禽流感病毒与新城疫病毒多靶标核酸质谱检测方法研究及应用

doi:10.11843/j.issn.0366-6964.2025.03.037

摘要/Abstract

摘要：

禽流感和新城疫是全球养禽业最具威胁的两种病毒性传染病，快速准确地病原检测和分型对于这两种动物疫病的早期防控和阻断传播具有至关重要的作用。本研究通过将微量多重RT-PCR、单碱基延伸和基于MALDI-TOF的质谱技术联合使用，针对禽流感和新城疫病毒建立了一种高通量核酸质谱检测方法，并对方法的灵敏度、特异性和重复性进行了评价。结果表明，建立的方法可即时同步检测禽流感病毒(A型通用)、H5、H7、H9亚型，新城疫病毒及其中强毒株，各个靶标的检测灵敏度为8.82~15.40 copies·μL^-1，与其他禽病病原核酸无交叉反应，对12 copies·μL^-1的6种混合靶标的检测重复性均在75%以上。对179份实验室收集的送检样品(包括拭子样品和组织脏器)检测结果表明，与荧光RT-PCR检测方法相比，禽流感病毒通用检测靶标符合率为98.9%；其他靶标的符合率均为100.0%。本研究为禽流感、新城疫病毒的快速鉴别检测及重要亚型/株型的同步快速分型鉴定提供了一种新的高通量替代方法。

关键词: 禽流感病毒, 新城疫病毒, 多靶标, 核酸质谱

Abstract:

Avian influenza (AI) and Newcastle disease (ND) are the two most threatening viral infectious diseases in the global poultry industry. Rapid and accurate pathogen detection and typing are of vital importance for the early control and transmission blocking of these two animal diseases. (Method) In this study, a high-throughput nucleic acid mass spectrometry method for detection of AIV and NDV was developed by combining micro multiplex RT-PCR, single base extension and MALDI-TOF mass spectrometry, and the sensitivity, specificity and repeatability of the method were evaluated. The results showed that the established method could detect AIV (type A), subtype H5, subtype H7, subtype H9, NDV and its velogenic/mesogenic strains synchronously. The analytical sensitivity of targets was 8.82-15.40 copies·μL^-1, and there was no cross-reaction with other avian pathogen nucleic acid. The reproducibility of 6 mixed targets at 12 copies·μL^-1 was above 75%. The detection results of 179 samples (including swab samples and tissue organs) collected in laboratory showed that the coincidence rate of universal target of AIV was 98.9%, and the other targets were 100.0% compared with real-time RT-PCR. So a new high-throughput alternative method is provided for differential detection of AIV, NDV, and their important subtypes/strains synchronously in this study.

Key words: avian influenza virus, Newcastle disease virus, multi-target, nucleic acid mass spectrometry

中图分类号:

S851.34

高志强, 赖平安, 宋悦谦, 种焱, 郭悠然, 白子龙, 郭惠民, 汪琳, 蒲静, 史喜菊, 任彤, 赵相鹏. 禽流感病毒与新城疫病毒多靶标核酸质谱检测方法研究及应用[J]. 畜牧兽医学报, 2025, 56(3): 1386-1395.

GAO Zhiqiang, LAI Ping'an, SONG Yueqian, CHONG Yan, GUO Youran, BAI Zilong, GUO Huimin, WANG Lin, PU Jing, SHI Xiju, REN Tong, ZHAO Xiangpeng. Studies and Application of Multi-target Nucleic Acid Mass Spectrometry Detection Method for Avian Influenza/Newcastle Disease Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1386-1395.

