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Identification of intron in ORF003 gene and its application for inactivation test of ISKNV.
Fu, Xiaozhe; Zhang, Lixi; Liu, Lihui; Lin, Qiang; Liang, Hongru; Niu, Yinjie; Huang, Zhibin; Li, Ningqiu.
Afiliação
  • Fu X; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
  • Zhang L; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
  • Liu L; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
  • Lin Q; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
  • Liang H; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
  • Niu Y; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
  • Huang Z; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China.
  • Li N; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Guangzhou, 510380, China. Electronic address: liningq@126.com.
Microb Pathog ; 138: 103822, 2020 Jan.
Article em En | MEDLINE | ID: mdl-31669501
The virus inactivation test is a critical skill in inactivated vaccine production. Active viruses produced viral mRNA in susceptible cells or the host can be used to infer whether a DNA virus is replicating by RT-PCR. But it is generally difficult to avoid genomic DNA contamination in the samples. However, the use of primers spanning an intron is an effective alternative for virus inactivation test. Therein, a nested RT-PCR was developed to detect active ISKNV in the inactivated vaccine. At first, the transcriptome analysis of CPB cell infected with ISKNV revealed several gaps in some viral transcripts compared to ISKNV genome. One intron in ORF003L with 80 bp (designated IN-3) was confirmed by PCR and sequencing analysis. Then, two primer sets (primer A and primer B) spanning the IN-3 intron were designed to detect ISKNV transcription. The nested RT-PCR conditions were optimized with 0.4 µM primer A and 0.2 µM primer B, and 68 °C and 55 °C for annealing temperature, respectively. The sensitivity results indicated that the nested RT-PCR could detect one copy of live ISKNV propagating in CPB cells for seven days. The nested RT-PCR method was more sensitive and accurate than the method of blind passages in cells and fish challenge experiments. Together, above results indicate that this assay is a time-saving, labor-extensive and cost-effective for inactivation test of ISKNV in killed vaccine production.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Íntrons / Fases de Leitura Aberta / Doenças dos Peixes / Iridoviridae Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Animals Idioma: En Revista: Microb Pathog Assunto da revista: DOENCAS TRANSMISSIVEIS / MICROBIOLOGIA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: China

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Íntrons / Fases de Leitura Aberta / Doenças dos Peixes / Iridoviridae Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Animals Idioma: En Revista: Microb Pathog Assunto da revista: DOENCAS TRANSMISSIVEIS / MICROBIOLOGIA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: China