非洲猪瘟病毒一步法巢式锁核酸荧光定量PCR方法的建立

doi:10.11924/j.issn.1000-6850.casb2022-0055

中国农学通报 ›› 2023, Vol. 39 ›› Issue (11): 143-151.doi: 10.11924/j.issn.1000-6850.casb2022-0055

• 畜牧·动物医学·蚕·蜂 • 上一篇下一篇

非洲猪瘟病毒一步法巢式锁核酸荧光定量PCR方法的建立

施科达¹^,²(), 徐民生¹^,², 李艳¹, 张昆丽¹, 翟少伦¹, 勾红潮¹, 宋帅¹, 杨冬霞¹, 臧莹安², 孙铭飞¹(), 李春玲¹()

¹ 广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站，广州 510640
² 岭南现代农业科学与技术广东省实验室茂名分中心/仲恺农业工程学院动物科技学院，广州 510225

收稿日期:2022-02-10 修回日期:2022-08-09 出版日期:2023-04-15 发布日期:2023-04-10
通讯作者: 李春玲，女，1965年出生，河南长葛人，研究员，博士，研究方向：动物传染病病原分子生物学。通信地址：510640 广州市天河区五山白石岗广东省农业科学院动物卫生研究所，Tel：020-85291976，E-mail：lclclare@163.com；孙铭飞，男，1978年出生，研究员，博士，研究方向：动物传染病。通信地址：510640 广州市天河区五山白石岗广东省农业科学院动物卫生研究所，E-mail：smf7810@126.com。
作者简介:
施科达，男，1994年出生，广东广州人，硕士，研究方向：动物传染病病原分子生物学。通信地址：510640 广州市天河区五山路白石岗广东省农业科学院动物卫生研究所猪病研究室，Tel：020-85291478，E-mail：541595557@qq.com。
基金资助:
广东省重点领域研发计划资助(2019B020211005); 广东省重点领域研发计划资助(2019B020217002); 广东省现代农业产业技术体系创新团队项目(2022KJ119); 广州市科技特派员项目(20212100027); 广东省农业科学院院长基金项目(202044)

Development of One-Step Nested-Locked Nucleic Acid Real-Time PCR Detection Method for African Swine Fever Virus

SHI Keda¹^,²(), XU Minsheng¹^,², LI Yan¹, ZHANG Kunli¹, ZHAI Shaolun¹, GOU Hongchao¹, SONG Shuai¹, YANG Dongxia¹, ZANG Ying'an², SUN Mingfei¹(), LI Chunling¹()

¹ Institute of Animal Health, Guangdong Academy of Agricultural Sciences/ Key Laboratory of Livestock Disease Prevention of Guangdong Province/ Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, P. R. China, Guangzhou 510640
² Maoming Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology/College of Animal Science & Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225

Received:2022-02-10 Revised:2022-08-09 Online:2023-04-15 Published:2023-04-10

摘要/Abstract

摘要：

试验基于非洲猪瘟病毒(African swine fever virus，ASFV)B646L(P72)蛋白基因序列，建立一种高灵敏度和高特异性的检测方法。采用Oligo 7软件设计一步法巢式锁核酸荧光定量PCR(One Step Nested-Locked Nucleic Acid real-time PCR，LNA-OSN-PCR)的嵌套引物以及探针，并对外引物进行锁核酸修饰。建立并优化LNA-OSN-PCR反应体系和条件，确立LNA-OSN-PCR标准曲线，并检验LNA-OSN-PCR方法的灵敏度、特异性和重复性。采用LNA-OSN-PCR方法，对96份临床疑似患病样品进行检测，同时与普通荧光探针PCR方法进行比较。试验确立并优化了LNA-OSN-PCR的反应条件和体系，建立的LNA-OSN-PCR标准曲线，具有较好的线性(R²=0.9945)。该方法的最低检测限为3×10^-1 copies/μL，变异系数小于3%，并且与其他病毒或细菌核酸无交叉反应。LNA-OSN-PCR方法与OIE推荐的荧光定量PCR方法比较，检测灵敏度提高10~100倍，在对96份临床疑似患病核酸样品进行检测中，检出55份阳性，41份阴性，比常规的荧光定量PCR具有更高的阳性检出率。并且能获得典型的扩增曲线。试验结合一步法巢式PCR和锁核酸修饰，建立了ASFV一步法巢式锁核酸荧光定量PCR，为检测ASFV提供一种高灵敏度和高特异性且经济实惠的检测方法。

关键词: 非洲猪瘟病毒, B646L(P72)蛋白基因, 锁核酸修饰, 一步法巢式荧光定量PCR

Abstract:

