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

Jing-Yi Xing; An-Qi Li; Xiao-Meng Guo; Meng Wang; Xin Guan; Liang Qiu; Qing-Li Zhang; Jie Huang

doi:10.1016/j.jip.2024.108212

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

J Invertebr Pathol. 2024 Nov:207:108212. doi: 10.1016/j.jip.2024.108212. Epub 2024 Sep 27.

Authors

Jing-Yi Xing¹, An-Qi Li², Xiao-Meng Guo³, Meng Wang⁴, Xin Guan⁴, Liang Qiu⁵, Qing-Li Zhang⁶, Jie Huang⁷

Affiliations

¹ State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Laboratory for Marine Fisheries Science and Food Production Processes, Laoshan Laboratory, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; College of Aquaculture, Tianjin Agricultural University, Tianjin 300384, China.
² College of Aquaculture, Tianjin Agricultural University, Tianjin 300384, China.
³ State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Laboratory for Marine Fisheries Science and Food Production Processes, Laoshan Laboratory, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
⁴ State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Laboratory for Marine Fisheries Science and Food Production Processes, Laoshan Laboratory, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; School of Agriculture, Ludong University, Yantai 264025, China.
⁵ State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Laboratory for Marine Fisheries Science and Food Production Processes, Laoshan Laboratory, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao, Shandong 266237, China; School of Agriculture, Ludong University, Yantai 264025, China. Electronic address: [email protected].
⁶ State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Laboratory for Marine Fisheries Science and Food Production Processes, Laoshan Laboratory, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao, Shandong 266237, China; College of Aquaculture, Tianjin Agricultural University, Tianjin 300384, China; College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China; School of Agriculture, Ludong University, Yantai 264025, China.
⁷ State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Laboratory for Marine Fisheries Science and Food Production Processes, Laoshan Laboratory, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao, Shandong 266237, China; College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.

PMID: 39343128
DOI: 10.1016/j.jip.2024.108212

Abstract

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.

Keywords: ATPase; Cherax quadricarinatus iridovirus; Decapod iridescent virus 1; Nested PCR; Shrimp hemocyte iridescent virus.

MeSH terms

Adenosine Triphosphatases / genetics
Animals
Aquaculture
DNA Primers / genetics
Decapoda / virology
Iridoviridae / genetics
Polymerase Chain Reaction* / methods

Substances

Adenosine Triphosphatases
DNA Primers