Identification of intron in ORF003 gene and its application for inactivation test of ISKNV

文献类型： 外文期刊

第一作者： Fu, Xiaozhe

作者： Fu, Xiaozhe;Zhang, Lixi;Liu, Lihui;Lin, Qiang;Liang, Hongru;Niu, Yinjie;Huang, Zhibin;Li, Ningqiu

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关键词： Infectious spleen and kidney necrosis virus; Intron; Inactivation test; Nested RT-PCR

期刊名称：MICROBIAL PATHOGENESIS （ 影响因子：3.738； 五年影响因子：3.663 ）

ISSN： 0882-4010

年卷期： 2020 年 138 卷

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收录情况： SCI

摘要： 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 mu M primer A and 0.2 mu M primer B, and 68 degrees C and 55 degrees 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 timesaving, labor-extensive and cost-effective for inactivation test of ISKNV in killed vaccine production.

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