多重PCR检测和鉴定五种猪腹泻病毒研究

发布时间：2018-07-28 16:14

【摘要】：近年来全球猪腹泻病频发,发病规模大,导致经济损失惨重。猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus, TGEV )、猪轮状病毒 A 型(Porcine group A rotavi ruses,GAR )、猪轮状病毒 C 型(Porcine group C rotaviruses, GCR)、猪圆环病毒 2 型(Porcine circovirus2,PCV2)被认为是最主要的猪病毒性腹泻病原。这5种病毒感染率高、危害大,且存在混合感染现象,现有检测手段存在一定缺陷且难以对混合感染情况进行有效检测。本研究分别建立了针对该5种腹泻病毒的普通多重PCR和EvaGreen多重实时荧光定量PCR方法并应用于临床样品检测。普通PCR在检测中有着其独特优势而被广泛使用。本实验首先通过GenBank下载5种病毒基因序列,经软件序列比对后,在保守区分别设计5对普通PCR引物。通过对反应体系和反应条件进行优化,单重和多重普通PCR均可产生大小分别为 126bp(GCR)、242bp(GAR)、319bp(TGEV)、394bp(PEDV)、508bp(PCV2)目的产物片段。而阴性对照和非靶标阴性对照没有相应的产物产生,表明建立的单重和多重普通PCR方法具有良好的特异性。单重PCR检测GCR、GAR、TGEV、PEDV 和 PCV2 的最低限分别为 5 copies、50 copies、5 copies、5 copies、5 copies。多重普通PCR检测混合病毒模板情况的灵敏度都可以达到50 copies,和单重PCR相同或低一个数量级,但相比于先前研究报道的多重反转录PCR灵敏度(104-103 copies)提高了约2个数量级。将建立的普通多重PCR方法用于172份临床样品检测。GCR、GAR、TGEV、PEDV 和 PCV2 阳性率分别为 5.81%、2.32%、8.72%、20.92%和 46.51%,总混合感染率为22.09%,其中主要是PCV2与其它病毒存在混合感染现象,PEDV和PCV2混合感染率较高,占16.86%。挑选64个样品进行多重PCR和单重PCR对比,结果显示两者检出率相近,多重PCR检出率略低。实时荧光定量PCR包括多重实时荧光定量PCR是通过实时监测荧光信号的一种核酸片段定性定量分析方法,近来被广泛用于病原微生物检测多重实时荧光定量PCR技术在猪病毒性腹泻上的应用很少。本研究根据5种腹泻病毒扩增产物Tm值,在保守区设计5对定量PCR引物,经反应体系和参数优化,建立了基于EvaGreen荧光染料和熔解曲线分析检测这5种病毒的单重和多重荧光定量PCR方法。结果显示设计的5对病毒引物能够形成特异的熔解峰,TGEV、GAR、GCR、PEDV 和 PCV2 对应产物 Tm 值为 75.07 ± 0.15℃、77.77± 0.06℃、79.63±0.25℃、83.60 ±0.17℃和88.63 ±0.06℃,和多重荧光定量PCR各病毒溶解峰Tm值类似,而非目的基因对照和阴性对照未形成影响实验分析的非特异峰,表明建立的单重和多重荧光定量PCR方法特异性良好。批内批间重复性实验结果显示Tm值变异系数小于0.5%,Ct值变异系数小于3.5%,表明重复性良好。TGEV、GAR、GCR、PEDV和PCV2单重荧光定量PCR的检测灵敏度分别是5、5、5、5和50copies。多重实时荧光定量PCR对单个病毒TGEV、GAR、GCR、PEDV和PCV2的检测灵敏度分别为5、5、50、5和50 copies,对5个病毒混合模板的检测灵敏度都达到50 copies。将建立的EvaGreen多重实时荧光定量PCR方法应用于172份临床样品检测。TGEV、GAR、GCR、PEDV 和 PCV2 阳性率分别为 5.23%、3.48%、7.56%、25.58%和44.76%。存在两种和三种病毒的混合感染,总混合阳性率为22.66%,其中PEDV和PCV2混合感染率较高,达到18.02%。检测结果和普通PCR相近。对其中90份临床样品分别进行各病毒单重实时荧光定量PCR检测,两者检出率比较接近。表明本研究建立的多重实时荧光定量PCR方法可较好地用于临床样品5种腹泻病毒检测。
[Abstract]:In recent years, the global swine diarrhoea has been frequently occurring, with large scale and heavy economic loss. Swine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), swine infectious gastroenteritis virus (Transmissible gastroenteritis virus, TGEV), porcine rotavirus A type (Porcine group), porcine rotavirus type Roup C rotaviruses, GCR), the porcine circovirus 2 (Porcine circovirus2, PCV2) is considered to be the most important pathogen of swine viral diarrhea. These 5 viruses have high infection rate, great harm and mixed infection. The existing detection methods have some defects and are difficult to detect the mixed infection effectively. This study established the needle respectively. The common multiplex PCR and EvaGreen multiple real-time fluorescent quantitative PCR methods for the 5 kinds of diarrhea viruses were applied to the detection of clinical samples. The common PCR has its unique advantages and is widely used. This experiment first downloads 5 virus gene sequences by GenBank. After the software sequence ratio, 5 pairs of ordinary PCR are designed in the conservative region. By optimizing the reaction system and reaction conditions, single weight and multiple common PCR can produce fragments of 126bp (GCR), 242bp (GAR), 319bp (TGEV), 394bp (PEDV), 508bp (PCV2), while negative and non target negative controls have no corresponding products, indicating that single and multiple common PCR methods are established. Good specificity. The minimum limits for single weight PCR detection of GCR, GAR, TGEV, PEDV and PCV2 are 5 copies, 50 copies, 5 copies, 5 copies, and 5 copies. multiple