Development of a Degenerate Primer RT-PCR Assay for Detection of Carmovirus

The objective of this study is to establish a rapid, sensitive, and broad-spectrum screening method for simultaneous detection and identification of the genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus using degenerate primer RT-PCR combined with sequence analysis.

Multiplexed analysis of genome sequences was aligned to search for suitable conserved regions for the design of the degenerate primers. One pair of degenerate primer Carmo-F2/Carmo-R2 was designed based on the RNA-dependent RNA polymerase (RdRp) gene sequences, and another pair primer Carmo-F2a/Carmo-R2a was formed by adding the non-complementary AT-rich sequences (AATAAATCATAA) to the 5′ end of the degenerate primers. The broad-spectrum, specificity, and sensitivity of RT-PCR method were analyzed. The sequencing, BLASTn analysis and phylogenetic analysis of PCR products were performed. The method was used to screen and detect viruses on Chinese hibiscus (Hibiscus rosa-sinensis) samples from Xiamen, China.

Degenerate primers Carmo-F2/Carmo-R2 and Carmo-F2a/Carmo-R2a were used to amplify partial RdRp gene of the members of genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus by RT-PCR. The fragment of approximately 500 and 550 bp was amplified, respectively. The developed RT-PCR assay was successfully used to detect angelonia flower break virus (AnFBV; Alphacarmovirus), calibrachoa mottle virus (CbMV; Alphacarmovirus), carnation mottle virus (CarMV; Alphacarmovirus), and pelargonium flower break virus (PFBV; Alphacarmovirus), hibiscus chlorotic ringspot virus (HCRSV; Betacarmovirus), melon necrotic spot virus (MNSV; Gammacarmovirus). The specificity test showed that no specific band could be obtained from maize chlorotic mottle virus (MCMV; Machlomovirus), pelargonium line pattern virus (PLPV; Pelarspovirus), carnation ringspot virus (CRSV; Dianthovirus), and healthy plants which including watermelon, melon, pumpkin, soybean, and pea. The sensitivity results showed that the primers Carmo-F2/Carmo-R2 could be detected up to 10^-2 dilution and the primers Carmo-F2a/Carmo-R2a could be detected up to 10^-3 dilution, which indicated that the non-complementary AT-rich sequences added at the 5′ end of the degenerate primers could increase the sensitivity of degenerate primer RT-PCR. BLAST analysis showed that the sequences determined had the highest sequence consistency with the corresponding virus species. Phylogenetic analysis based on partial amino acid sequences of the RdRp gene showed that it is consistent with the current classification of the subfamily Procedovirinae, and the viruses could be identified at the species level. HCRSV was detected in all 13 H. rosa-sinensis samples with suspected virus symptoms.

The RT-PCR method based on the degenerate primers Carmo-F2/Carmo-R2 and Carmo-F2a/Carmo-R2a can be used for the screening and detection of viruses of the genera Alphacarmovirus, Betacarmovirus, and Gammacarmovirus, and can be used for rapid identification of virus species in combination with sequence analysis and phylogenetic analysis, and may be used to discover the new virus. H. rosa-sinensis plants were infested with HCRSV in Xiamen, China.