (GenBank accession No. OP328809, OP328810, OP328811, OP328812). These sequences were also subjected to BLASTn search to determine their homology and showed the highest sequence identity to CiaRV, EAPV, PLV, and TeMV isolates (Gen-Bank accession No. MT302542, MT450870, MH379331, MK340754), respectively. The results further confirmed that the correct fragments had been amplified. The primer  TTC ACC CCG ATA ACC AAGG  597  This study  CiaRV-R  GAC ACC CTG ATA GAA TCG AG  EAPV-F  GAA TGG TCA CAA CCC CAA C  529  This study  EAPV-R  TAT CTA GGC ATA TAC GGC

Optimization and establishment of multiplex RT-PCR
Different amplification efficiencies indicated by different band intensities were obtained when equal concentrations (0.15 pM) of the four primer pairs were used in multiplex RT-PCR (Fig. 3A, Lane 1). To optimize the multiplex PCR system, combinations of different concentrations of the four primer pairs were tested in multiplex RT-PCR. The results showed that the amplification efficiencies for EAPV varied with different combinations of primer concentrations and that those for CiaRV, PLV, and TeMV remained stable. Balanced amplification was achieved when the primer concentrations were 0.3 pM for EAPV and 0.15 pM for CiaRV, PLV, and TeMV (Fig. 3A, Lane 4). Gradient RT-PCR was performed to determine the optimal annealing temperature. When the annealing temperature was set as 56 °C, it obtained the best amplification result. Therefore, 56 °C was used as the optimal annealing temperature for the multiplex RT-PCR (Fig. 3B). In testing various combinations of extension time and number of cycles, the results were obtained when the extension time was 60 s and the cycle number was 35 (Fig. 3C).

Sensitivities of the simplex and multiplex RT-PCR assays
The results showed that the detection limits of the respective simplex RT-PCR for CiaRV were equivalent to 100 pg of total RNA, those for EAPV and PLV were 10 pg of total RNA, and those for TeMV were 1.0 ng of total RNA (Fig. 4A-D). On the other hand, in the multiplex RT-PCR assay, the detection limit was 100 pg of total RNA for CiaRV and EAPV, 10 pg of total RNA for PLV, and 1.0 ng of total RNA for TeMV (Fig. 4E). These results indicated that multiplex RT-PCR was only tenfold less sensitive than simplex RT-PCR for the detection of EAPV.

Discussion
Passion fruit is an economically important fruit crop, and the planting area of passion fruit in southern China has gradually increased in recent years. Viral diseases are a major constraint for passion fruit production (Baker et al. 2011), and viruses mixed infection often caused more severe symptoms. Thus, the development of reliable, fast, and cost-effective detection method for the viral disease management remains a necessity. This paper describes a multiplex RT-PCR assay for the simultaneous detection of four viruses infecting passion fruit (Baker et al. 2011). The assay described above provides a sensitive tool for the simultaneous detection of CiaRV, EAPV, PLV, and TeMV in single or mixed infections and was successfully tested on 59 field samples. One frequent problem encountered in multiplex RT-PCR is the unbalanced amplification of viruses due to the presence of multiple gene targets in one reaction and to primers conferring compatibility with their targets, which may result in competition for enzymes and nucleotides (Wei et al. 2008). In our study, weak amplification of the EAPV fragment was obtained when using equal concentrations of the four primer sets in the multiplex RT-PCR assay. To balance the amplification efficiency and ensure similar band intensities of the four viruses, eventually, relatively higher primer concentrations (0.30 pM) for EAPV were used for the production of similar intensities of the amplicons in the same reaction.
In addition, the sensitivity of the multiplex RT-PCR compared to that of simplex RT-PCR was approximately tenfold lower for the detection of EAPV. Similar findings have been reported in other studies (Wei et al. 2009;Xue et al. 2021;Yao et al. 2014), which is due to some factors affecting multiplex RT-PCR efficiency, such as high oligonucleotide and primer concentrations.
To validate the reliability of the multiplex RT-PCR assay in field applications, 59 passion fruit field samples were collected from three counties in the Gannan district of China and tested by the multiplex RT-PCR assay. The results of the multiplex RT-PCR assay indicated that the single infection rates of the four viruses were 4/59 (6.8%), 0/59 (0.0%), 0/59 (0.0%), and 26/59 (44.1%), respectively. TeMV had the highest detection rate. Furthermore, 27/59 (45.8%) of the samples were coinfected with 2-4 viruses. In the 29 coinfected samples, TeMV was also detected in each sample. The TeMV total detection rate was 53/59 (89.8%). According to this result, TeMV has a high prevalence and is the major passion fruit virus pathogen of Gannan district. Additionally, 57/59 (96.6%) of the collected samples were infected with at least one virus. These results suggested that virus infection may be very frequent in this passion fruit production area and represent a significant threat to passion fruit production. Passion fruit growers likely face important challenges and must take appropriate precautions to manage the spread of the viral disease.
To our knowledge, this is the first report of the simultaneous detection of CiaRV, EAPV, PLV, and TeMV using a multiplex RT-PCR assay. The optimized multiplex RT-PCR assay developed in this study is a convenient and sensitive method that can be used to detect coinfections of CiaRV, EAPV, PLV, and TeMV in diseased fruit plants. This assay should be more time and cost saving, especially for detecting coinfecting viruses in large-scale surveys, than the previous single-virus detection methods for these four viruses.
Author contribution The author Aijun Huang devised the experiment and drafted the manuscript. Peipei Gu, Ying Wang, Jiali Li, and Zhixun Yang performed the experiments. The corresponding author Long Yi did critical revision of drafted manuscript.
Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations
Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest The authors declare no competing interests.
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