禽痘病毒微滴数字PCR检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2026.01.029

Abstract

Abstract:

A microdroplet digital polymerase chain reaction （ddPCR） method for the detection of fowlpox virus （FWPV） was established. In this study， a more complete ddPCR reaction system was established through the optimization of the reaction system， including the optimization of primer and probe concentrations， the optimization of annealing temperature and other steps， and reproducibility， sensitivity， and specificity experiments. In this method， the optimal primer concentration was determined to be 900 nmol·L^-1， the optimal probe concentration was 200 nmol·L^-1， and the results of ddPCR were optimized under the reaction condition of 55 ℃. The method showed no cross-reactivity with other common avian viruses and is highly specific. The reproducibility of the method is good， and the coefficients of variation between and within batches are less than 10%. The lowest detection limit of this ddPCR method was 2.37 copies per reaction. Using the established ddPCR detection method and the industry standard （SN/T 1226—2015） detection method for 80 samples， the coincidence rate was 100%， which was higher than the coincidence rate of 75% of the industry standard nested PCR method. In 25 artificially inoculated samples， the detection rate of ddPCR for positive samples reached 100%， while the detection rate of the nested PCR method specified in the industry standard for positive samples was 33.3%. The establishment of this method helps the relevant inspection and quarantine departments at ports to detect the FWPV in a timely manner and reduces the risk of the domestic poultry breeding industry by providing a more accurate and efficient detection method.

Key words: fowlpox virus, droplet digital PCR, quantitation assay

CLC Number:

S852.659.1

ZHANG Yifan, HAN Xiao, ZHANG Yena, KONG Pengli, SONG Houhui, ZHANG Xiaofeng, WANG Xiaodu, SHUAI Jiangbing. Establishment and Application of a Droplet Digital PCR Assay for Fowlpox Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 337-348.

