基于纳米孔测序的十种猪病原检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.09.033

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4546-4558.doi: 10.11843/j.issn.0366-6964.2025.09.033

基于纳米孔测序的十种猪病原检测方法的建立

邱话龙传¹^,²(), 金芊芊¹^,², 许潇涵¹^,², 周静², 蔡承志², 李龙¹^,²^,*()

1. 浙江农林大学动物医学院, 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, 浙江省动物医学与健康管理国际科技合作基地, 同一健康和食品安全"一带一路"国际联合实验室, 中澳动物健康大数据分析联合实验室, 杭州 311300
2. 浙江家禾泰弘生物科技有限公司, 杭州 311112

收稿日期:2024-12-04 出版日期:2025-09-23 发布日期:2025-09-30
通讯作者: 李龙 E-mail:2022608031031@stu.zafu.edu.cn;lilong@zafu.edu.cn
作者简介:邱话龙传(1999-)，男，江苏南通人，硕士生，主要从事纳米孔测序在临床方面的应用研究，E-mail: 2022608031031@stu.zafu.edu.cn

Establishment of Ten Swine Pathogens Detection Method based on Nanopore Sequencing

QIU Hualongchuan¹^,²(), JIN Qianqian¹^,², XU Xiaohan¹^,², ZHOU Jing², CAI Chengzhi², LI Long¹^,²^,*()

1. Key Laboratory of Applied Biotechnology on Animal Science & Veterinary Medicine of Zhejiang Province, Zhejiang Engineering Research Center for Veterinary Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, Belt and Road International Joint Laboratory for One Health and Food Safety, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Veterinary Medicine of Zhejiang A&F University, Hangzhou 311300, China
2. Zhejiang Jiahetaihong Biotechnology Co, Ltd, Hangzhou 311112, China

Received:2024-12-04 Online:2025-09-23 Published:2025-09-30
Contact: LI Long E-mail:2022608031031@stu.zafu.edu.cn;lilong@zafu.edu.cn

摘要/Abstract

摘要：

旨在研究一套用于纳米孔靶向测序(nanopore targeting sequencing, NTS)的同时检测10种猪病原的引物组，并以此探索后续在此平台上检测全部猪病原的可能性。用Primer Premier 6软件设计10种病原的引物；用PCR及琼脂糖凝胶电泳方法验证10种引物的特异性；用纳米孔测序技术探索多重PCR体系中的最优引物浓度，通过比对各浓度梯度下的病原检出数量确定最优引物浓度；用纳米孔测序技术验证多重PCR的敏感性，用最优引物浓度对不同浓度梯度的病原模板进行扩增，确定检测敏感性；最后用临床样本(n=201)用荧光定量PCR对NTS进行符合性测试。结果显示：10种猪病病原引物特异，对其他10种猪病原均未扩出条带；反应体系中的引物终浓度在3和5 μmol·L^-1时，能检测到模板中的10种病原；在加入5 μL浓度为9×10² copies·mL^-1的核酸模板时，可检出4组重复中的所有阳性样本；在临床样本的检测上，NTS检测出的病原阳性数量更多。经卡方检验，两者对5种病原的检测结果存在统计学差异(P < 0.05)，另5种不存在统计学差异(P>0.05)；经卡帕值分析，6种病原检测的一致性极好，4种较好。结果显示，NTS检测方法可以同时检测出10种不同的猪病原，对于两种检测方法结果不符合样本需经第三方检测方法进一步验证。

关键词: 纳米孔靶向测序, PCR, 一致性, 猪病原

Abstract:

This study was conducted to develop a primer set for Nanopore targeting sequencing (NTS) to simultaneously detect ten porcine pathogens and explore the potential for detecting all porcine pathogens on this platform. Primers for ten pathogens were designed using Primer Premier 6 software. The specificity of the ten primers was verified by PCR and agarose gel electrophoresis. The optimal primer concentration in the multiplex PCR system was explored using nanopore sequencing technology by comparing the detection numbers of pathogens at different concentration gradients. The sensitivity of multiplex PCR was verified using the optimal primer concentration obtained in the previous step. The detection sensitivity was determined by amplifying pathogen templates at different concentration gradients and analyzing the corresponding results. Finally, a conformity test between fluorescence quantitative PCR and NTS was performed using clinical samples (n=201). The agarose gel electrophoresis images for verifying primer specificity all showed a single, bright, and clear band, and no bands were amplified for the other ten porcine pathogens not selected in this study. All ten pathogens could be detected in the reaction system when the final concentration of primers was 3 and 5 μmol·L^-1, while all other tested concentrations failed to detect all ten pathogens. All positive samples in the four replicates could be detected when 5 μL of the nucleic acid template with a concentration of 9×10² copies·mL^-1 was added. In the detection of clinical samples, more pathogen-positive samples were detected by NTS. After the chi-square test, there were statistically significant differences in the detection results of five pathogens (P < 0.05), while no statistically significant differences were found in the detection results of the other five pathogens (P>0.05). After kappa value analysis, excellent consistency was observed in the detection of six pathogens, and good consistency was observed in the detection of four pathogens. The results indicate that the NTS detection method can simultaneously detect ten different porcine pathogens. For samples with inconsistent detection results compared to the control method, further verification by a third-party detection method is required.

