摘要
副猪嗜血杆菌(HPS)、胸膜肺炎放线杆菌(APP)、多杀性巴氏杆菌(Pm)和猪链球菌(SS)的单独或混合感染是全球养猪生产中常见的健康问题之一。由于传统的单一检测方法无法满足多病原混合感染的检测要求,为了缩短检测时间,降低检测成本,本研究根据HPS的16S r RNA基因、APP的apx IVA基因、Pm的KMT1基因以及SS的gdh基因序列设计特异性引物和Taq Man探针,PCR扩增相应基因后,将其分别克隆至p MD19-T载体中,构建重组质粒标准品p MD19T-HPS、p MD19T-APP、p MD19T-Pm和p MDT19-SS。经各反应条件的优化建立了同时检测HPS、APP、Pm和SS的多重Taq Man荧光定量PCR(q PCR)检测方法。利用该方法检测HPS、APP、Pm、SS、大肠杆菌、金黄色葡萄球菌、都柏林沙门氏菌、枯草芽孢杆菌、乳酸菌等常见细菌,结果显示,该方法对HPS、APP、Pm和SS均有扩增反应,与其他常见细菌均无交叉反应,特异性较强。对10倍倍比稀释后等浓度等体积混合的4种重组质粒标准品混合物的检测结果显示,该方法对p MD19T-HPS、p MD19T-APP、p MD19T-SS的检测限均为1×10^(1)拷贝/μL,对p MD19T-Pm的检测限为1×10^(2)拷贝/μL,敏感性较高;选取3个浓度的p MD19T-HPS、p MD19T-APP、p MD19T-Pm和p MD19T-SS(1×10^(6)拷贝/μL、1×10^(5)拷贝/μL、1×10^(4)拷贝/μL)等浓度等体积混合物为模板,进行组内和组间重复性试验,结果显示组内与组间重复性试验的变异系数均小于3%,重复性较好。利用该方法与中华人民共和国农业农村部公告中HPS常规PCR方法(GB/T34750-2017)、APP常规PCR方法(NY/T 537-2023)、Pm常规PCR方法(NY/T 564-2016)、SS常规PCR方法(GB/T19915.3-2005)分别检测62份临床样品,结果显示,本研究建立的多重Taq Man q PCR检测方法对HPS的检出率为69.35%(43/62),对APP的检出率为14.52%(9/62),对Pm的检出率为25.81%(16/62),对SS的检出率为80.65%(50/62);常规PCR对HPS的检出率为59.68%(37/62),对APP的检出率为8.06%(5/62),对Pm的检出率为22.58%(14/62),对SS的检出率为77.42%(48/62),其中HPS和APP混合感染率为1.61%(1/62),HPS和Pm混合感染率为6.45%(4/62),HPS和SS混合感染率为20.97%(13/62),APP和SS混合感染率为3.22%(2/62),HPS、APP和SS混合感染率为1.61%(1/62)。经计算,HPS、APP、Pm和SS的多重Taq Man qPCR检测方法与常规PCR的阳性符合率为100%,总符合率分别为:90.32%、96.77%、100%、96.77%。本研究建立的多重Taq Man qPCR检测方法特异性强、敏感性高、重复性好,为HPS、APP、Pm和SS临床样品的快速鉴别检测提供了有力技术支持。
Single or mixed infections with Haemophilus parasuis(HPS),Actinobacillus pleuropneumoniae(APP),Pasteurella multocida(Pm)and Streptococcus suis(SS)are among the most common health problems in pig production worldwide.However,conventional single detection methods cannot meet the detection requirements of multi-pathogen mixed infection.To shorten the detection time and reduce the detection cost,specific primers and TaqMan probes were designed using the 16S rRNA gene of HPS,the apxIVA gene of APP,the KMT1 gene of Pm and the gdh gene of SS,and a multiplex TaqMan qPCR method for simultaneous detection of HPS,APP,Pm and SS was established by optimizing the reaction conditions.The method was used to detect HPS,APP,Pm,SS,Escherichia coli,Staphylococcus aureus,Salmonella Dublin,Bacillus subtilis,Lactic acid bacteria and other common bacteria,and it showed only the fragments of HPS,APP,Pm and SS were amplified,and there was no cross-reaction with other common bacteria,showing strong specificity.The detection results of the mixture of HPS,APP,Pm and SS plasmid standards after 10-fold dilution showed that the detection limit of HPS,APP and SS plasmid standards was 1×10^(1) copies/μL,while for Pm plasmid standard was 1×10^(2) copies/μL,indicating the high sensitivity.Three concentrations of HPS,APP,Pm and SS plasmid standard(1×10^(6) copies/μL,1×10^(5) copies/μL,1×10^(4) copies/μL)were selected as the template for intra-and inter-group repeatability tests.The results showed that the coefficient of variation in both intra-and inter-group repeatability tests was less than 3%,demonstrating good repeatability.Comparison of the assay with the routine PCR detection method for HPS(GB/T 34750-2017),APP(NY/T 537-2023),Pm(NY/T 564-2016),and SS(GB/T19915.3-2005)published by the Ministry of Agriculture and Rural Affairs of the People's Republic of China in detecting 62 clinical samples showed that the detection rate of multiplex TaqMan qPCR assay established in this study was 69.35%(43/62)for HPS,14.52%(9/62)for APP,25.81%(16/62)for Pm and 80.65%(50/62)for SS,respectively.The detection rate of conventional PCR was 59.68%(37/62)for HPS,8.06%(5/62)for APP,22.58%(14/62)for Pm and 77.42%(48/62)for SS.The rates of mixed infection of HPS and APP were 1.61%(1/62),6.45%(4/62),20.97%(13/62),3.22%(2/62),and 1.61%(1/62)respectively.The positive coincidence rate of multiplex TaqMan qPCR of HPS,APP,Pm and SS with conventional PCR was 100%,and the total coincidence rate was 90.32%,96.77%,100%and 96.77%,respectively.The multiplex TaqMan qPCR assay established in this study has strong specificity,high sensitivity and good repeatability,which provides strong technical support for rapid differential detection of HPS,APP,Pm and SS clinical samples.
作者
毛茵茗
汤德元
曾智勇
王彬
黄涛
何松
周飘
胡雯雯
周敏
MAO Yin-ming;TANG De-yuan;ZENG Zhi-yong;WANG Bin;HUANG Tao;HE Song;ZHOU Piao;HU Wen-wen;ZHOU Min(College of Animal Science,Guizhou University,Guiyang 550025,China)
出处
《中国预防兽医学报》
北大核心
2025年第9期913-919,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
贵州大学重点项目(黔科合平台人才[2018]5781-8)
国家自然科学基金(31860716)。
