基于毛细管电泳的7种猪源性疫病的多重PCR检测方法的建立

doi:10.3969/j.issn.1004-1524.2021.04.07

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (4): 618-631.DOI: 10.3969/j.issn.1004-1524.2021.04.07

基于毛细管电泳的7种猪源性疫病的多重PCR检测方法的建立

涂藤¹^,²(), 尹清清¹^,², 张鹏飞¹^,², 王印¹^,²^,^*(), 杨泽晓¹^,², 姚学萍¹^,², 罗燕¹^,²

1.四川农业大学动物医学院,四川成都 611130
2.动物疫病与人类健康四川省重点实验室,四川成都 611130

收稿日期:2020-05-22 出版日期:2021-04-25 发布日期:2021-04-25
通讯作者: 王印
作者简介:*王印,E-mail:yaanwangyin@tom.com
涂藤(1997—),男,四川成都人,硕士研究生,主要从事动物传染病病原分子生物学研究。E-mail:309349398@qq.com
基金资助:
国家农业产业技术体系四川兽药创新团队专项(CARS-SVDIP);国家重点研发计划(2018YFD0501102)

Establishment and application of multiplex PCR-capillary electrophoresis for 7 swine diseases

TU Teng¹^,²(), YIN Qingqing¹^,², ZHANG Pengfei¹^,², WANG Yin¹^,²^,^*(), YANG Zexiao¹^,², YAO Xueping¹^,², LUO Yan¹^,²

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611130, China

Received:2020-05-22 Online:2021-04-25 Published:2021-04-25
Contact: WANG Yin

摘要/Abstract

摘要：

为建立一种基于毛细管电泳技术,可同时高效地检测7种常见猪源性疫病(猪链球菌、猪瘟病毒、日本脑炎病毒、猪繁殖与呼吸综合征病毒、副猪嗜血杆菌、猪轮状病毒、沙门氏菌)的高通量快速检测方法,本研究将多重PCR与毛细管电泳相结合,针对各毒株基因保守序列分别设计7对特异性引物,在每条引物的5'端加上通用引物标签序列,构成特异性嵌合引物。进行梯度PCR扩增以优化退火温度,使用正交试验以进行特异性嵌合引物浓度的优化。取10⁷~10^-1 copies·μL^-1浓度梯度的标准品进行检测,以评价该方法的灵敏性。选取7种病原的DNA或cDNA,进行随机分组作为模板进行扩增,检测交叉反应性。以猪流行性腹泻病毒、猪传染性胃肠炎病毒、构建的非洲猪瘟质粒、大肠埃希菌等其他重要病原的DNA或cDNA进行扩增,评价该方法的特异性。使用梯度稀释的7重混合样品进行扩增,每个稀释度进行3次扩增,以评价该方法的重复性。结果显示:该方法的最优退火温度为58.3 ℃,对7种病原进行同时检出的检测限度为10³ copies·μL^-1,各种对照样品均出现了特异性峰值,并且所有的阴性对照样品均未扩增,也未出现交叉反应性,重复试验之间数据差异变化微小,63份临床样品检测结果与国标或行业检测标准的检测结果完全一致。以上实验结果显示,所建立的方法具有高通量、特异性好、灵敏度高、方便快速等优点,可用于临床鉴别诊断和流行病学调查,有较高的临床应用价值。

关键词: 猪源性疫病, 毛细管电泳, 高通量检测

Abstract:

The aim of this study was to develop a multiple PCR assay on a basis of capillary electrophoresis for simultaneous detection of seven pathogens of swine infectious diseases, including Streptococcus suis(SS), Classical swine fever virus(CSFV), Japanese encephalitis virus(JEV), Porcine reproductive and respiratory syndrome virus(PRRSV), Haemophilus parasuis(HPS), Porcine rotavirus(PoRV), Salmonella(SE). Firstly the conservative sequences of the 7 reference pathogens were analyzed to design 7 pairs of specific primers. Subsequently, the specific chimeric primers were obtained by adding universal primer sequences in 5' end of each specific primer. Gradient PCR amplification was adopted to optimize annealing temperature. The primer concentrations were optimized by orthogonal test. The sensitivity test was performed by using serially diluted standards (10 ⁷-10^-1 copies·μL^-1). Cross reactivity test was performed by using random mixed templates that orginate from cDNA or DNA of these 7 pathogens. Specificity test was performed by detection of some other common pathogens, including PEDV, TGEV, ASFV plasmid, E. coli. Repeatability test was performed by using serially diluted standards as templates to be amplified triplicates. The results indicated that the optimum annealing temperature was 58.3 ℃. Detection limit of this assay was 10 ³ copies·μL^-1, when 7 positive results were visible from identical lane. With a variety of control samples, the results of positive samples showed specificity peak in contrast of the negative results of all the negative control samples. In addition, neither cross reactivity nor unspecific amplification were detected within these 7 kinds of random mixed templates. the differences among triplicate experiments were small and low. The test results of the 63 clinical samples were exactly the same with the results obtained by national standards. As is indicated in the results, it is an effective toll applied to differential diagnosis rapidly for clinical samples and epidemiological investigation.

