A Novel Multi-Gene Combined RT-PCR Assay for Rapid and Sensitive Detection of Maize Dwarf Mosaic Virus

Maize, a staple food and cash crop worldwide, also serves as a critical industrial raw material. However, it is significantly threatened by viral pathogens, particularly maize dwarf mosaic virus (MDMV), the primary cause of maize dwarf mosaic disease, a debilitating condition affecting maize cultivation. This study aims to establish a multi-gene combined RT-PCR assay for the rapid specific, sensitive, and reliable detection of MDMV without the need for special expensive equipment. Samples of imported maize, sorghum, and barley were collected from ports in Fujian and Shanghai. Primers targeting the coat protein (CP) and cytoplasmic inclusion protein (CI) genes of MDMV were designed and optimized. Through the design and screening of primers, as well as the optimization of reaction conditions and primer concentrations, a multi-gene combined RT-PCR assay was established to simultaneously detect both genes. Additionally, a real-time fluorescent-based RT-PCR (RT-qPCR) assay was developed using the CP gene to confirm the accuracy of multi-gene combined RT-PCR assay. The sensitivity of the optimized multi-gene combined RT-PCR assay enables the detection of MDMV in infected maize leaf crude extracts at dilutions of 5.37 pg/μL. This assay exhibited excellent specificity, high sensitivity, and robust repeatability, providing swift and accurate detection of MDMV. The multi-gene combined RT-PCR assay offers precise and efficient technical support for MDMV detection and contributes to improved maize production practices.