牛副流感3型病毒Nano-PCR、LAMP方法的建立及初步应用

试验旨在建立牛副流感3型病毒（bovine parainfluenza type-3 virus,BPIV3）纳米PCR（Nano-PCR）与环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）新型快速分子检测技术,对其进行特异性和敏感性对比试验,并对10份临床样品进行了检测。特异性与敏感性试验结果显示,建立的Nano-PCR和LAMP方法只对BPIV3特异,而对牛呼吸道合胞体病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒无交叉反应;建立的Nano-PCR和LAMP方法具有相同的敏感性,均是普通PCR的10倍,最低核酸检出量均为4.16×10²拷贝/μL。临床检测结果显示,建立的两种方法阳性符合率为100%,且阳性检出率均高于普通PCR。因此,本试验建立的Nano-PCR和LAMP方法为BPIV3的临床诊断提供了更快速、敏感、可靠的工具。     

鸡传染性支气管炎病毒纳米PCR检测方法的建立及初步应用

根据鸡传染性支气管炎病毒(IBV)的3′端非编码区保守区域,设计并合成一对特异性引物,建立IBV纳米PCR检测方法,并进行了特异性、灵敏性和重复性试验,然后将建立的方法进行初步应用。结果显示:该方法特异性良好,能从4种不同基因型IBV核酸样品中检测到约346bp的目的条带,而对禽偏肺病毒、H9亚型禽流感病毒、新城疫病毒、传染性喉气管炎病毒、传染性法氏囊病毒、马立克氏病病毒、禽白血病病毒、鸭肝炎病毒等其他病原检测结果均为阴性;敏感性试验表明,纳米PCR的最低检测浓度为1.01×10^-4ng/μL,其敏感性是普通PCR的10倍;重复性试验显示3次均能检出IBV核酸。纳米PCR方法的临床应用表明,送检样品及攻毒鸡样品的检出率分别达到50%(10/20)和60%(30/50),与普通PCR方法检测结果符合率为100%。研究建立的IBV纳米PCR检测方法具有较高的敏感性和良好的特异性,为IBV的临床诊断、病原检测和流行病学调查提供了新的技术手段。     

Establishment and Preliminary Application of Nano-PCR and LAMP Methods for Bovine Parainfluenza Type-3 Virus

This study was aimed to establish Nano-PCR and LAMP which were new rapid type of molecular detection technologies of bovine parainfluenza type-3 virus (BPIV3).The comparative specificity and sensitivity of BPIV3 Nano-PCR and LAMP PCR were tested, and the assay was applied to detect 10 clinical samples. The specificity and sensitivity tests showed that Nano-PCR and LAMP were only sensitive to BPIV3,without cross reaction to other viruses, such as bovine respiratory syncytial virus,infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. In the sensitivity test, Nano-PCR and LAMP showed 10 times sensitivity than that of the traditional PCR technology,with the minimum detection of 4.16×10² copies/μL. Clinical test results showed that the coincidence rate of Nano-PCR and LAMP could reach to 100%, and the positive detection rate was much higher than that of normal PCR.Therefore, the Nano-PCR and LAMP methods established in this study provided a faster, sensitive and reliable tool for the clinical diagnosis of BPIV3.