Zhang Lu, Liu Kexin, Liu Mengran, Hu Jingjie, Bao Zhenmin, Wang Mengqiang
MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao 266003, China; Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institution, Ocean University of China, Sanya 572024, China.
MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Qingdao 266003, China; Key Laboratory of Tropical Aquatic Germplasm of Hainan Province, Sanya Oceanographic Institution, Ocean University of China, Sanya 572024, China; Hainan Yazhou Bay Seed Laboratory, Sanya 572024, China.
J Invertebr Pathol. 2023 Nov;201:108024. doi: 10.1016/j.jip.2023.108024. Epub 2023 Nov 20.
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is classified as a reportable crustacean disease by the World Organisation for Animal Health (WOAH), which causes poor growth in Penaeus vannamei. According to genome sequence alignment analysis, enzymatic recombinase amplification (ERA) primers and probe were designed based on the ORF1 region of IHHNV, and a real-time ERA assay for IHHNV detection (IHHNV-ERA) was established. The experimental results show that IHHNV-F2/IHHNV-R2 and IHHNV-Probe can effectively amplify the target gene, and the sensitivity is 1.4 × 10 copies/μL within 14.97 ± 0.19 min, while the qPCR using primers 309F/309R could reach the detection limit of 1.4 × 10 copies/μL within 21.76 ± 0.63 min, and the sensitivity results of one-step PCR could be as low as 1.4 copies/μL with expense of time and false positives. The IHHNV-ERA system can effectively amplify the target gene at 42 ℃ within 20 min, and has no cross-reaction with white spot syndrome virus (WSSV), Ecytonucleospora hepatopenaei (EHP), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), and healthy shrimp genomic DNA. Test results of practical samples showed that the detection rate of IHHNV-ERA (18/20) was better than the industrial standard qPCR assay (17/20). Compared with the existing technology, the useful results of this detection assay are: (1) get rid of the dependence on the thermal cycle instrument in the PCR process; (2) the experimental procedure is simple, time-consuming and fast; (3) the detection sensitivity is high. This study provides an ERA based detection assay for IHHNV, which can be used not only for the rapid detection of IHHNV infection, but also for the field screening of pathogens. This assay can also be applied to clinical inspection, customs detection, enterprise quality inspection and other fields, and has obvious practical application value.
传染性皮下和造血组织坏死病毒（IHHNV）被世界动物卫生组织（WOAH）列为应报告的甲壳类疾病，该病毒会导致凡纳滨对虾生长不良。通过基因组序列比对分析，基于IHHNV的ORF1区域设计了酶促重组酶扩增（ERA）引物和探针，并建立了用于检测IHHNV的实时ERA检测方法（IHHNV-ERA）。实验结果表明，IHHNV-F2/IHHNV-R2和IHHNV-探针能够有效扩增靶基因，在14.97±0.19分钟内灵敏度可达1.4×10拷贝/μL，而使用引物309F/309R的qPCR在21.76±0.63分钟内可达到1.4×10拷贝/μL的检测限，一步法PCR的灵敏度结果低至1.4拷贝/μL，但耗时且存在假阳性。IHHNV-ERA系统能够在42℃下20分钟内有效扩增靶基因，且与白斑综合征病毒（WSSV）、肝肠胞虫（EHP）、引起急性肝胰腺坏死病的副溶血性弧菌（VpAHPND）以及健康对虾基因组DNA无交叉反应。实际样品检测结果显示，IHHNV-ERA的检出率（18/20）优于行业标准qPCR检测方法（17/20）。与现有技术相比，该检测方法的有益结果为：（1）摆脱了PCR过程中对热循环仪的依赖；（2）实验操作简单、耗时短且快速；（3）检测灵敏度高。本研究提供了一种基于ERA的IHHNV检测方法，不仅可用于快速检测IHHNV感染，还可用于病原体的现场筛查。该方法还可应用于临床检验、海关检测、企业质检等领域，具有明显的实际应用价值。