图/表 8

表 1

图 1

图 2

图 3

图 4

图 5

表 2

表 3

参考文献 11

1	World Organisation for Animal Health. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals[S]. Paris: The World Organisation for Animal Health. https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.03.04_AI.pdf, 2021: 1-26.
2	丛彦龙, 钟颖, 孙艺学等. H9N2禽流感流行病学及其疫苗的研究进展[C]//中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集. 中国畜牧兽医学会, 2016: 35-38.
	CONG Y L, ZHONG Y, SUN Y X, et al. Advance on epidemiology and vaccine of H9N2 avian influenza virus[C]//Proceedings of the Fifth Academic Symposium of the Veterinary Public Health Branch of Chinese Association of Animal Science and Veterinary Medicine. Chinese Association of Animal Science and Veterinary Medicine, 2016: 35-38. (in Chinese)
3	连聪,温肖会,吕殿红,等.我国新城疫分子流行病学研究进展[J].广东农业科学,2023,50(2):115-124.
	LIANC,WENX H,LVD H,et al.Progress of molecular epidemiology of Newcastle disease in China[J].Guangdong Agricultural Sciences,2023,50(2):115-124.
4	TOSTJ,GUTI G.DNA analysis by mass spectrometry-past, present and future[J].J Mass Spectrom,2006,41(8):981-995.
5	张阳东,宋秀军,高峥,等.核酸质谱仪检测单核苷酸多态性的临床应用[J].医疗卫生装备,2023,44(1):87-92.
	ZHANGY D,SONGX J,GAOZ,et al.Clinical application of nucleic acid mass spectrometer for detection of single nucleotide polymorphism[J].Chinese Medical Equipment Journal,2023,44(1):87-92.
6	朱雪松.禽流感和新城疫的流行防控现状[J].兽医导刊,2014(03):23-31.
	ZHUX S.Epidemic prevention and control of avian influenza and Newcastle disease[J].Vet Orientation,2014(03):23-31.
7	GIOVANNELLII,CICCONEN,VAGGELLIG,et al.Utility of droplet digital PCR for the quantitative detection of polyomavirus JC in clinical samples[J].J Clin Virol,2016,82,70-75. doi: 10.1016/j.jcv.2016.07.008
8	高志强,汪琳,刘环,等.纳米孔测序技术对一份禽流感病毒阳性样品的分子溯源[J].中国动物检疫,2024,41(2):86-91.
	GAOZ Q,WANGL,LIUH,et al.Molecular tracing of a positive sample with avian influenza virus by nanopore sequencing[J].China Animal Health Inspection,2024,41(2):86-91.
9	ZHAOF,LUJ,LUB,et al.A Novel strategy for the detection of SARS-CoV-2 variants based on multiplex PCR-mass spectrometry minisequencing technology[J].Microbiol Spectrum,2021,9(3):e0126721. doi: 10.1128/Spectrum.01267-21
10	SJÖHOLMM I,DILLNERJ,CARLSONJ.Multiplex detection of human herpesviruses from archival specimens by using matrix-assisted laser desorption ionization-time of flight mass spectrometry[J].J Clin Microbiol,2008,46(2):540-545.
11	宋士琦,裘慧,陈吴健,等.基于基质辅助激光解吸/电离飞行时间质谱的猪呼吸道病毒多目标鉴定方法的建立和应用[J].微生物学报,2023,63(7):2713-2727.
	SONGS Q,QIUH,CHENW J,et al.Establishment and application of a multi-target detection method for porcine respiratory viruses based on MALDI-TOF MS[J].Acta Microbiologica Sinica,2023,63(7):2713-2727.

