猪札幌病毒荧光定量RT⁃PCR检测方法建立及遗传进化分析  

Development and Application of A SYBR Green I Real⁃time Fluorescent Quantitative RT⁃PCR Method for Detection of Swine Sapporo Virus,Along with Analysis of the Virus Variation and Evolution

作　　者：杜松 舒相华[1] 姚俊 袁红 张雪 宋春莲[1] DU Song;SHU Xianghua;YAO Jun;YUAN Hong;ZHANG Xue;SONG Chunlian(Yunnan Agricultural University,Kunming 650201,China;Yunnan Academy of Animal Husbandry and Veterinary Sciences,Kunming 650224,China)

机构地区：[1]云南农业大学,昆明650201 [2]云南省畜牧兽医科学院,昆明650224

出　　处：《病毒学报》2024年第4期811-822,共12页Chinese Journal of Virology

基　　金：国家重点研发专项省部联动项目(项目号:2022YFD1601901),题目:香格里拉市迪庆藏猪产业提质增效关键技术集成与示范。

摘　　要：猪札幌病毒(Porcine sapovirus,PoSaV)是一种引起猪急性胃肠炎的肠道病毒,对于生猪养殖业造成严重威胁。PoSaV感染猪只表现出的症状与其他腹泻病毒引起的症状相似,难以区分。为了检测该病毒并了解其在猪场中的流行情况,我们建立了一种PoSaV检测方法。本研究根据GenBank中GIII基因型,设计引物,优化反应条件,通过特异性评价,灵敏度和重复性检测,建立荧光定量RT‐PCR方法。本试验从贵州省获取20份腹泻母猪肛拭子样品,通过RT‐PCR进行检测,并进行VP1全基因扩增测序,使用MEGA7.0进行系统进化树构建和同源性分析。结果显示,建立的荧光定量RT‐PCR检测方法具有较强特异性和高灵敏度,在拷贝值为1×101·μL‐1时可以达到普通RT‐PCR方法100倍以上的检出限。此外,该方法具有良好的重复性和稳定性,批内变异系数与批间变异系数均小于1%;在检测猪腹泻临床样本中发现阳性率为85%(17/20)。将获得的17条VP1全基因序列与国内外14条参考毒株进行比对,结果显示:所有测序毒株与参考序列核苷酸同源性介于81.0%‐99.2%,氨基酸同源性介于80.8%‐97.6%,并且均与美国经典Cowden毒株亲缘性较远。所有测序毒株与YNAN、YNAN2020亲缘性较近,其中5株(GZ‐VP1‐3、GZ‐VP1‐5、GZ‐VP1‐6、GZ‐VP1‐9、GZ‐VP1‐11)与云南省分离株YNAN核苷酸及氨基酸同源性为99.2%、96.9%‐97.6%,亲缘性最为亲近,表明可能由一个原始毒株进化而来。测序毒株均在同一分支上亲缘性较近,但核苷酸及氨基酸同源性在91.8%‐100%,显示VPl基因存在一定程度变异。本研究成功建立一种PoSaV检测方法,深入分析了当前毒株的遗传变异情况,对PoSaV防控有着一定的启示作用。The porcine sapovirus(PoSaV)is classified as an enterovirus and is known to induce acute gastroenteritis in pigs,presenting a significant threat to the swine breeding industry.The PoSaV‐infected pigs exhibited symptoms similar to those caused by other diarrheal viruses and were difficult to distinguish.We designed a PoSaV assay to detect the virus and assess its prevalence in pig farms.According to the GIII genotype in GenBank,primers were designed,the reaction conditions were optimized,and the fluorescence quantitative RT‐PCR method was established by specificity evaluation,sensitivity,and repeatability detection.In this study,20 anal swab samples were collected from diarrhea sows in Guizhou province and detected by RTPCR.The whole VP1 gene was sequenced.MEGA 7.0 was used for phylogenetic tree construction and homology analysis.The results showed that the established fluorescence quantitative RT‐PCR detection method had high specificity and sensitivity.At a viral copy number of 1×101·μL‐1 the method showed excellent repeatability and stability.Both intra‐assay and inter‐assay coefficients of variation were less than 1%.The positive rate of porcine diarrhea in clinical samples was determined to be 85%(17/20)during testing.The 17 VP1 gene sequences obtained were compared with 14 reference strains in China and abroad.The results revealed that the nucleotide homology between all sequenced strains and the reference sequence ranged from 81.0%to 99.2%,while the amino acid homology ranged from 80.8%to 97.6%.Furthermore,it was observed that all strains were significantly different from the classic Cowden strain.All the sequenced strains showed close genetic similarity to YNAN and YNAN2020,with five of them(GZ‐VP1‐3,GZ‐VP1‐5,GZ‐VP1‐6,GZ‐VP1‐9,GZ‐VP1‐11)sharing 99.2%and 96.9–97.6%in nucleotide and amino acid homology with the YNAN strain in Yunnan province.This suggests that they may have evolved from an original strain.All sequenced strains were clustered closely on the same branch,wit

关 键 词：猪札幌病毒 荧光定量PCR VP1基因 遗传进化分析 

分 类 号：S854.4[农业科学—临床兽医学]