Key words: nanopore targeted sequencing, PCR, consistency, porcine pathogens

中图分类号:

S854.4

邱话龙传, 金芊芊, 许潇涵, 周静, 蔡承志, 李龙. 基于纳米孔测序的十种猪病原检测方法的建立[J]. 畜牧兽医学报, 2025, 56(9): 4546-4558.

QIU Hualongchuan, JIN Qianqian, XU Xiaohan, ZHOU Jing, CAI Chengzhi, LI Long. Establishment of Ten Swine Pathogens Detection Method based on Nanopore Sequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4546-4558.

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病原体名称 Pathogen name	引物序列(5′→3′) Primer sequence	GenBank号 GenBank Number	基因名称 Gene name	引物位置 Primer position	长度/bp Length
APP	F: AAATAATGGGAATAACCAAAATA R: TAAAACGGGTTAAAACTGAAATT	GQ332268.1	CVCC 263 ApxIVA	1 818-1 840 2 413-2 435	618
MHP	F: CTGATAACATCCGAATTATCCGGG R: TATTCTCCTCGTAAATTTGCTC	KY696715.1	TZ-1 L-lactate dehydrogenase (ldh)	236-259 722-743	508
PRRSV-2	F: GTACTGCGGCGCGGAATCGAGC R: CTCTGGCCCACATTTTATCAAGC	PQ306310.1	SHEU-2022	2 582-2 603 3 068-3 090	511
PRRSV-1	F: CCTTGGTCGCTACACCAATGTTGCC R: GCTTGTTTGACCCCGTGTGGACGC	MK057532.1	KU-N1712	4 986-5 010 5 567-5 590	604
PCV2	F: CTCCTAGATCTCAAGGACAGCGGAG R: AGAGAGCTTCTACAGCTGGGAC	MK347388.1	BX3109	376-400 852-873	497
PRV	F: CCATCTGGTGAACGTGTCCGAGG R: AGTCGCCCATGTCCGAGACCACG	MT459823.1	SD-H glycoprotein E (gE)	252-274 744-766	514
Li	F: GGTATACCATAGGCAAATCGC R: ATACGCTACGAGATGAATGG	CP004029.1	N343	1 261 490-1 261 510 1 262 219-1 262 200	710
PCV3	F: CCGCGTGATTTTAAAACTGAAGTTT R: GGGGCTGCCCACCCACCCACCCA	OR758934.1	PCV3/CH/ Guangxi-21	678-702 1 156-1 178	500
Er	F: TGCTTGTGTTGTGATTTCTTGACG R: ACAGGACTCGCATGTTTTACAGGT	CP017116.1	WH13013	946 023-946 046 946 506-946 529	506
TGEV	F: CAACAAATCTTTTTGAAGGTGATAA R: TCTTAGAAACTGAAAAGTTGTGAA	MZ368889.1	HB-1	8 653-8 677 9 133-9 156	503

