[1]吴双,张聪,袁慧莎,等.血清4型禽腺病毒TaqMan探针实时荧光定量PCR检测方法的建立与应用[J].江苏农业学报,2023,(01):134-141.[doi:doi:10.3969/j.issn.1000-4440.2023.01.016]
　WU Shuang,ZHANG Cong,YUAN Hui-sha,et al.Establishment and application of quantitative real-time PCR detection method for fowl aviadenovirus serotype 4 based on TaqMan probe[J].,2023,(01):134-141.[doi:doi:10.3969/j.issn.1000-4440.2023.01.016]
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血清4型禽腺病毒TaqMan探针实时荧光定量PCR检测方法的建立与应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2023年01期
页码:
134-141
栏目:
畜牧兽医·水产养殖
出版日期:
2023-02-28

文章信息/Info

Title:
Establishment and application of quantitative real-time PCR detection method for fowl aviadenovirus serotype 4 based on TaqMan probe
作者:
吴双1;  张聪1; 2;  袁慧莎1; 2;  戴利霞2;  林梦舟1;  吴植1;  朱善元1
（1.江苏农牧科技职业学院江苏省兽用生物制药高技术研究重点实验室，江苏泰州225300；2.和盛食品集团有限公司，江苏泰州225300）
Author(s):
WU Shuang1; ZHANG Cong1; 2; YUAN Hui-sha1; 2; DAI Li-xia2; LIN Meng-zhou1; WU Zhi1; ZHU Shan-yuan1
（1.Jiangsu Provincial Key Laboratory of Veterinary Bio-pharmaceutical High-Tech Research， Jiangsu Agri-animal Husbandry Vocational College， Taizhou 225300，China；2.Hesheng Food Group Co., Ltd., Taizhou 225300，China）
关键词:
血清 4 型禽腺病毒; 荧光定量PCR; 临床应用
Keywords:
fowl aviadenovirus serotype 4; fluorescent quantitative PCR; clinical application
分类号:
S831.7
DOI:
doi:10.3969/j.issn.1000-4440.2023.01.016
文献标志码:
A
摘要:
为建立快速检测血清4型禽腺病毒病（Fowl aviadenovirus serotype 4，FAdV-4）的基于TaqMan探针的Quantitative real-time PCR（qPCR）检测方法，本研究根据 Hexon基因序列，设计相关的qPCR特异性引物和探针，经特异性、敏感性和重复性试验以及反应程序等系列优化后，最终建立荧光定量检测方法并应用于临床样品的病原检测。FAdV-4的qPCR结果表明，优化后标准曲线决定系数（R2）为0.998，扩增效率（E）为95.226%，该方法效果良好。利用该方法，除FAdV-4外其他禽类常见病原均未检测到，该方法具有良好的特异性。该qPCR方法检出阳性的最低拷贝数为100个拷贝，而普通PCR方法检出阳性的最低拷贝数为10 000个拷贝，说明其具有良好的灵敏度。组内和组间变异系数均≤0.22%，该方法的重复性较高。对180份从江苏、安徽、江西和广西等省份采集的临床疑似样品进行检测，结果显示，qPCR方法检出率为51.1%，而普通PCR方法检出率为32.8%；同时基于qPCR引物的检出符合率为100.00%，而基于普通PCR特异性引物检出的符合率为82.35%，表明该qPCR方法针对临床疑似样品的检测准确性更高。该方法的建立有助于推动禽腺病毒病的分子流行病学研究和防控。
Abstract:
To establish rapid detecting method for fowl aviadenovirus serotype 4 (FAdV-4) detection based on quantitative real-time PCR (qPCR) with TaqMan probe, specific primers and TaqMan probe for qPCR were designed based on Hexon gene sequence of FAdV-4 in GenBank. After a series of optimization operations of specificity, sensitivity, repetitive experiments and reaction procedure, the fluorescence quantitative detection method was finally established and was applied to the pathogen detection of clinical samples. qPCR results of FAdV-4 showed that, coefficient of determination (R2) of the optimized standard curve was 0.998, and the amplification efficiency (E) was 95.226%, which indicated that the method had good effect. Other common avian pathogens were not detected except for FAdV-4 by using the detecting method. The lowest copy for detecting sensitivity by qPCR method was 100, while the lowest copy for detecting sensitivity by conventional PCR method was 10 000, which indicated that the qPCR method had good sensitivity. The variation coefficients between and within groups were all ≤0.22%, which showed high repeatability of the method. 180 clinical suspected samples collected from Jiangsu province, Anhui province, Jiangxi province and Guangxi Zhuang Autonomous Region were detected. The results showed that, positive detection rate of qPCR method was 51.1%, while the value of conventional PCR method was 32.8%. The positive detection rate based on qPCR primers was 100.00%, and the value based on conventional PCR specific primers was 82.53%, which revealed that the qPCR method had higher positive detection rate for clinical suspected FAdV-4 sample detection. The establishment of quantitative real-time PCR with TaqMan probe detection method is conducive to promote molecular epidemiological investigation and control of fowl aviadenovirus.

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备注/Memo

备注/Memo:
收稿日期：2022-05-22基金项目：江苏高校“青蓝工程”项目[苏教师函（2020）10号]；江苏省高等学校自然科学研究重大项目（21KJA230001）；江苏省2019年度高校优秀科技创新团队项目作者简介：吴双(1983-), 女, 江苏徐州人, 博士, 教授, 主要从事动物传染病的防控工作。（Tel）0523-86356818；（E-mail）108570936@qq.com。张聪为共同第一作者通讯作者：朱善元，（E-mail）jstzzsy@126.com
更新日期/Last Update: 2023-03-21