Optimizing the nested PCR method for Decapod iridescent virus 1 (DIV1) targeting ATPase gene by reselecting the inner primers

DIV1 has the characteristics of fast transmission and a broad host range. Its infection leads to a high mortality rate, posing a serious threat to the global crustacean aquaculture industry. In order to increase the accuracy of DIV1 detection and reduce the difficulty of result interpretation, this study modified the original nested PCR method targeting the DIV1 ATPase gene. The internal primers for the nested PCR were redesigned to produce a 338 bp amplification product in the second step PCR, effectively distinguishing the target band from primer dimers. The newly established nested PCR method exhibits strong specificity and high sensitivity, with a detection limit as low as 1.37 × 10¹ copies/reaction. The developed nested PCR assay provides new technical support for the accurate detection of DIV1 in global crustacean aquaculture.