Development and application of an immunocapture real-time PCR for the detection of Spiroplasma citri, the causal agent of citrus stubborn disease

Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), causes significant losses in citrus crops. An efficient pathogen detection system is critical for epidemiology studies, particularly when a large sample size is involved. In this study, we report the development of an immunomolecular assay, immunocapture real-time polymerase chain reaction (IC-qPCR), targeting the spiralin gene for direct detection of S. citri without DNA isolation. This method can use either plant sample extracts or media in which S. citri was cultivated. The IC-qPCR protocol demonstrated a limit of detection for pure S. citri culture at a Ct value of 36.523 with a 10³-fold dilution factor, making it equally sensitive as qPCR, which exhibited signal disappearance at a 10^–3 dilution (Ct value of 37.484). In contrast, the immunological double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test produced positive results up to a 10^–2 dilution only. For S. citri-infected citrus samples, the established IC-qPCR protocol had a limit of detection at 36.46 Ct with a 1/64-fold dilution factor, matching the sensitivity of qPCR, where signal disappearance occurred at a 1/64 dilution (Ct value of 37.21). On the other hand, the immunological DAS-ELISA test yielded positive results only up to a 1/16 dilution, with optical density (OD) values of 0.364 and 0.113 for 1/16 and 1/32 dilutions, respectively. The IC-qPCR assay shows no cross-reaction for any other highly related spiroplasma species and bacteria affecting citrus trees including Candidatus liberibacter, Xylella fastidiosa, and Xanthomonas campestris pv. citri. Therefore, IC-qPCR assay provides an alternative quick and very sensitive method to screening S. citri, with the advantage of not requiring any concentration or DNA purification steps while still allowing an accurate diagnosis of CSD.