Key words: swine infectious diseases, capillary electrophoresis, high throughput detection

中图分类号:

S852.3

涂藤, 尹清清, 张鹏飞, 王印, 杨泽晓, 姚学萍, 罗燕. 基于毛细管电泳的7种猪源性疫病的多重PCR检测方法的建立[J]. 浙江农业学报, 2021, 33(4): 618-631.

TU Teng, YIN Qingqing, ZHANG Pengfei, WANG Yin, YANG Zexiao, YAO Xueping, LUO Yan. Establishment and application of multiplex PCR-capillary electrophoresis for 7 swine diseases[J]. Acta Agriculturae Zhejiangensis, 2021, 33(4): 618-631.

图/表 15

表1 毛细管电泳多重PCR所用引物

Table 1 Primers used in capillary electrophoresis multiplex PCR

病原 Pathogen	引物 Primer sequences (5'-3')	靶基因 Target gene	大小 Size/bp
SS CSFV JEV PRRSV HPS PoRV SE	F:AGGTGACACTATAGAATAATAATCCGCCAGCAACAAC R:GTACGACTCACTATAGGGATCAGCACGCAATAAAGCAC F:AGGTGACACTATAGAATATGCCCGTTTGATACGAGTCC R:GTACGACTCACTATAGGGAGGTCTTTACCACTTCTGTCC F:AGGTGACACTATAGAATAGCAAACGACAAACCAACACTA R:GTACGACTCACTATAGGGACAATGCTTCCTTTCCCGA F:AGGTGACACTATAGAATAACCTCCAGATGCCGTTTGT R:GTACGACTCACTATAGGGAACAAGGTTTACCACTCCCT F:AGGTGACACTATAGAATAGGGATTAGGTAGTTGGTGGGG R:GTACGACTCACTATAGGGACTGTGACTAACGTCAATGTAATGCT F:AGGTGACACTATAGAATATATCCTACCGAAGCATCAACTC R:GTACGACTCACTATAGGGAAAGATTGTCCCATCGATATCCA F:AGGTGACACTATAGAATACCCTTTGCGAATAACATCC R:GTACGACTCACTATAGGGATTGGCTATGTGTTGCGGA	SLY E2 E M 16S VP7 Inva	160 193 235 261 292 324 348

表2 嵌合引物浓度优化正交试验方案

Table 2 Orthogonal experimental scheme of chimeric primer concentrations

因素 Elements	嵌合引物使用浓度Concentration of chimeric primers/(μmol·L^-1)
因素 Elements	SS	CSFV	JEV	PRRSV	HPS	PoRV	SE
实验1 Test 1	1	1	1	1	1	1	1
实验2 Test 2	1	1.25	1	1	0.75	1.25	1.25
实验3 Test 3	1	1.5	1	1	0.75	1.5	1.5
实验4 Test 4	1.25	1	1	1	0.75	1.5	1.5
实验5 Test 5	1.25	1.25	1	1	0.75	1	1
实验6 Test 6	1.25	1.5	1	1	1	1.25	1.25
实验7 Test 7	1.5	1	1	1	0.75	1.25	1.5
实验8 Test 8	1.5	1.25	1	1	1	1.5	1
实验9 Test 9	1.5	1.5	1	1	0.75	1	1.25
实验10 Test 10	1	1	1	1	0.75	1.25	1
实验11 Test 11	1	1.25	1	1	0.75	1.5	1.25
实验12 Test 12	1	1.5	1	1	1	1	1.5
实验13 Test 13	1.25	1	1	1	1	1.5	1.25
实验14 Test 14	1.25	1.25	1	1	0.75	1	1.5
实验15 Test 15	1.25	1.5	1	1	0.75	1.25	1
实验16 Test 16	1.5	1	1	1	0.75	1	1.25
实验17 Test 17	1.5	1.25	1	1	1	1.25	1.5
实验18 Test 18	1.5	1.5	1	1	0.75	1.5	1

