A multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees

A multiplex polymerase chain reaction (mPCR) assay was developed to detect six RNA and one DNA citrus virus: Citrus leaf rugose virus (CLRV), Citrus psorosis virus (CPsV), Citrus tatter leaf virus (CTLV), Citrus tristeza virus (CTV), Citrus variegation virus (CVV), Citrus yellow mosaic virus (CYMV), and Indian citrus ringspot virus (ICRSV) from citrus plants. These seven viruses are classified in six different virus genera. Degenerate primers were designed based on the respective virus isolate sequence data available from the GenBank and were used for reliable detection of the different viruses by simplex- and mPCR. The sensitive and simultaneous detection of RNA and DNA viruses using the mPCR decreases the risk of contamination, saves time and reduces the cost as compared to other conventional methods for citrus virus detection. Seven different fragments (245-942 bp) specific to the viruses were simultaneously amplified using mPCR and were identified on the basis of their molecular sizes. The consistent results of the mPCR were compared with simplex PCR for detection of each virus pathogen. The mPCR results were confirmed with sequencing analysis. The mPCR provides a useful rapid method for detecting multiple viruses in citrus plants that will aid in the production of virus-free citrus plants for certification programs.