靶标 Target	基因 Gene	扩增引物(5′→3′) Amplification primers	延伸引物(5′→3′) Extension primers	延伸引物质量/u Mass of unextended primers	延伸产物质量/u Mass of extended primers
AIV	M	F: ACGTTGGATGCCRATCYTGTCACCTCTGACTAA	TCACTGGGCACGGTGA	4 922.24	5 209.44
		R: ACGTTGGATGCGTCTACGCTGCAGTCCTC
H5	HA	F1: ACGTTGGATGATTTAYTCAACAGTGGCGRGTT	GCCTAGCACTGGCAATC	5 468.64	5 715.84(C)
		F2: ACGTTGGATGATTTAYTCAACAGTGGCGAGCT			5 795.84(T)
		R1: CTACAATCTGAACTCACAAGTTTARATGC	TCCCTAGTGCTGGCAATCA	5 763.84	6 011.04(C)
		R2: CTACAATCTGAACTCACAGCTCTARATGC			6 091.04(T)
			TGGATGTGCTCCAATGG	5 241.44	5 512.64(A)
					5 528.64(G)
H7	HA	F: ACGTTGGATGAAAATAGAATACAGATWRACCCRGT	CTTCGGGGCATCATGTTT	5 496.64	5 823.84(T)
		R: ACGTTGGATGGTGCACYGCATGTTTCC			5 743.84(C)
H9	HA	F: CGGATAACAATTTCACACAGGATGGGGTTTGCTGCC	GACATGGCCCAGAA	4 281.84	4 553.04(T)
		R: ACGTTGGATGAATTATATACAAATGTTGCAYCTG			4 529.04(G)
					4 609.04(A)
			GGTGTTGGACATAGCCCAGAA	6 495.24	6 766.44(T)
					6 742.44(G)
					6 822.44(A)
NDV	M	F: ACGTTGGATGTATGRTGRTAACWTGCAAGAAGAG	AAGGTGCCTGCACTA	4 577.04	4 824.44(G)
		R: ACGTTGGATGGCTTTCACRTGCTTVACTG			4 904.24(A)
NDV	F	F: ACGTTGGATGTCCGGAGGATACAAGRGTCYG	CCGGAGGAAGGAGACAGA	5 631.64	5 918.84(G)
中强毒株		R: ACGTTGGATGAGCTGTTGCRACCCCAAG			5 902.84(A)
NDV			CGGAGGAAGGAGACAAA	5 326.44	5 613.64(G)
					5 597.64(A)
mesogenic strains			CCGGAGGAAGGAGGCAGA	5 976.84	6 264.04(G)
					6 248.04(A)
			GTCTGGAGGAAGGAGACGGA	6 296.04	6 583.24(G)
					6 567.24(A)
IC	β-actin	F: ACGTTGGATGCCTTCAACACCCCTGCCATG	GTGCTGTCCCTGTACGCCTCTGG	6 998.60	7 325.8(T)
		R: ACGTTGGATGGTCCATCACGATRCCRGTGGT			7 245.80(C)
			CTGCTGTCCCTGTATGCCTCTGG	6 973.60	7 300.80(T)
					7 220.80(C)
			CTGCTGTCTCTGTACGCCTCTGG	6 973.60	7 300.80(T)
					7 220.80(C)

混合RNA浓度/(copies·μL^-1) Mixed RNA concentration	靶标名称 Target name	检测结果(Pos/Total) Detection result	阳性率/% Positive rate
50	AIV-M	8/8	100
	H5-HA	8/8	100
	H7-HA	8/8	100
	H9-HA	8/8	100
	NDV-M	8/8	100
	NDV-VF	8/8	100
25	AIV-M	8/8	100
	H5-HA	8/8	100
	H7-HA	8/8	100
	H9-HA	8/8	100
	NDV-M	8/8	100
	NDV-VF	8/8	100
12	AIV-M	7/8	87.5
	H5-HA	8/8	100
	H7-HA	7/8	87.5
	H9-HA	6/8	75
	NDV-M	7/8	87.5
	NDV-VF	8/8	100

检测靶标 Test target	数量 Number	检测结果 Test results				符合率/% Coincidence	Kappa
检测靶标 Test target	数量 Number	RRT-PCR(—)/MS(—)	RRT-PCR(+)/MS(+)	RRT-PCR(—)/MS(+)	RRT-PCR(+)/MS(—)	符合率/% Coincidence	Kappa
AIV 通用	179	122	55	0	2	98.9	0.974
AIV general
H5	179	167	12	0	0	100	1.0
H7	179	175	4	0	0	100	1.0
H9	179	161	18	0	0	100	1.0
NDV通用	179	174	5	0	0	100	1.0
NDV general
NDV中强毒株	179	174	5	0	0	100	1.0
NDV mesogenic strains

