鸭甲肝病毒1和3型一步法双重RT-PCR检测方法的建立及应用

发布时间：2018-05-30 23:33

本文选题：鸭病毒性肝炎 + DHAV-1　；参考：《四川农业大学》2015年硕士论文

【摘要】：鸭病毒性肝炎(DVH)是雏鸭的一种急性传染性病,主要侵害4周龄以内的雏鸭。该病由至少三种不同的病原引起,包括鸭甲肝病毒(DHAV).1型鸭星状病毒(DastV-1)口2型鸭星状病毒(DastV-2)。分布范围最广的D IAV属于小RNA病毒科禽肝病毒属。基于中和试验和系统发育分析,DHAV已被分为三种基因型(DHAV-1、 DHAV-2、DHAV-3)。其中,DHAV-1分布范围最广泛, DHAV-2仅在台湾发现报道,而近几年DHAV-3已经给我国大陆地区和韩国的养鸭业造成严重危害。DHAV-1与DHAV-2之间无交叉中和反应,DHAV-1和 DHAV-3之间交叉保护作用不明显。通过对雏鸭的临床症状和剖检病变可以初步对DVH进行诊断,但是该方法不能够区分DHAV-1 和 DHAV-3.目前血清学诊断技术和分子生物学诊断技术已经用DHAV的检测和诊断,血清学诊断技术主要包括中和试验、间接血凝试验、琼脂扩散试验和免疫荧光抗体技术等,但是由于其繁琐耗时和成本高,不能用于快速诊断。分子生物学诊断技术包括RT-PCR口RT-LAMP方法等,能够快速特异地对DHAV分离株和临床样品进行诊断,因此建立一种能够快速鉴别诊断DHAV-1和DHAV-3的一步法双重RT-PCR方法对我国DVH的防治有积极的作用。1.建立一步法双重RT-PCR方法用于检测DHAV-1和DHAV-3为了能够快速鉴别诊断DHAV-1和DHAV-3,本研究根据GenBank中DHAV-1和DHAV-3的全基因组序列,在DHAV-1和DHAV-3的基因保守区域分别设计了2对特异性引物,并成功建立一步法双重RT-PCR方法鉴别诊断DHAV-1和DHAV-3。该一步法双重RT-PCR方法能够分别针对DHAV-1和DHAV-3特异性地扩增出992bp和1533bp的目的片段,而对健康雏鸭肝脏组织、番鸭细小病毒、鸭瘟病毒、鸭圆环病毒、禽流感病毒、鸭疫里默氏菌、巴氏杆菌、金黄色葡萄球菌、大肠杆菌和沙门氏菌核酸的检测结果为阴性。该方法最低能够检测出lOpg DHAV-(?)DHAV-3的RNA,具有较高的敏感性。2.应用一步法双重RT-PCR方法检测DHAV-1和D HAV-3本研究中使用建立好的一步法双重RT-PCR方法DHAV人工感染雏鸭和DHAV分离株进行检测,结果显示该一步法双重RT-PCR方法的检测结果和病毒分离实验完全一致。应用建立好的一步法双重RT-PCR方法对从2012～2014年在四川、山东、湖北和广西省采集的52份疑似DVH病料进行检测,结果显示52份病料中有41份为DHAV-1阳性,11份为DHAV-3阳性,这些材料未发现混合感染情况。以上实验证明本研究中建立的一步法双重RT-PCR检测方法能够用于DHAV-1(?)DHAV-3的临床诊断和流行病学调查,并且检测结果表明我国四川、山东、湖北和广西省养鸭地区均有DHAV-1和DHAV-3的流行。综上所述,本研究中建立的一步法双重RT-PCR检测方法可以快速地鉴别诊断DHAV-1和DHAV-3的感染情况。
[Abstract]:Duck viral hepatitis (DVH) is an acute sexually transmitted disease of ducklings, mainly infecting ducklings within 4 weeks of age. The disease is caused by at least three different pathogens, including DHAV1. 1 duck stellate virus (DastV-1) and DastV-2 stellate virus (DastV-2). The most widely distributed D IAV belongs to the genus Avian liver virus of small RNA virus family. Based on neutralization test and phylogenetic analysis, DHAV has been divided into three genotypes: DHAV-1, DHAV-2, DHAV-3. The distribution of DHAV-1 was the most widespread, and DHAV-2 was only reported in Taiwan. In recent years, DHAV-3 has caused serious harm to duck industry in mainland China and South Korea. There is no cross neutralization reaction between DHAV-1 and DHAV-2. The cross protection between DHAV-1 and DHAV-3 is not obvious. DVH can be diagnosed by clinical symptoms and pathological changes of ducklings, but this method can not distinguish DHAV-1 from DHAV-3. At present, serological diagnostic techniques and molecular biological diagnostic techniques have been used for detection and diagnosis of DHAV. Serological diagnostic techniques mainly include neutralization test, indirect hemagglutination test, Agar diffusion test and immunofluorescence antibody technique. However, because of its tedious, time-consuming and high cost, it can not be used for rapid diagnosis. Molecular biological diagnostic techniques, including RT-PCR oral RT-LAMP, can be used to diagnose DHAV isolates and clinical samples quickly and specifically. Therefore, the establishment of a one step double RT-PCR method for rapid differential diagnosis of DHAV-1 and DHAV-3 has a positive effect on the prevention and treatment of DVH in China. A one step double RT-PCR method was established to detect DHAV-1 and DHAV-3. In order to differentiate DHAV-1 and DHAV-3 quickly, two pairs of specific primers were designed in the conserved region of DHAV-1 and DHAV-3 according to the whole genome sequence of DHAV-1 and DHAV-3 in GenBank. One step double RT-PCR method was successfully established for differential diagnosis of DHAV-1 and DHAV-3. The two-step RT-PCR method could specifically amplify the target fragments of 992bp and 1533bp against DHAV-1 and DHAV-3, respectively, and could be applied to the liver tissue of healthy ducklings, Muscovy duck parvovirus, duck plague virus, duck circovirus, avian influenza virus and Riemer's disease virus. The nucleic acids of Pasteurella, Staphylococcus aureus, Escherichia coli and Salmonella were negative. This method can detect lOpg DHAV-(?)DHAV-3 at least, and has high sensitivity. 2. 2. One step double RT-PCR method was used to detect DHAV-1 and D HAV-3. In this study, a one step double RT-PCR method was used to detect artificially infected ducklings and DHAV isolates. The results show that the double RT-PCR method is in good agreement with the virus isolation test. A one step double RT-PCR method was used to detect 52 suspected DVH samples collected from 2012 to 2014 in Sichuan, Shandong, Hubei and Guangxi provinces. The results showed that 41 of 52 samples were DHAV-1 positive and 11 were DHAV-3 positive. No mixed infection was found in these materials. The results show that the two-step RT-PCR detection method can be used in the clinical diagnosis and epidemiological investigation of DHAV-1(?)DHAV-3, and the results show that DHAV-1 and DHAV-3 are prevalent in duck breeding areas of Sichuan, Shandong, Hubei and Guangxi provinces in China. In conclusion, the one-step double-step RT-PCR detection method established in this study can quickly differentiate the infection between DHAV-1 and DHAV-3.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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