Development of multiplex polymerase chain reaction (M-PCR) for simultaneous detection of White spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP) and Vibrio parahaemolyticus AHPND from cultured shrimp, Litopenaeus vannamei

Abstract

Abstract White spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), and Acute hepatopancreatic necrosis disease (AHPND) are the major threats to the whiteleg shrimp, Litopenaeus vannamei. This study was conducted to develop multiplex polymerase chain reaction (M-PCR) for the detection of shrimp pathogens. This study was conducted between January 2020 to July 2022. Total of 328 samples were collected from shrimp farms of Karnataka and they were screened for WSSV, EHP, and AHPND by M-PCR. Several parameters were optimized for the standardization of M-PCR. The specificity and sensitivity of the reaction were evaluated. The results showed that this technique can detect WSSV, AHPND, and EHP in a single reaction with high specificity. Sensitivity result showed it can detect WSSV (0.3 pg/µl), AHPND (0.1 pg/µl), and EHP (0.2 pg/µl). Out of 328 samples, 72 (21.9%) samples were found to be positive for EHP, 16 (4.8%) samples were found to have been infected by WSSV and 5 (1.5%) samples were found to have multiple infections with WSSV, and EHP. In consideration of the specificity and sensitivity of this technique, we conclude that M-PCR could be used instead of a conventional PCR assay targeting individual genes for the rapid detection of WSSV, AHPND, and EHP simultaneously.