猪圆环病毒2型和3型双重荧光定量PCR检测方法的建立

doi:10.3969/j.issn.1004-1524.2022.03.05

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (3): 457-463.DOI: 10.3969/j.issn.1004-1524.2022.03.05

猪圆环病毒2型和3型双重荧光定量PCR检测方法的建立

牟泓晔¹(), 周小杰¹, 杨永春¹, 王晓杜¹, 周莹珊¹^,²^,^*(), 宋厚辉¹^,^*

1.浙江农林大学动物科技学院·动物医学院,浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,动物健康互联网检测技术浙江省工程研究中心,浙江杭州 311300
2.浙江农林大学动物科技学院·动物医学院,浙江省动物医学与健康管理国际科技合作基地,中澳动物健康大数据分析联合实验室, 浙江杭州 311300

收稿日期:2020-11-06 出版日期:2022-03-25 发布日期:2022-03-30
通讯作者: 周莹珊,宋厚辉
作者简介:*宋厚辉,E-mail: songhh@zafu.edu.cn
牟泓晔(1995—),女,山东威海人,硕士研究生,主要从事猪病毒病的研究。E-mail: 1014475543@qq.com
基金资助:
国家自然科学基金(31902249);浙江省基础公益研究计划(LQ19C180003);浙江省重点研发计划(2019C02043);浙江省重点研发计划(2019C02052);浙江省重点研发计划(2018C02028)

Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3

MOU Hongye¹(), ZHOU Xiaojie¹, YANG Yongchun¹, WANG Xiaodu¹, ZHOU Yingshan¹^,²^,^*(), SONG Houhui¹^,^*

1. Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China
2. Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China

Received:2020-11-06 Online:2022-03-25 Published:2022-03-30
Contact: ZHOU Yingshan,SONG Houhui

摘要/Abstract

摘要：

为建立一种同时检测猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)的方法,参照GenBank上已登录的PCV2 Cap基因和PCV3 Cap基因的保守序列,设计特异性引物和TaqMan探针,通过优化反应条件,建立了同时检测PCV2和PCV3的双重荧光定量PCR检测方法,并对其进行特异性、灵敏性、可重复性检验。特异性试验结果显示,该方法除了对PCV2和PCV3的检测结果为阳性外,对猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪细小病毒(PPV)、伪狂犬病病毒(PRV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)检测均呈阴性,无交叉反应,特异性较强。敏感性试验结果显示,该检测方法同时检测猪圆环病毒2型和3型质粒标准品的最低检测极限均可达到10 拷贝·μL^-1,敏感性较高;该方法组内和组间的变异系数均小于2%,重复性较好。采集浙江省181份猪肉及全血样品,分别利用本文建立的检测方法与标准普通荧光PCR检测方法进行检测,结果显示,PCV2的阳性率为50.83%(92/181),PCV3的阳性率为37.57%(68/181),PCV2和PCV3共感染率为12.15%(22/181);而普通荧光PCR的上述检测结果分别为50.28%(91/181)、36.46%(66/181),共感染率为11.60%(21/181),两种方法对PCV2和PCV3的检测符合率分别可达98.91%和97.06%,对PCV2和PCV3混合感染符合率为95.45%。综上所述,本试验建立的双重荧光定量PCR检测方法可同时对PCV2和PCV3进行快速鉴别检测,可用于病原学检测和流行病学调查等。

关键词: 猪圆环病毒2型, 猪圆环病毒3型, TaqMan探针, 双重荧光定量PCR

Abstract:

To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3),specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested.Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies·μL^-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181),the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181).The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181).The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.

Key words: porcine circovirus 2, porcine circovirus 3, TaqMan probe, duplex fluorescence quantitative PCR

中图分类号:

S852.65

牟泓晔, 周小杰, 杨永春, 王晓杜, 周莹珊, 宋厚辉. 猪圆环病毒2型和3型双重荧光定量PCR检测方法的建立[J]. 浙江农业学报, 2022, 34(3): 457-463.

MOU Hongye, ZHOU Xiaojie, YANG Yongchun, WANG Xiaodu, ZHOU Yingshan, SONG Houhui. Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3[J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 457-463.

图/表 7

图1 目的基因的PCR扩增结果 M,DL2000 DNA分子质量标记;1,PCV3基因;2,PCV2基因。

Fig.1 Amplification of the target genes by PCR M, DL2000 DNA Marker;1,PCV3 gene; 2,PCV2 gene.

图2 TaqMan荧光定量PCR检测标准曲线

Fig.2 The standard curve of TaqMan fluorescence quantitative PCR

图3 双重荧光定量PCR特异性检测结果 1,猪圆环病毒2型;2,猪圆环病毒3型;3,猪繁殖与呼吸综合征病;4,伪狂犬病病毒;5,猪细小病毒;6,猪瘟病毒(CSFV);7,猪流行性腹泻病毒;8,猪传染性胃肠炎病毒;9,ddH2O。

Fig.3 Specificity detection results of duplex fluorescence quantitative PCR 1,PCV2; 2, PCV3; 3,PRRSV; 4,PRV; 5,PPV; 6,CSFV; 7,PEDV; 8,TGEV; 9,ddH2O.