表3 临床样品说明

Table 3 Statement of Clinical specimens

分组 Specimen groups	感染情况 Infection conditions	数量 Total No.	采集地点 Collecting place
1	JEV	5	四川遂宁 Suining,Sichuan
2	CSFV	10	四川浦江 Pujiang,Sichuan
3	PRRSV	11	四川雅安 Yaan,Sichuan
4	HPS	8	四川遂宁Suining,Sichuan
5	SS	7	四川雅安Yaan,Sichuan
6	SE	4	四川遂宁Suining,Sichuan
7	JEV+PRRSV	3	四川浦江Pujiang,Sichuan
8	PRRSV+CSFV	2	四川遂宁Suining,Sichuan
9	CSFV+HPS	2	四川成都Chengdu,Sichuan
10	PRRSV+HPS	4	四川雅安Yaan,Sichuan
11	SS+SE	3	四川遂宁Suining,Sichuan
12	CSFV+HPS	2	四川浦江Pujiang,Sichuan
13	CSFV+JEV+PRRSV	1	四川遂宁Suining,Sichuan
14	SE+HPS+SS	1	四川成都 Chengdu,Sichuan
15	Uninfected	3	四川绵阳 Mianyang,Sichuan

图1 特异性嵌合引物单重PCR验证结果 M,DNA相对分子质量标准; 1,SE; 2,PoRV; 3,HPS; 4,PRRSV; 5,JEV; 6,CSFV; 7,SS。

Fig.1 Verified results of specific chimeric primers M, DNA Marker B (100-600 bp); 1, SE; 2, PoRV; 3, HPS; 4, PRRSV; 5, JEV; 6, CSFV; 7, SS.

图2 毛细管电泳单重PCR 1,SS; 2,CSFV; 3,JEV; 4,PRRSV; 5,HPS; 6,PoRV; 7,SE; 8,ddH2O阴性对照; M,QIAxcel Size Marker(25~500 bp)。

Fig.2 Results of capillary electrophoresis simplex PCR 1, SS; 2, CSFV; 3, JEV; 4, PRRSV; 5, HPS; 6, PoRV; 7, SE; 8, ddH2O as negative control; M, QIAxcel Size Marker(25-500 bp).

图3 单重毛细管电泳检测峰图结果 A~D分别为SS、CSFV、JEV、PRRSV的单重检测峰图。横坐标为bp数,纵坐标为相对荧光强度。

Fig.3 Result of capillary electrophoresis simplex PCR A-D indicated detection peak figures of SS, CSFV, JEV, PRRSV. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.

图4 单重毛细管电泳检测峰图结果 A~C分别为HPS、PoRV、SE的单重检测峰图;D,Size Marker对照。横坐标为bp数,纵坐标为相对荧光强度。

Fig.4 Result of capillary electrophoresis simplex PCR A-C indicated detection peak figures of HPS, PoRV, SE. D indicated detection peak figures of Size Marker as control. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.

图5 毛细管电泳多重PCR退火温度优化凝胶图 1,50.7 ℃; 2,52.0 ℃; 3,53.9 ℃; 4,56.3 ℃; 5,58.3 ℃; 6,59.4 ℃; 7,60.0 ℃; 8,阴性对照; M,QIAxcel Size Marker (25~500 bp)。

Fig.5 Gel figure of capillary electrophoresis multiplex PCR annealing temperature test Lane 1, 50.7 ℃; Lane 2, 52.0 ℃; Lane 3, 53.9 ℃; Lane 4, 56.3 ℃; Lane 5, 58.3 ℃; Lane 6, 59.4 ℃; Lane 7, 60.0 ℃; Lane 8, Negative control; Lane M, QIAxcel Size Marker (25-500 bp).

图6 毛细管电泳多重PCR退火温度优化峰图

Fig.6 Result of capillary electrophoresis multiplex PCR annealing temperature test

图7 毛细管电泳多重PCR灵敏性试验结果 M,QIAxcel Size Marker (25~500 bp); 1,107 copies·μL-1; 2,106 copies·μL-1; 3,105 copies·μL-1; 4,104 copies·μL-1; 5,103 copies·μL-1; 6,102 copies·μL-1; 7,101 copies·μL-1; 8,100 copies·μL-1; 9,10-1 copies·μL-1; 10,阴性对照。

Fig.7 Results of capillary electrophoresis multiplex PCR sensitivity test Lane M, QIAxcel Size Marker (25-500 bp); Lane 1, 107 copies·μL-1; Lane 2, 106 copies·μL-1; Lane 3, 105 copies·μL-1; Lane 4, 104 copies·μL-1; Lane 5, 103 copies·μL-1; Lane 6, 102 copies·μL-1; Lane 7, 101 copies·μL-1; Lane 8, 100 copies·μL-1; Lane 9, 10-1 copies·μL-1; Lane 10, Negative control.

