Development and validation of a loop-mediated isothermal amplification technique (LAMP) for the detection of Spiroplasma citri, the causal agent of citrus stubborn disease

Abstract

Citrus stubborn disease (CSD) is caused by a Gram-positive bacterium, Spiroplasma citri, and is an endemic, but manageable, citrus disease. CSD-affected trees are low yielding but a key problem is that its symptoms are similar to and easily mistaken for Huanglongbing (HLB), a devastating citrus disease controlled by quarantine and eradication. Therefore, a rapid and simple test for S. citri is needed to readily distinguish CSD from HLB. To this end, a Loop-Mediated Isothermal Amplification technique (LAMP) was developed to detect S. citri, targeting the spiralin gene. The protocol was optimized for crude plant extracts from infected trees to allow on-site field testing. The LAMP assay showed high specificity to S. citri and detected DNA to a level of 100 fg/μl with no inhibition by crude plant extracts. Although the LAMP assay was 9 times less sensitive than qPCR with purified DNA templates, it performed well in field validations using a portable BioRanger device with citrus crude extracts. The LAMP assay showed detection efficiency and percentage yes/no calls similar to those obtained by real time PCR conducted with DNA extracted and purified from the same sample. The LAMP procedure allows growers, pest control or diagnostic services to rapidly test for S. citri in the field without a laboratory or DNA purification.