图4 双重荧光定量PCR敏感性检测结果 1~8,pMD-PCV2质粒浓度为1.0×108~1.0×101拷贝·μL-1;9,PCV2阴性对照;a~h,pMD-PCV3质粒浓度为1.0×108~1.0×101拷贝·μL-1;i,PCV3阴性对照。下同。

Fig.4 Sensitivity detection results of duplex fluorescence quantitative PCR 1-8,1.0×108-1.0×101 copies·μL-1 of pMD-PCV2; 9,PCV2 negative control; a-h,1.0×108-1.0×101 copies·μL-1 of pMD-PCV3; i, PCV3 negative control.The same as below.

图5 普通PCR敏感性检测结果 1~8,pMD-PCV2质粒浓度为1.0×108~1.0×101拷贝·μL-1;9,PCV2阴性对照;a~h,pMD-PCV3质粒浓度为1.0×108~1.0×101拷贝·μL-1;i,PCV3阴性对照。

Fig.5 Sensitivity detection results of ordinary PCR 1-8, 1.0×108-1.0×101copies·μL-1 of pMD-PCV2; 9,PCV2 negative control; a-h,1.0×108-1.0×101copies·μL-1 of pMD-PCV3; i, PCV3 negative control.

表1 PCV2与PCV3标准品可重复性试验

Table 1 Reproducibility test of PCV2 and PCV3 standard products

质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	组内重复试验(Ct) Intra-assay Ct value		组间重复试验(Ct) Inter-assay Ct value
质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	平均值 $x -$ ±SD	变异系数CV/%	平均值 $x -$ ±SD	变异系数CV/%
PCV2	10⁴	28.24±0.15	0.53	27.90±0.19	0.68
	10³	31.67±0.37	1.17	32.06±0.11	0.34
	10²	34.49±0.29	0.84	34.02±0.44	1.29
PCV3	10⁴	26.53±0.13	0.49	27.27±0.16	0.59
	10³	29.15±0.12	0.41	29.21±0.12	0.41
	10²	32.19±0.09	0.28	32.59±0.14	0.43

表1 PCV2与PCV3标准品可重复性试验

Table 1 Reproducibility test of PCV2 and PCV3 standard products

质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	组内重复试验(Ct) Intra-assay Ct value		组间重复试验(Ct) Inter-assay Ct value
质粒标准品 Plasmid standard	终浓度 Concentration/ (copies·μL^-1)	平均值 $x -$ ±SD	变异系数CV/%	平均值 $x -$ ±SD	变异系数CV/%
PCV2	10⁴	28.24±0.15	0.53	27.90±0.19	0.68
	10³	31.67±0.37	1.17	32.06±0.11	0.34
	10²	34.49±0.29	0.84	34.02±0.44	1.29
PCV3	10⁴	26.53±0.13	0.49	27.27±0.16	0.59
	10³	29.15±0.12	0.41	29.21±0.12	0.41
	10²	32.19±0.09	0.28	32.59±0.14	0.43

表2 临床样品的检测结果

Table 2 Test results of clinical samples

检测方法 Method	PCV2		PCV3		共感染 Co- infection	共感染率 Co-infection rate/%
检测方法 Method	阳性样品 Number of positive samples	检出率 Detection rate/%	阳性样品 Number of positive samples	检出率 Detection rate/%	共感染 Co- infection	共感染率 Co-infection rate/%
双重荧光定量PCR Double fluorescence quantitative PCR	92	50.83	68	37.57	22	12.15
普通荧光定量PCR Ordinary fluorescence quantitative PCR	91	50.28	66	36.46	21	11.60
符合率Coincidence rate/%	98.91	97.06	95.45