病原体名称 Pathogen name	引物序列(5′→3′) Primer sequence	方法来源 Method source
APP	F: GGGGACGTAACTCGGTGATT R: GCTCACCAACGTTTGCTCAT P: FAM-CGGTGCGGACACCTATATCT-BHQ1	NY/T 537—2023
MHP	/	产品货号：BTN15-74120 Product item number： BTN15-74120 猪肺炎支原体荧光定量 PCR试剂盒(探针法) Mycoplasma hyopneumoniae fluorescence quantitative PCR kit (probe method)
PRRSV-2	F: GCACTGATTGACAYTGTGCC R: CGCATGGTTCTCGCCAAT P: FAM-AGTCACCTATTCAATTAGGGCGACCG-TAMRA	GB/T 18090—2023
PRRSV-1	F: CAGATGCAGAYTGTGTTGCCT R: TGGAGDCCTGCAGCACTTTC P: FAM-ATACATTCTGGCCCCTGCCCAYCACGT-TAMRA	GB/T 18090—2023
PCV2	F: GGAGTCTGGTGACCGTTGC R: CCAATCACGCTTCTGCATTTT P: FAM-CCGCTCACTTTCAAAAGTTCAGCCA-BHQ1	GB/T 35901—2018
PRV	F: GCTGTACGTGCTCGTGAT R: TCAGCTCCTTGATGACCGTGA P: HEX-CACAACGGCCACGTCGCCACCTG-BHQ1	GB/T 35911—2018
Li	F: GAGCACGATTACAAATTGCTTCA R: TGCTATCTCTGCTGCATGTAATGA P: FAM-TCCCTGCACCTCCTTGAATACAATCCACA-TAMRA	SN/T 3488—2013
PCV3	F: GGGAAAGCCCGAAACACA R: ACTGCCTCCACACTCCACAAT P: HEX-TACGATAAACAACTGGACCCCGACCGA-BHQ1	DB 31/T 955—2022
Er	F: GTGGCGCATATAATCCCGTA R: GCAAACGCAATGGCTTCT	DB 34/T 3284—2018
TGEV	F: AAGGAAGATGGCGACCAGATAG R: CACTTCTGATGGGCGAGCAT P: HEX-CACGTTCACACACAAAT-MGB	DB33/T 2254—2020

病原名称 The name of the pathogen	NTS与qPCR共同检出阳性占比 The positive detection rate of NTS and qPCR combined	仅通过NTS检出阳性占比 Proportion of positives detected by NTS only	仅通过qPCR检出阳性占比 Proportion of positives detected by qPCR only	NTS与qPCR共同检出阴性占比 The proportion of negative NTS and qPCR co-detection
APP	24.4(49/201)	2.5(5/201)	2.5(5/201)	70.6(142/201)
MHP	20.4(41/201)	2.0(4/201)	1.5(3/201)	76.1(153/201)
PRRSV-2	10.0(20/201)	6.0(12/201)	1.5(3/201)	82.6(166/201)
PRRSV-1	1.0(2/201)	2.0(4/201)	0.5(1/201)	96.5(194/201)
PCV2	23.9(48/201)	6.5(13/201)	1.0(2/201)	68.7(138/201)
PRV	1.5(3/201)	0.5(1/201)	0.0(0/201)	98.0(197/201)
Li	4.5(9/201)	8.5(17/201)	2.0(4/201)	85.1(174/201)
PCV3	58.7(118/201)	9.0(18/201)	1.0(2/201)	31.3(63/201)
Er	54.2(109/201)	4.5(9/201)	4.0(8/201)	37.3(75/201)
TGEV	2.5(5/201)	4.5(9/201)	0.0(0/201)	93.0(187/201)

病原名称 The name of the pathogen	阳性符合率/% Positive coincidence rate	阴性符合率/% Negative coincidence rate	总符合率/% Total coincidence rate	卡方检验 Chi-square test	卡帕值 Kappa value
APP	90.74 (49/54)	96.60 (142/147)	95.02 (191/201)	P＞0.05	0.873 4
MHP	93.18 (41/44)	97.45 (153/157)	96.52 (194/201)	P＞0.05	0.899 0
PRRSV-2	86.96 (20/23)	93.26 (166/178)	92.54 (186/201)	P＜0.05	0.685 4
PRRSV-1	66.67 (2/3)	97.98 (194/198)	97.51 (196/201)	P＞0.05	0.433 2
PCV2	96.00 (48/50)	91.39 (138/151)	92.54 (186/201)	P＜0.05	0.814 0
PRV	100.00 (3/3)	99.49 (197/198)	99.50 (200/201)	P＞0.05	0.854 7
Li	69.23 (9/13)	90.96 (171/188)	89.55 (180/201)	P＜0.05	0.410 7
PCV3	98.33 (118/120)	77.78 (63/81)	90.05 (181/201)	P＜0.05	0.786 4
Er	93.16 (109/117)	89.29 (75/85)	91.54 (184/201)	P＞0.05	0.825 9
TGEV	100.00 (5/5)	95.41 (187/196)	95.52 (192/201)	P＜0.05	0.508 3

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基于纳米孔测序的十种猪病原检测方法的建立

Establishment of Ten Swine Pathogens Detection Method based on Nanopore Sequencing

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