摘要
【背景】猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)、新型猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome coronavirus,SADS-CoV)与塞内卡病毒A型(Seneca virus A,SVA)均为猪的新发病原,严重危害养猪业的发展。猪感染这3种新发病原难以根据临床症状诊断,因此亟须建立多重RT-PCR检测方法对疑似患病猪进行快速诊断,以降低经济损失。【目的】建立能同时检测PDCoV、SADS-CoV和SVA单一或混合感染的三重RT-PCR检测方法。【方法】参考GenBank中登录的PDCoV和SADS-CoV N基因、SVA L/P1基因保守区域,设计3对特异性引物,以温度梯度PCR法确定最适退火温度(Tm);采用方阵法优化其引物浓度;构建重组质粒PMD-PDCoV、PMD-SADS-CoV和PMD-SVA作为标准品确定最小检测量(limits of detection,LOD);以猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪繁殖与呼吸综合征病毒等6种常见感染猪的病毒核酸样本为模板,确定三重RT-PCR法的特异性;以批间和批内实验验证其重复性;经检测疑似感染的临床样本并与已报道的检测方法进行比较,评估其临床应用效果。【结果】最佳退火温度为58.3℃;引物最佳浓度分别为0.5、0.25和0.25μmol/L;其敏感性高,PMD-PDCoV、PMD-SADS-CoV与PMD-SVA最低检测限分别为1、1和10 copies/μL;其特异性强,仅对PDCoV、SADS-CoV和SVA有特异性条带,对其他病毒均无扩增条带;该方法重复性好,批间和批内实验检测结果均一致。经临床样本检测PDCoV、SADS-CoV和SVA的阳性率分别为65.85%、30.49%和57.32%,与已报道的检测方法一致。最后从13份PDCoV、SADS-CoV和SVA均为阳性的样品中随机选取5份样品进行测序,并做同源性及进化树分析,结果显示5份阳性病料的PCR扩增序列之间相似性较高,而且和参考序列之间也具有较高的相似性,均可达到96%以上。表明本研究建立的方法在应用于临床样品检测中具有较高的准确性和可靠性。【结论】
[Background]Porcine delta coronavirus(PDCoV),swine acute diarrhea syndrome coronavirus(SADS-CoV),and Seneca virus A(SVA)are new pathogens which seriously endanger the development of pig industry.The clinical symptoms of pigs infected with the three pathogens are difficult to distinguish.Therefore,it is urgent to establish a multiplex RT-PCR detection method for rapid diagnosis of suspected pigs and reduce economic losses.[Objective]To establish a triplex RT-PCR method for simultaneous detection of single or mixed infection of PDCoV,SADS-CoV,and SVA.[Methods]Three pairs of specific primers were designed according to the conserved regions of the N genes of PDCoV and SADS-CoV and the L/P1 genes of SVA registered in GenBank,and the optimal annealing temperature(Tm)was determined by temperature gradient PCR method.The primer concentration was optimized by array method.The recombinant plasmids PMD-PDCoV,PMD-SADS-CoV,and PMD-SVA were constructed as standards to determine the limits of detection(LOD).The specificity of the triplex RT-PCR method was determined with the nucleic acid samples of 6 common pig viruses including porcine transmissible gastroenteritis virus,porcine epidemic diarrhea virus,and porcine reproductive and respiratory syndrome virus.The repeatability of the established method was verified by inter-batch and intra-batch tests.Finally,we employed the triplex RT-PCR method to detect the clinical samples suspected of infection and compared the results with those obtained with the reported detection methods,thus evaluating the clinical application performance of the method.[Results]The optimal Tm was 58.3℃,and the optimal primer concentrations were 0.5μmol/L,0.25μmol/L,and 0.25μmol/L,respectively.The established method had high sensitivity,with the LODs of 1 copy/μL,1 copy/μL,and 10 copies/μL for PMD-PDCoV,PMD-SADS-CoV,and PMD-SVA,respectively.It had strong specificity,with specific bands only for PDCoV,SADS-CoV,and SVA and no bands for other viruses.Moreover,the method had good repeatability as the
作者
陈见兴
王豪杰
潘喻
刘弘毅
林欢
朱良全
陈洪岩
谢金鑫
夏长友
张贺
王玉娥
CHEN Jianxing;WANG Haojie;PAN Yu;LIU Hongyi;LIN Huan;ZHU Liangquan;CHEN Hongyan;XIE Jinxin;XIA Changyou;ZHANG He;WANG Yu’e(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,Xinjiang,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,Heilongjiang,China;China Institute of Veterinary Drug Control,Beijing 102600,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2022年第12期5100-5111,共12页
Microbiology China
基金
国家重点研发计划青年科学家项目(2021YFF0703100)。
关键词
猪丁型冠状病毒
新型猪急性腹泻综合征冠状病毒
塞内卡病毒A型
三重RT-PCR
porcine deltacoronavirus(PDCoV)
swine acute diarrhea syndrome coronavirus(SADS-CoV)
Seneca virus A(SVA)
triplex reverse transcription-polymerase chain reaction