图8 毛细管电泳多重PCR特异性试验结果 1,含有7种病原DNA/cDNA的混合液; 2,HPS、PoRV、SE; 3,SS、CSFV; 4,CSFV、JEV; 5,CSFV、PRRSV、HPS; 6,SS、CSFV、PRRSV、HPS; 7,CSFV、PRRSV; 8,阴性对照; M,QIAxcel Size Marker (25~500 bp); 11,E. coli; 12,PPV; 13,ASFV; 14,PEDV; 15,TGEV。

Fig.8 Results of capillary electrophoresis multiplex PCR specificity test Lane 1, Mixed template containing DNA/cDNA of 7 pathogens; Lane 2, HPS, PoRV and SE; Lane 3, SS and CSFV; Lane 4, CSFV and JEV; Lane 5, CSFV, PRRSV and HPS; Lane 6, SS, CSFV, PRRSV and HPS; Lane 7, CSFV and PRRSV; Lane 8, Negative control; Lane M, QIAxcel Size Marker (25- 500 bp); Lane 11, E. coli; Lane 12, PPV; Lane 13, ASFV; Lane 14, PEDV; Lane 15, TGEV.

图9 毛细管电泳多重PCR交叉反应性试验结果 A~C分别为SS+CSFV、CSFV+JEV、CSFV+PRRSV的交叉反应性检测峰图。横坐标为bp数,纵坐标为相对荧光强度。

Fig.9 Results of capillary electrophoresis multiplex PCR cross reactivity test A-C indicated cross reactivity peak figures of SS+CSFV, CSFV+JEV and CSFV+PRRSV. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.

图10 毛细管电泳多重PCR交叉反应性试验结果 A~D分别为SS+CSFV+PRRSV+HPS、CSFV+PRRSV+HPS、HPS+PoRV+SE、HPS+SE的交叉反应性检测峰图。横坐标为bp数,纵坐标为相对荧光强度。

Fig.10 Results of capillary electrophoresis multiplex PCR cross reactivity test A-D indicated cross reactivity peak figures of SS+CSFV+PRRSV+HPS, CSFV+PRRSV+HPS, HPS+PoRV+SE, HPS+SE. The abscissas represented the bp numbers and the ordinates represented the relative intensity of fluorescence.

图11 重复性试验叠加峰图

Fig.11 Overlaided peak figure of repeatability test

表4 毛细管电泳多重PCR临床样品检测结果

Table 4 Results of clinical specimens by capillary electrophoresis multiplex PCR

分组 Specimen groups	国标/行业检测结果Results identified by national standard/insdustry standard							毛细管电泳多重PCR结果 Results of multiplex PCR-CE
分组 Specimen groups	SS	CSFV	JEV	PRRSV	HPS	PoRV	SE	毛细管电泳多重PCR结果 Results of multiplex PCR-CE
1	-	-	+4	-	-	-	-	+5 JEV
2	-	+9	-	-	-	-	-	+10 CSFV
3	-	-	-	+10	-	-	-	+11 PRRSV
4	-	-	-	-	+8	-	-	+8 HPS
5	+7	-	-	-	-	-	-	+7 SS
6	-	-	-	-	-	-	+4	+4 SE
7	-	-	-	-	-	+3	-	+3 PoRV
7	-	-	+3	+3	-	-	-	+3 JEV, PRRSV
8	-	+2	-	+2	-	-	-	+2 PRRSV, CSFV
9	-	+2	-	-	+2	-	-	+2 CSFV, HPS
10	-	-	-	+4	+4	-	-	+4 PRRSV, HPS
11	+3	-	-	-	-	-	+3	+3 SS,SE
12	+2	-	-	-	-	+2	-	+2 PoRV, HPS
13	-	+1	+1	+1	-	-	-	+1 CSFV,JEV,PRRSV
14	+1	-	-	-	+1	-	+1	+1 SE,HPS,SS
3份阴性样品	-	-	-	-	-	-	-	Negative
3 negative samples

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基于毛细管电泳的7种猪源性疫病的多重PCR检测方法的建立

Establishment and application of multiplex PCR-capillary electrophoresis for 7 swine diseases

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