common PCR tests can reach 50, the same or one order of magnitude as single or low, but compared to the multiple reverse transcripts reported previously. The PCR sensitivity (104-103 copies) increased by about 2 orders of magnitude. The common multiple PCR method will be established for 172 clinical samples to detect.GCR, GAR, TGEV, PEDV and PCV2, respectively, 5.81%, 2.32%, 8.72%, 20.92% and 46.51%, and the total mixed infection rate is 22.09%, mainly the presence of mixed infection between PCV2 and other viruses, PEDV and PCV2. The rate of mixed infection was high, and 64 samples were selected for 16.86%. to compare multiple PCR and single PCR. The results showed that the detection rate was similar, and the detection rate of multiple PCR was slightly lower. Real time fluorescence quantitative PCR including multiple real-time quantitative PCR was a qualitative and quantitative analysis method of nucleic acid segments through real-time monitoring of fluorescence signals, and recently it was widely used for disease. The application of multiple real-time fluorescence quantitative PCR in the detection of viral diarrhea in swine was very few. This study was based on the Tm value of 5 kinds of diarrhea virus, and designed 5 pairs of quantitative PCR primers in the conservative region. Through the optimization of the reaction system and parameters, a single and multiple detection of these 5 viruses was established based on the EvaGreen fluorescent dye and the fusion curve analysis. The results of heavy fluorescence quantitative PCR showed that the designed 5 pairs of primers could form a specific fusion peak. The Tm values of the products of TGEV, GAR, GCR, PEDV and PCV2 were 75.07 + 0.15, 77.77 + 0.06, 79.63 + 0.25, 83.60, 0.17 and 88.63 + 75.07, similar to those of the multiple fluorescence quantitative PCR viruses, but not the target genes. The control and negative control did not form the non specific peak of the experimental analysis, indicating that the established single weight and multiple fluorescence quantitative PCR method had good specificity. The results of interbatch reproducibility test showed that the Tm value variation coefficient was less than 0.5% and the Ct value variation coefficient was less than 3.5%, indicating that the reproducibility of good.TGEV, GAR, GCR, PEDV and PCV2 single heavy fluorescence quantitative PCR were detected. The sensitivity of 5,5,5,5 and 50copies. multiplex real-time fluorescent quantitative PCR for single virus TGEV, GAR, GCR, PEDV and PCV2 are 5,5,50,5 and 50 copies respectively. The sensitivity of the 5 virus mixed templates is 50 copies. and the EvaGreen multiple realtime fluorescence quantitative method is applied to 172 clinical samples. The positive rates of.TGEV, GAR, GCR, PEDV and PCV2 were 5.23%, 3.48%, 7.56%, 25.58% and 44.76%. were mixed with two and three viruses, and the total mixed positive rate was 22.66%. The mixed infection rate of PEDV and PCV2 was higher, and the result of 18.02%. detection was similar to that of ordinary PCR. The virus single weight of 90 clinical samples was carried out in real time respectively. The detection rate of fluorescence quantitative PCR was close. It showed that the multiple real-time fluorescence quantitative PCR method established in this study could be better used for the detection of 5 kinds of diarrhoea virus in clinical samples.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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