為了明確PCR產(chǎn)物直接測(cè)序與克隆后測(cè)序結(jié)果的差異，驗(yàn)證PCR產(chǎn)物直接測(cè)序的“準(zhǔn)確性”，將從兩個(gè)規(guī)?；i場(chǎng)采集的組織樣品（A、B）PRRSV ORF5 PCR陽性擴(kuò)增產(chǎn)物膠回收后連接至pMD19-T載體，分別取15個(gè)陽性亞克隆單菌落，同原始PCR產(chǎn)物進(jìn)行測(cè)序，對(duì)兩個(gè)樣品各獲得的16條序列進(jìn)行比對(duì)分析。結(jié)果顯示：A、B兩組樣品16條序列間的核苷酸同源性分別為84.2%~100%、87.4%~100%，PCR產(chǎn)物與其15個(gè)亞克隆測(cè)序獲得序列的核苷酸同源性分別為87.7%~97.3%、87.9%~100%。遺傳演化分析方面：樣品A序列與其亞克隆序列被劃分在譜系1、5、8，占比分別為6.25%、87.50%、6.25%，與其12個(gè)亞克隆序列同屬于譜系5；樣品B與其亞克隆序列被劃分在譜系5、8，占比分別為68.75%、31.25%，與其10個(gè)亞克隆序列同屬于譜系5。結(jié)果表明，同一樣品中PRRSV ORF5序列具有多樣性，不同樣品的PRRSV ORF5序列豐富度不同。結(jié)果提示，在生產(chǎn)實(shí)際中，通過PCR對(duì)PRRSV ORF5產(chǎn)物進(jìn)行直接測(cè)序，對(duì)于了解場(chǎng)內(nèi)流行毒株具有一定的指示意義。
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.
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