参考文献 15

[1]	XIAO C T, HALBUR P G, OPRIESSNIG T. Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d[J]. The Journal of General Virology, 2015, 96(Pt 7): 1830-1841. DOI URL
[2]	DARWICH L, SEGALÉS J, MATEU E. Pathogenesis of postweaning multisystemic wasting syndrome caused by porcine circovirus2: an immune riddle[J]. Archives of Virology, 2004, 149(5): 857-874. DOI URL
[3]	PALINSKI R, PIÑEYRO P, SHANG P C, et al. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J]. Journal of Virology, 2017, 91(1): DOI: 10.1128/jvi.01879-16. DOI
[4]	沈顺新, 和玉丹. 猪圆环病毒病的危害及其防控[J]. 今日畜牧兽医, 2020, 36(4): 27-28.
	SHEN S X, HE Y D. The harm of porcine circovirus disease and its prevention and control[J]. Animal Husbandry and Veterinary Today, 2020, 36(4): 27-28. (in Chinese)
[5]	杨威, 田青, 赵兴华, 等. 猪圆环病毒3型PCR检测方法的建立及应用[J]. 中国动物传染病学报, 2018, 26(2):9-14.
	YANG W, TIAN Q, ZHAO X H, et al. Establishment and application of PCR detection method for porcine circovirus type 3[J]. Chinese Journal of Zoological Infectious Diseases, 2018, 26(2):9-14. (in Chinese with English abstract)
[6]	邓同炜, 薛亚南, 卢建洲, 等. 猪繁殖与呼吸综合征病毒、猪圆环病毒2型与副猪嗜血杆菌混合感染的诊断及病原分析[J]. 中国畜牧兽医, 2020, 47(10): 3401-3409.
	DENG T W, XUE Y N, LU J Z, et al. Diagnosis and pathogen analysis of mixed infection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and Haemophilusparasuis[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(10): 3401-3409. (in Chinese with English abstract)
[7]	季程远, 王蔚怡, 黄欣, 等. 猪圆环病毒2型和3型双重PCR检测方法的建立与应用[J]. 中国预防兽医学报, 2018, 40(10): 913-916.
	JI C Y, WANG W Y, HUANG X, et al. Establishment and application of a duplex PCR for detection of porcine circovirus 2 and porcine circovirus 3[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(10): 913-916. (in Chinese with English abstract)
[8]	吕素芳, 李峰, 郭广君, 等. 猪圆环病毒3型SYBR greenⅠ荧光定量PCR方法建立及初步应用[J]. 中国兽医学报, 2019, 39(7): 1250-1255.
	LYU S F, LI F, GUO G J, et al. Preliminary application and development of SYBR green Ⅰ fluorescent quantitative PCR method for detection of type 3 porcine circovirus[J]. Chinese Journal of Veterinary Science, 2019, 39(7): 1250-1255. (in Chinese with English abstract)
[9]	庄金秋, 梅建国, 张颖, 等. 猪圆环病毒2型Cap蛋白琼脂扩散试验检测方法的建立[J]. 中国动物检疫, 2020, 37(9): 113-117.
	ZHUANG J Q, MEI J G, ZHANG Y, et al. Establishment of an agar-gel precipitation test for PCV2 Cap protein[J]. China Animal Health Inspection, 2020, 37(9): 113-117. (in Chinese with English abstract)
[10]	张禹, 张韵, 孙世琪, 等. 猪圆环病毒2型抗体胶体金免疫层析检测方法的建立[J]. 中国预防兽医学报, 2019, 41(7): 705-709.
	ZHANG Y, ZHANG Y, SUN S Q, et al. Establishment of colloidal gold immunochromatography for porcine circovirus type 2 antibody[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(7): 705-709. (in Chinese with English abstract)
[11]	刘雨丰, 马攀攀, 陈陆, 等. 猪圆环病毒2型特异性IgA间接ELISA检测方法的建立与初步应用[J]. 中国预防兽医学报, 2012, 34(4): 301-304.
	LIU Y F, MA P P, CHEN L, et al. Development and primary application of indirect ELISA method for detecting the IgA against porcine circovirus type 2[J]. Chinese Journal of Preventive Veterinary Medicine, 2012, 34(4): 301-304. (in Chinese with English abstract)
[12]	ROSELL C, SEGALÉS J, RAMOS-VARA J A, et al. Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome[J]. The Veterinary Record, 2000, 146(2): 40-43. DOI URL
[13]	GUO Z H, RUAN H Y, QIAO S L, et al. Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) in pigs with watery diarrhea in Henan Province, central China[J]. Microbial Pathogenesis, 2020, 142: 104047. DOI URL
[14]	XIA D L, HUANG L P, XIE Y X, et al. The prevalence and genetic diversity of porcine circovirus types 2 and 3 in Northeast China from 2015 to 2018[J]. Archives of Virology, 2019, 164(10): 2435-2449. DOI URL
[15]	WANG Y, NOLL L, LU N Y, et al. Genetic diversity and prevalence of porcine circovirus type 3 (PCV3) and type 2 (PCV2) in the Midwest of the USA during 2016-2018[J]. Transboundary and Emerging Diseases, 2020, 67(3): 1284-1294. DOI URL

猪圆环病毒2型和3型双重荧光定量PCR检测方法的建立

Establishment of a duplex fluorescent quantitative PCR assay for detection of porcine circovirus type 2 and type 3

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 15

相关文章 4

编辑推荐

Metrics

本文评价

[1]	徐丽华, 蓝胜芝, 余斌, 李军星, 张鹏超, 李宝臣, 苏菲, 袁秀芳. 浙江地区猪圆环病毒2型检测及遗传变异分析[J]. 浙江农业学报, 2020, 32(11): 1970-1977.
[2]	赵青. 猪圆环病毒疫苗对仔猪生产性能的效果评估[J]. 浙江农业学报, 2018, 30(10): 1662-1664.
[3]	王净修;高章照;姜永厚;*;吴海港;饶品彬;全滟平;徐辉. 猪圆环病毒2型ORF4基因的原核表达及多克隆抗体的制备[J]. , 2014, 26(1): 0-39.
[4]	申世川;王一成;*;袁秀芳;徐丽华;李军星. 猪圆环病毒2型LAMP检测方法的建立[J]. , 2013, 25(1): 0-26.