蟲媒病病原多重液相芯片檢測方法的建立
發(fā)布時間：2018-11-22 16:42
【摘要】：目的：蟲媒病是指以昆蟲作為媒介通過吸血傳播造成人畜感染的一類疾病。本研究利用液相芯片技術(shù)建立蟲媒病多重病原的檢測方法。方法：根據(jù)藍(lán)舌病病毒(BTV)、鹿流行性出血熱病毒(EHDV)、Q熱貝氏柯克斯體(Coxiella burnetii)、非洲豬瘟病毒(ASFV)、西尼羅病毒(WNV)、萊姆病伯氏疏螺旋體(BB)、水泡性口炎病毒(VSV)、裂谷熱病毒(RVFV)、埃博拉病毒(EBV)和施馬倫貝格病毒(SBV)的高度保守區(qū)域分別設(shè)計了10對引物與10種探針。選用不同編碼的磁性微球分別與10種蟲媒病的特異性探針進(jìn)行有效偶聯(lián)。在此基礎(chǔ)上,通過上下游引物比例、液相雜交溫度、液相雜交時間等關(guān)鍵因素的優(yōu)化,建立了10種蟲媒病原的多重液相芯片檢測體系。隨后對該多重液相體系的特異性、靈敏度和重復(fù)性進(jìn)行驗證。結(jié)果：通過PCR/RT-PCR擴(kuò)增得到的片段與預(yù)期大小相吻合。將純化后的PCR產(chǎn)物與載體連接構(gòu)建陽性質(zhì)粒,經(jīng)過DNAMAN分析比對,其同源性均大于97%。當(dāng)引物比例為1：2,雜交溫度為50℃,雜交時間為30min時,每種蟲媒病的熒光中位值明顯高于背景值,能有效、特異的檢測出目的片段。結(jié)果顯示整個體系對口蹄疫、小反芻獸疫等疫病無交叉反應(yīng),最小檢出量是102拷貝數(shù)并且具有良好的重復(fù)性。利用建立的多重液相芯片體系對已知蟲媒病原混合樣品和300份蚊、蜱、蠓的未知核酸進(jìn)行檢測,發(fā)現(xiàn)病原均為陽性且內(nèi)蒙古呼倫貝爾6只蜱中攜帶伯氏疏螺旋體。結(jié)論：本研究成功建立了能同時檢測十種蟲媒病病原的液相芯片方法,為多種蟲媒病原高通量快速篩查搭建了技術(shù)平臺,這對動物蟲媒性疫病的防控以及鑒定等都將具有重要意義。
[Abstract]:Objective: insecticidal disease is a kind of disease which is transmitted by sucking blood by insects. In this study, a liquid-phase microarray technique was used to detect multiple pathogens of insect-borne diseases. Methods: according to bluetongue virus (BTV), deer epidemic hemorrhagic fever virus (EHDV), Q Rebekovskii virus (Coxiella burnetii), African classical swine fever (ASFV), West Nile virus (WNV), Lyme disease spirochetes (BB), 10 pairs of primers and 10 probes were designed for highly conserved regions of vesicular stomatitis virus (VSV),) Rift Valley Fever virus (RVFV),) Ebola virus (EBV) and Schmalenberger virus (SBV), respectively. Magnetic microspheres with different codes were effectively coupled with 10 specific probes of insect-borne diseases. On this basis, the multiplex liquid-phase microarray detection system of 10 kinds of insect-borne pathogens was established by optimizing the key factors such as the proportion of primers, the temperature of liquid phase hybridization and the time of liquid phase hybridization. The specificity, sensitivity and repeatability of the multiplex liquid system were then verified. Results: the fragments amplified by PCR/RT-PCR coincided with the expected size. The purified PCR product was ligated with the vector to construct the positive plasmid. The homology of the purified PCR product was more than 97 by DNAMAN analysis. When the primer ratio was 1: 2, the hybridization temperature was 50 鈩，
本文編號：2349919
[Abstract]:Objective: insecticidal disease is a kind of disease which is transmitted by sucking blood by insects. In this study, a liquid-phase microarray technique was used to detect multiple pathogens of insect-borne diseases. Methods: according to bluetongue virus (BTV), deer epidemic hemorrhagic fever virus (EHDV), Q Rebekovskii virus (Coxiella burnetii), African classical swine fever (ASFV), West Nile virus (WNV), Lyme disease spirochetes (BB), 10 pairs of primers and 10 probes were designed for highly conserved regions of vesicular stomatitis virus (VSV),) Rift Valley Fever virus (RVFV),) Ebola virus (EBV) and Schmalenberger virus (SBV), respectively. Magnetic microspheres with different codes were effectively coupled with 10 specific probes of insect-borne diseases. On this basis, the multiplex liquid-phase microarray detection system of 10 kinds of insect-borne pathogens was established by optimizing the key factors such as the proportion of primers, the temperature of liquid phase hybridization and the time of liquid phase hybridization. The specificity, sensitivity and repeatability of the multiplex liquid system were then verified. Results: the fragments amplified by PCR/RT-PCR coincided with the expected size. The purified PCR product was ligated with the vector to construct the positive plasmid. The homology of the purified PCR product was more than 97 by DNAMAN analysis. When the primer ratio was 1: 2, the hybridization temperature was 50 鈩，
本文編號：2349919
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