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首页> 外文期刊>Microbial Pathogenesis >Identification of intron in ORF003 gene and its application for inactivation test of ISKNV
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Identification of intron in ORF003 gene and its application for inactivation test of ISKNV

机译：ISW003基因内含子的鉴定及其在ISKNV灭活试验中的应用

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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.
机译：病毒失活试验是灭活疫苗生产的关键技能。活性病毒在易感细胞中产生病毒mRNA，或者宿主可用于推断DNA病毒是否通过RT-PCR复制。但通常难以避免样品中的基因组DNA污染。然而，跨越内含子的引物的使用是病毒失活试验的有效替代方案。其中，开发了一种嵌套的RT-PCR以在灭活的疫苗中检测活性ISKNV。首先，与IsknV基因组相比，用IsknV感染的CPB细胞的转录组分析显示出一些病毒转录物中的几个间隙。通过PCR和测序分析证实了具有80bp（指定为-3中的orf003l的一个内含子。然后，设计了跨越3个内含子的两个引物组（引物A和引物B）以检测IsknV转录。嵌套的RT-PCR条件用0.4μm引物A和0.2μm引物B和68℃和55摄氏度进行优化，以进行退火温度。敏感性结果表明嵌套RT-PCR可以检测在CPB小区中传播的一份活性ISKNV七天。嵌套的RT-PCR方法比细胞和鱼攻击实验中的盲通道的方法更敏感和准确。在一起，上面的结果表明，该测定是一种令人惊叹，劳动 - 广泛且具有成本效力，可用于杀死疫苗生产中Isknv的失活试验。

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