内蒙古猪病毒性腹泻流行病学调查及猪流行性腹泻病毒M、S基因序列分析

发布时间：2018-03-15 00:35

本文选题：猪病毒性腹泻　切入点：多重RT-PCR　出处：《内蒙古农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)是目前造成猪病毒性腹泻的三种主要病原,且在临床症状、病理变化以及流行病学上极为相似。传统检测方法虽能对三者进行鉴别诊断,但因操作复杂、检测周期长、灵敏度低等缺点而在实践中并不常用。本试验针对三种病毒的保守序列分别设计特异性引物,并在明确各病毒单项RT-PCR最优反应条件的前提下,逐步优化多重RT-PCR反应条件,最终建立了可同时检测上述三种病毒的多重RT-PCR检测方法。特异性、灵敏性及一致性试验结果表明,多重RT-PCR方法特异性强,灵敏度高,与常规RT-PCR方法的整体符合率为97.5%,适用于三种病毒的鉴别诊断和流行病学调查。应用已建立的多重RT-PCR方法,对2013～2015年间内蒙古9个盟市的413份腹泻样品和115份无腹泻症状样品进行PEDV、TGEV、PoRV三种病原的检测。结果显示,受检的413份腹泻病料中有260份呈现PEDV阳性,阳性率为62.95%；未发现TGEV感染病例；PoRV仅于个别猪场与PEDV昆合感染,阳性率为1.94%。在未发生腹泻的猪群中同样存在PEDV感染,阳性率为41.74%,未发现TGEV感染,仅发现一例PoRV感染猪只。不同日龄猪群均存在PEDV感染,以哺乳仔猪阳性率最高,达72.03%；其次是母猪。此外,不同类型样品的病原检出率有所差异,肠道样品中PEDV检出率最高,达87.50%,其次是肛门拭子,粪便中PEDV阳性率最低,为52.50%。检测结果表明,PEDV是引起内蒙古地区猪群病毒性腹泻的主要病原,并主要危害哺乳仔猪。对各盟市PEDV检测为阳性的病料进行M、S基因的克隆、测序,并与国内外已报道的57条基因序列进行比对和遗传进化分析。同源性及遗传进化分析结果表明,CH/NMG/HLBE株、CH/NMG/BYNE株、CH/NMG/CF株和CH/NMG/EEDS株、CH/NMG/BT株、CH/NMG/HHHT株同源性较高,并与近年来流行的“亚美型”毒株亲缘关系较近,同位于G1分支上；CH/NMG/WLCB株和CH/NMG/XLGL株与疫苗株Vaccine CV777同源性较高,同位于G3分支上；CH/NMG/TL株则与吉林CH/JLGZ/2011株亲缘关系最为密切,同位于G2分支。核苷酸及氨基酸序列比对结果表明,PEDV的M基因较为保守,但其S基因变异明显,且主要集中于S1区的N’端。
[Abstract]:Porcine epidemic diarrhea virus (PEDVV), transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) are the three main pathogens causing porcine viral diarrhea. The pathological changes and epidemiology are very similar. Although the traditional detection method can differentiate the three, but because of the complexity of the operation, the detection period is long. In this experiment, specific primers were designed for the conserved sequences of three viruses, and the conditions of multiple RT-PCR reaction were optimized step by step on the premise of determining the optimal reaction conditions of individual RT-PCR of each virus. Finally, a multiplex RT-PCR detection method was established for simultaneous detection of the above three viruses. The results of specificity, sensitivity and consistency test showed that the multiplex RT-PCR method had strong specificity and high sensitivity. The overall coincidence rate with the conventional RT-PCR method is 97.50.It is suitable for the differential diagnosis and epidemiological investigation of three viruses. 413 diarrhea samples and 115 diarrhea free samples from 9 cities in Inner Mongolia between 2013 and 2015 were tested for three pathogens, PEVV TGEV and PoRV. The results showed that 260 of 413 samples were positive for PEDV. The positive rate of TGEV infection was 62.95, and the positive rate was 1.94.The positive rate was 1.94.The positive rate of PEDV infection was 41.74 in the pigs without diarrhea, and no TGEV infection was found. Only one PoRV infected pig was found. The positive rate of PEDV was 72.03 in suckling piglets, 72.03 in suckling piglets, and 72.03 in sows. In addition, the positive rate of PEDV was the highest in different types of samples and the highest in intestinal samples. The positive rate of PEDV in feces was the lowest (52.50). The results showed that PEDV was the main pathogen causing viral diarrhea in pigs in Inner Mongolia. The PEDV positive samples were cloned and sequenced. The results of homology and genetic evolution analysis showed that CHP / NMG- / BYNE and CH/NMG/EEDS / NMG- / BT were highly homologous to CHR / NMG- / HHT, respectively, and the results of homology and genetic evolution analysis showed that CHR / NMG- / HLBE was highly homologous to CHP / NMG- BYNE and CHP / NMG- / BYNE, respectively, and there was a high homology between CHR / NMGP-HHT and CH-NMGP-HHT. It was closely related to the prevalent "Asian American" virus strains in recent years, and had the highest homology with Vaccine CV777 in G _ 1 branch and CH/NMG/XLGL strain, and was the most closely related to Jilin CH/JLGZ/2011 strain on the same G3 branch. The results of nucleotide and amino acid sequence alignment showed that the M gene of PEDV was conserved, but its S gene mutation was obvious, and it was mainly concentrated at the N' end of S1 region.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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