科研动态

为了明确PCR产物直接测序与克隆后测序结果的差异，验证PCR产物直接测序的“准确性”，将从两个规模化猪场采集的组织样品（A、B）PRRSV ORF5 PCR阳性扩增产物胶回收后连接至pMD19-T载体，分别取15个阳性亚克隆单菌落，同原始PCR产物进行测序，对两个样品各获得的16条序列进行比对分析。结果显示：A、B两组样品16条序列间的核苷酸同源性分别为84.2%~100%、87.4%~100%，PCR产物与其15个亚克隆测序获得序列的核苷酸同源性分别为87.7%~97.3%、87.9%~100%。遗传演化分析方面：样品A序列与其亚克隆序列被划分在谱系1、5、8，占比分别为6.25%、87.50%、6.25%，与其12个亚克隆序列同属于谱系5；样品B与其亚克隆序列被划分在谱系5、8，占比分别为68.75%、31.25%，与其10个亚克隆序列同属于谱系5。结果表明，同一样品中PRRSV ORF5序列具有多样性，不同样品的PRRSV ORF5序列丰富度不同。结果提示，在生产实际中，通过PCR对PRRSV ORF5产物进行直接测序，对于了解场内流行毒株具有一定的指示意义。

Comparison of Sequencing Results between PRRSV ORF5 Amplification Products and Its Cloning

In order to clarify the difference between direct sequencing and post cloning sequencing of PCR products，and verify the“accuracy”of the former，PRRSV ORF5 PCR positive amplified product glue of the tissue samples（A，B）collected from two intensive swine farms were recycled and then connected to pMD19-T vector，15 positive subclone single colonies were respectively taken and sequenced with the original PCR products，then 16 sequences obtained from each sample were compared and analyzed. The results showed that the nucleotide homology between the 16 sequences in the two groups were from 84.2% to 100% and from 87.4% to 100%，respectively，and that of PCR products and their 15 subclone sequences were from 87.7% to 97.3% and from 87.9% to 100%，respectively. For genetic evolution analysis，the sequence of sample A and its subclone sequences were divided into lineages 1，5 and 8，accounting for 6.25%，87.50% and 6.25%，respectively，sample A together with its 12 subclone sequences belonged to lineages 5；sample B and its subclone sequences were divided into lineages 5 and 8，accounting for 68.75% and 31.25%，respectively，sample B together with its 10 subclone sequences belonged to lineages 5. In conclusion，PRRSV ORF5 sequence were diverse in the same sample but with different abundance in different samples. Therefore，direct sequencing of PRRSV ORF5 products by PCR contributed to identifying the prevalent strains within a farm.

原文链接：https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&dbname=CJFDAUTO&filename=ZGDW202210018&uniplatform=NZKPT&v=vPzqR5W2eDAEVlhxy0NZDKQP-gdbE3_LRVcH1XBBAG-1w0LB30uKKsNOJK7fdfEC