Figures/Tables 10

Table 1

Fig.1

Fig.2

Fig.3

Table 2

Fig.4

Fig.5

Fig.6

Table 3

Table 4

References 40

[1]	GIOTIS E S，LAIDLAW S M，BIDGOOD S R，et al.Modulation of early host innate immune response by an avipox vaccine virus’ lateral body protein［J］.Biomedicines，2020，8（12）：634.
[2]	SONG H S，KIM H S，KIM S H，et al.Research note：simultaneous detection of infectious laryngotracheitis virus，fowlpox virus，and reticuloendotheliosis virus in chicken specimens［J］.J Poult Sci，2021，100（4）：100986.
[3]	李亮亮，由玉岩，任倩俐.褐马鸡常见疾病的预防与治疗［J］.畜牧业环境，2024（13）：87-88.
	LI L L，YOU Y Y，REN J L.Prevention and treatment of common diseases in crossoptilon mantchuricum［J］.Animal Industry and Environment，2024（13）：87-88.（in Chinese）
[4]	田如玉.斑鸠源禽痘病毒的分离鉴定及检测方法的初步建立［D］.武汉：华中农业大学，2022.
	TIAN R Y.Isolation，identification and detection method of avian pox virus from turtledove［D］.Wuhan：Huazhong Agricultural University，2022.（in Chinese）
[5]	GIOTIS E S，SKINNER M A.Spotlight on avian pathology：fowlpox virus［J］.Avian Pathol，2019，48（2）：87-90.
[6]	宋玉红.禽痘病毒的诊断与防控［J］.兽医导刊，2021（17）：32-33.
	SONG H Y.Diagnosis and control of fowlpox virus［J］.Veterinary Orientation，2021（17）：32-33.（in Chinese）
[7]	YEHIA N，ELSAYED S，AL-SAEED F A，et al.Current situation and genomic characterization of fowlpox virus in lower egypt during 2022［J］.J Poult Sci，2023，102（8）：102769.
[8]	SARMA G，KERSTING B A，SPINA G.Field safety and efficacy of a unique live virus vaccine for controlling avian encephalomyelitis and fowlpox in poultry［J］.Vet World，2019，12（8）：1291-1298.
[9]	VAN DER MEER C S，PAULINO P G，JARDIM T H A，et al.Detection and molecular characterization of Avipoxvirus in Culex spp.（Culicidae） captured in domestic areas in Rio de Janeiro，Brazil［J］.Sci Rep，2022，12（1）：13496.
[10]	MAPACO L P，LACERDA Z，MONJANE I V A，et al.Molecular characterization of avipoxviruses circulating in Mozambique，2016-2018［J］.Arch Virol，2018，163（8）：2245-2251.
[11]	MURER L，WESTENHOFEN M，KOMMERS G D，et al.Identification and phylogenetic analysis of clade C Avipoxvirus in a fowlpox outbreak in exotic psittacines in southern Brazil［J］.J Vet Diag Investig，2018，30（6）：946-950.
[12]	MATOS M，BILIC I，PALMIERI N，et al.Epidemic of cutaneous fowlpox in a naïve population of chickens and turkeys in Austria：detailed phylogenetic analysis indicates co-evolution of fowlpox virus with reticuloendotheliosis virus［J］.Transbound Emerg Dis，2022，69（5）：2913–2923.
[13]	TADESE T，FITZGERALD S，REED W M.Detection and differentiation of re-emerging fowlpox virus （FWPV） strains carrying integrated reticuloendotheliosis virus （FWPV-REV） by real-time PCR［J］.Vet Microbiol，2008，127（1-2）：39-49.
[14]	BRANDOLINI M，TADDEI F，MARINO M M，et al.Correlating qRT-PCR，dPCR and viral titration for the identification and quantification of SARS-CoV-2：a new approach for infection management［J］.Viruses，2021，13（6）：1022.
[15]	杨国平，周彪，苏小建，等.猴痘病毒数字PCR检测方法的建立［J］.中国国境卫生检疫杂志，2024，47（4）：340-344.
	YANG G P，ZHOU B，SU X J，et al.Establishment of digital PCR method for detection of monkeypox virus［J］.Chinese Journal of Frontier Health and Quarantine，2024，47（4）：340-344.（in Chinese）
[16]	VYNCK M，TRYPSTEEN W，THAS O，et al.The future of digital polymerase chain reaction in virology［J］.Mol Diagn Ther，2016，20（5）：437-447.
[17]	FANG W B，LIU X D，MAIGA M，et al.Digital PCR for single-cell analysis［J］.Biosensors （Basel），2024，14（2）：64.
[18]	田国忠，姜海，薛盼盼，等.布鲁氏菌微滴式数字PCR方法的建立与应用［J］.中国人兽共患病学报，2021，37（4）：301-305.
	TIAN G Z，JIANG H，XUE P P，et al.Establishment and application of droplet digital PCR in the detection of Brucela［J］.Chinese Journal of Zoonoses，2021，37（4）：301-305.（in Chinese）
[19]	原霖，董浩，倪建强，等.非洲猪瘟病毒微滴数字PCR检测方法的建立［J］.畜牧与兽医，2019，51（7）：81-84.
	YUAN L，DONG H，NING J Q，et al.Development of droplet digital PCR for detection of African swine fever virus［J］.Animal Husbandry & Veterinary Medicine，2019，51（7）：81-84.（in Chinese）
[20]	郑子繁，柳方方，刘卫晓，等.数字PCR技术在核酸标准物质研制中的应用［J］.生物技术进展，2020，10（6）：579-584.