[1]	赵康宁, 杨忠龙, 陈怡, 朱春成, 郭云飞, 印云聪, 秦涛, 陈素娟, 彭大新. 16株新型H3N3亚型禽流感病毒的遗传变异分析[J]. 畜牧兽医学报, 2024, 55(9): 4029-4040.
[2]	吕亚迪, 杨洁, 谢文婷, 徐婷, 陈瑞爱. 共表达膜结合型与可溶性H9N2亚型禽流感病毒HA蛋白的重组基因Ⅶ型新城疫病毒的构建及免疫效果评价[J]. 畜牧兽医学报, 2024, 55(5): 2123-2134.
[3]	毛秋艳, 周淑宁, 刘朔, 彭程, 尹馨, 张雅馨, 周婉婷, 李金平, 侯广宇, 蒋文明, 宋厚辉, 刘华雷. H3亚型禽流感病毒荧光定量RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2024, 55(3): 1137-1146.
[4]	张高峰, 魏家阳, 冯贺龙, 李丽, 曾哲, 田光明, 聂仁锋, 罗青平, 温国元, 魏红波, 商雨. 生物矿化对新城疫病毒LaSota株生物学特性及免疫原性的影响[J]. 畜牧兽医学报, 2024, 55(12): 5663-5671.
[5]	杨芷翊, 王新凯, 史玉婷, 付思源, 张钰炘, 曹琛福, 贾伟新. 基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立[J]. 畜牧兽医学报, 2023, 54(9): 3803-3811.
[6]	周勇, 李知新, 鲁宏伟, 孙燕, 李甜, 杜凡姝, 蒲娟. 我国H5和H7N9亚型高致病性禽流感的监测及疫情暴发分析[J]. 畜牧兽医学报, 2022, 53(9): 3093-3106.
[7]	崔明仙, 王星博, 黄彦铭, 卞希一, 冯梦珂, 颜焰, 董伟仁, 周继勇. 3株H3N2亚型禽流感病毒的基因组特征与演化分析[J]. 畜牧兽医学报, 2022, 53(11): 4116-4122.
[8]	段志强, 谢玲玲, 陈佳琪, 唐宏, 王燕碧, 赵采芹, 赵佳福. 新城疫病毒M蛋白与宿主蛋白相互作用的研究进展[J]. 畜牧兽医学报, 2022, 53(1): 32-42.
[9]	孙华鹏, 崔新鑫, 潘亮奇, 许丰祥, 李硕, 吴梅花, 朱旭辉, 于亚南, 李明亮, 刘杨, 瞿孝云, 廖明, 孙海亮. 中国H9N2亚型禽流感病毒的流行现状[J]. 畜牧兽医学报, 2021, 52(5): 1218-1229.
[10]	李静云, 连朋敬, 白玉, 奚柳青, 张子卉, 牛小飞, 杨俊琦, 乔健. H9N2亚型禽流感病毒感染对小鼠肠道菌群的影响[J]. 畜牧兽医学报, 2021, 52(5): 1359-1368.
[11]	段志强, 谢玲玲, 周迪, 王燕碧, 赵采芹, 唐宏. 新城疫病毒M蛋白细胞核定位的机制与功能研究进展[J]. 畜牧兽医学报, 2021, 52(4): 891-898.
[12]	栗永华, 刘伟, 徐智凯, 刘炜玮, 孙英杰, 仇旭升, 谭磊, 宋翠萍, 廖瑛, 丁铲, 孟春春. 稳定表达TIGAR基因的BHK-21细胞系的构建及其对新城疫病毒增殖效果的评价[J]. 畜牧兽医学报, 2021, 52(2): 440-449.
[13]	李丽, 唐国毅, 冯贺龙, 薛玉涵, 任助, 王国康, 贾妙妙, 商雨, 罗青平, 邵华斌, 温国元. 基于马赛克HA序列的H9亚型禽流感灭活疫苗的免疫效力分析[J]. 畜牧兽医学报, 2021, 52(12): 3569-3577.
[14]	邵琪, 屈阳, 朱子晨, 孟春春, 仇旭升, 廖瑛, 谭磊, 宋翠萍, 刘炜玮, 孙英杰, 丁铲. 应用G3BP1稳转细胞系监测应激状态下的应激颗粒形成[J]. 畜牧兽医学报, 2020, 51(9): 2275-2283.
[15]	赵冰倩, 罗畅, 刘健新, 李慧子, 张彭涛, 于相龙, 刘博洋, 宁章勇. 连翘水提液体外对禽流感病毒增殖及炎症因子表达的抑制效应[J]. 畜牧兽医学报, 2020, 51(6): 1466-1474.