In this study, a highly sensitive and specific detection method was established based on the B646L (P72) protein gene of African swine fever virus (ASFV). The nested primers and probes of LNA-OSN-PCR (One Step Nested-Locked Nucleic Acid real-time PCR) were designed with Oligo 7 software; external primers were modified with locked nucleic acids. The reaction system and conditions of LNA-OSN-PCR were established and optimized, and the standard curve of LNA-OSN-PCR was established. Then, the sensitivity, specificity and repeatability of LNA-OSN-PCR were tested. A total of 96 clinical suspected diseased samples were detected by LNA-OSN-PCR and compared with the samples detected by conventional fluorescent PCR method. The reaction conditions and system of LNA-OSN-PCR were established and optimized, and the standard curve of LNA-OSN-PCR had good linearity (R²=0.9945). The LNA-OSN-PCR had a minimum detection limit of 3×10^-1 copies/μL, with a coefficient of variation of less than 3% and no cross-reaction with other viral or bacterial nucleic acids. Compared with the qPCR method recommended by OIE, the detection sensitivity of the LNA-OSN-PCR method was increased by 10-100 times. Among the 96 samples tested by LNA-OSN-PCR, 55 were positive and 41 were negative, with a higher positive detection rate than traditional qPCR and a better typical amplification curve. By combining the one-step nested PCR and lock-in nucleic acid modification, the LNA-OSN-PCR method for ASFV was established to provide a highly sensitive, specific and economical method for ASFV detection.

Key words: African swine fever virus, B646L (P72) protein gene, locked nucleic acid modified, one-step nested real-time PCR

施科达, 徐民生, 李艳, 张昆丽, 翟少伦, 勾红潮, 宋帅, 杨冬霞, 臧莹安, 孙铭飞, 李春玲. 非洲猪瘟病毒一步法巢式锁核酸荧光定量PCR方法的建立[J]. 中国农学通报, 2023, 39(11): 143-151.

SHI Keda, XU Minsheng, LI Yan, ZHANG Kunli, ZHAI Shaolun, GOU Hongchao, SONG Shuai, YANG Dongxia, ZANG Ying'an, SUN Mingfei, LI Chunling. Development of One-Step Nested-Locked Nucleic Acid Real-Time PCR Detection Method for African Swine Fever Virus[J]. Chinese Agricultural Science Bulletin, 2023, 39(11): 143-151.

图/表 8

图1. 非洲猪瘟病毒B646L(P72)基因的LNA-OSN-PCR嵌套内、外引物和探针设计示意图

	引物和探针	序列(5’-3’)
LNA-OSN-PCR	内引物F1	ATAGATGAACATGCGTCTGG
	内引物R1	CAAAATCCTCATCAACACCG
	外引物F2	TG(+)^*GTATTCC(+)TCCC(+)GTG(+)GC(+)TTC
	外引物R2	C(+)CCCAGT(+)AGA(+)CGC(+)AATA(+)TACGC
	探针	FAM-CTGAAAGCTTATCTCTGCGTGGT-BHQ1
荧光定量PCR	VP72-F1	GCTTTCAGGATAGAGATACAGCTCT
	VP72-R1	CCGTAGTGGAAGGGTATGTAAGAG
	VP72-T1	FAM-CCGTAACTGCTCATGGTATCAATCTTATCG-BHQ1

表1. LNA-OSN-PCR嵌套内、外引物，锁核酸修饰位点和探针核酸序列

图2. 内、外引物梯度退火温度凝胶电泳成像 A：LNA修饰的外引物不同退火温度PCR凝胶电泳结果；B：内引物不同退火温度PCR凝胶电泳结果

图3. 内引物和探针不同浓度的扩增曲线 A：200~600 nmol/L各浓度引物的荧光PCR扩增曲线；B：100~500 nmol/L各浓度探针的荧光PCR扩增曲线

图4. LNA-OSN-PCR和荧光探针PCR标准曲线 A：LNA-OSN-PCR荧光扩增曲线；B：荧光探针PCR扩增曲线；C：LNA-OSN-PCR和荧光探针PCR标准曲线；NC，阴性对照

P72蛋白标准质粒/ (copies/μL)	组内		组间(Ct值)		置信区间(95%)
P72蛋白标准质粒/ (copies/μL)	$\bar{x}±s$ (Ct值)	变异系数/%	$\bar{x}±s$ (Ct值)	变异系数/%	置信区间(95%)
3×10³	18.56±0.12	0.62	18.79 ± 0.46	2.45	1.84~18.9
3×10²	21.51±0.10	0.48	21.67 ± 0.64	2.95	21.3~21.9
3×10¹	24.84±0.05	0.21	24.97 ± 0.48	1.90	24.7~25.1
3×10⁰	27.83±0.19	0.67	28.21 ± 0.14	0.50	27.9~28.2
3×10^-1	30.08±0.06	0.21	30.66 ± 0.77	2.50	29.9~30.8

表2. LNA-OSN-PCR重复性试验

非洲猪瘟检测	一步法巢式锁核酸荧光定量PCR (LNA-OSN-PCR)	荧光探针PCR
阳性^*/份	55	47
阴性/份	41	49
合计/份	96	96
阳性检出率	57.3%	49.0%

表3. LNA-OSN-PCR的临床样品检测

图5. LNA-OSN-PCR和荧光探针PCR的临床样品检测比较 A：临床样品LNA-OSN-PCR与荧光探针PCR的Ct值比较；蓝色圆点为荧光探针PCR，红色三角形为LNA-OSN-PCR；上下点为同一样品；B：部分临床样品的LNA-OSN-PCR的扩增曲线；C：部分临床样品的荧光探针PCR扩增曲线

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非洲猪瘟病毒一步法巢式锁核酸荧光定量PCR方法的建立

Development of One-Step Nested-Locked Nucleic Acid Real-Time PCR Detection Method for African Swine Fever Virus

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