Recently, unanticipated Moko disease-related strains from Brazil were reported that clustered into the previously described Solanaceae-related sequevars IIA-41 and IIB-25, which are not related to historic Moko lineages, and into a newly proposed sequevar, IIA- 53. This finding highlights the fact that the Moko ecotype benefits from a broad genetic basis and harbors far more genetic diversity than anticipated. Based on the whole-genome sequences of Moko strains, we developed a robust, simple, and affordable duplex PCR assay that is specific for phylotype II Rssc strains that can be retrieved from banana and plantain tissues. Here, we present an extensive characterization of the performance of the duplex PCR assay. The diagnostic method presented in this study for detecting Moko disease-causing strains and variant IIB-4NPB strains was developed to fit with the requirements of officially accredited diagnostic laboratories. The proposed protocol was fully evaluated, as required by the ISO 17025 standard, by following EPPO protocols, thus ensuring the highest confidence in the method development and validation. The focus of this study was on the development of a method for the detection of the Moko lineage, which is capable of causing wilt in Musaceae plants. This duplex PCR assay was able to detect the historical diversity of Moko strains and also the newly discovered Moko-related Brazilian strains that cluster into sequevars IIB-25, IIA-41, and IIA-53. The epidemiologically variant IIB-4NPB strains that cause latent infection within the vascular system of plantains were also detected in this duplex PCR assay Fenofibrate framework. The protocol developed in this work appears suitable for Flecainide acetate research and diagnostic laboratories and showed reliable accuracy, detectability, and repeatability. Currently, no specific diagnostic protocol related to Rssc strains that are pathogenic to Musaceae plants has been defined by the European Commission. In addition, it is known that some strains within phylotype II that cause banana and Musaceae wilt are also able to wilt Solanaceae and may represent a threat to both plant families; phylotype II Musaceae-adapted strains might be carried by Solanaceae plants. Therefore, a general assay to detect Rssc strains and a specific Brown rot detection protocol for Solanaceae should be used along with a specific Rs banana wilt detection protocol to detect both types of strains. The performance assessment of the duplex PCR assay was fully compliant with its use as a reference method for diagnostic laboratories. Moko and the epidemiologically variant IIB- 4NPB strains were both specifically detected with confidence.