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To establish a multiplex PCR method for simultaneous and rapid detection of Spiroplasma eriocheiris and white spot syndrome virus (WSSV) in Procambarus clarkii with recommendations for application to other crustacea.Three primer sets were mixed at a ratio of 1: 3: 1 to amplify specific fragments of the S. eriocheiris, WSSV, P. clarkii crayfish (control organism) genomes, respectively. S. eriocheiris and WSSV were used to challenge the susceptible crustacea in the experimental groups. Total DNA of the samples was purified and detected by multiplex PCR. The PCR-amplified products produced four groups of results as follows. One fragment of 1195 bp, amplified by the primer set ITS-crayfish/28S-crayfish, served as an internal control, showed no pathogen detection, thus confirming the specificity of our positive tests. Two groups represented by: (i) samples challenged by S. eriocheiris alone, or (ii) challenged by WSSV alone, yielded two fragments each; i.e. those from S. eriocheiris (271 bp) plus the internal control and those from WSSV (530 bp) plus the internal control. Finally, for the fourth group, in cases of double challenged treatments, all three amplified products were detected simultaneously.Simultaneous and rapid detection of two pathogens in P. clarkii is important to maintain productive and healthy crayfish in aquaculture. The direct detection of S. eriocheiris and WSSV from P. clarkii is practicable with multiplex PCR.This study shows that the two pathogens are simultaneously and rapidly detected in P. clarkii by multiplex PCR, thus increasing the efficiency of pathogen detection.