	ZHENG Z F，LIU F F，LIU W X，et al.Application of digital PCR technology in the development of nucleic acid reference materials［J］.Current Biotechnology，2020，10（6）：579-584.（in Chinese）
[21]	冯杰，聂洪波，曹乾大，等.微滴式数字PCR技术在动物疫病检测中的应用研究进展［J］.中国动物检疫，2024，41（4）：58-63，90.
	FENG J，NIE H B，CAO Q D，et al.Research progress in the application of droplet-based digital PCR assay to animal disease detection［J］.China Animal Health Inspection，2024，41（4）：58-63，90.（in Chinese）
[22]	蒋文明，李金平，尹馨，等.H7亚型禽流感病毒微滴式数字PCR检测方法的建立及应用［J］.中国兽医杂志，2023，59（2）：39-43.
	JIANG W M，LI J P，YIN X，et al.Development and application of droplet digital PCR for detection of H7 subtype avian influenza virus［J］.Chinese Journal of Veterinary Medicine，2023，59（2）：39-43.（in Chinese）
[23]	HOPKEN M W，PIAGGIO A J，PABILONIA K L，et al.Predicting whole genome sequencing success for archived avian influenza virus （Orthomyxoviridae） samples using real-time and droplet PCRs［J］.J Virol Methods，2020，276：113777.
[24]	LI Q C，ZHANG Y B，MENG F F，et al.A New strategy for the detection of chicken infectious anemia virus contamination in attenuated live vaccine by droplet digital PCR［J］.Bio Med Res Int，2019，2019：2750472.
[25]	CAO Y Q，YU M，DONG G H，et al.Digital PCR as an emerging tool for monitoring of microbial biodegradation［J］.Molecules （Basel，Switzerland），2020，25（3）：706.
[26]	ZHU H L，ZHANG H Q，XU Y，et al.PCR past，present and future［J］.Biotechniques，2020，69（4）：317-325.
[27]	GRIFFITHS K R，MCLAUGHLIN J L H，HALL F，et al.Development of seven new dPCR animal species assays and a reference material to support quantitative ratio measurements of food and feed products［J］.Foods （Basel，Switzerland），2023，12（20）：3839.
[28]	QUAN P L，SAUZADE M，BROUZES E.dPCR：a technology review［J］.Sensors （Basel），2018，18（4）：1271.
[29]	HINDSON C M，CHEVILLET J R，BRIGGS H A，et al.Absolute quantification by droplet digital PCR versus analog real-time PCR［J］.Nat Methods，2013，10（10）：1003-1005.
[30]	王艺凯，齐玮，杨诣，等.数字PCR技术在国门生物安全保障领域的应用［J］.分析仪器，2023 （6）：109-116.
	WANG Y K，QI W，YANG Y，et al.Application of digital PCR technology in the field of entry and exit port biosecurity assurance［J］.Analytical Instrumentation，2023（6）：109-116.（in Chinese）
[31]	SHARIF H M.Molecular properties of serum thymidine kinase 1 and use of the canine enzyme in disease monitoring［D］.Uppsala：Swedish University of Agricultural Sciences，2012.
[32]	DENG L C，LIU C X，LI L T，et al.Genomic characteristics of an avipoxvirus 282E4 strain［J］.Virus Res，2023，336：199218.
[33]	国家质量监督检验检疫总局.禽痘检疫技术规范：SN/T 1226-2015［S］.北京：中国标准出版社，2015.
	General Administration of Quality Supervision，Inspection and Quarantine.Quarantine protocol for fowlpox：SN/T 1226-2015［S］.Beijing：Standards Press of China，2015.（in Chinese）
[34]	罗海峰，杜红心，彭必江，等.基层医院生化分析仪的使用现状与需求调查［J］.检验医学与临床，2015，12 （16）：2309-2310，2430.
	LUO H F，DU H X，PENG B J，et al.Investigation and analysis on curent situation and demands of biochemical analyzers in primary hospitals［J］.Laboratory Medicine and Clinic，2015，12（16）：2309-2310，2430.（in Chinese）
[35]	史光霞.基层检验的现状和改进建议［J］.检验医学与临床，2007（1）：62-63.
	SHI G X.The Current Status and Improvement Proposals for Grassroots Inspection［J］.Laboratory Medicine and Clinic，2007 （1）：62-63.（in Chinese）
[36]	郭婷，王磊，李小飞.非小细胞肺癌EGFR突变检测技术的研究进展［J］.临床与病理杂志，2017，37（9）：1936-1942.
	GUO T，WANG L，LI X F.Research progress in the detection technology of EGFR mutations in non-small cell lung cancer［J］.Journal of Clinical and Pathological Research，2017，37（9）：1936-1942.（in Chinese）
[37]	SHI J Z，JIN Q Y，ZHANG X Z，et al.The development of a sensitive droplet digital polymerase chain reaction test for quantitative detection of goose astrovirus［J］.Viruses，2024，16（5）：765.
[38]	秦爱，王娟，邓方进，等.数字聚合酶链式反应技术在食品安全核酸检测领域中的研究进展及标准化现状［J］.食品科学，2024，45（18）：350-360.
	QIN A，WANG J，DENG F J，et al.Progress in the application of digital polymerase chain reaction in food safety detection and current status of its standardization［J］.Food Science，2024，45（18）：350-360.（in Chinese）
[39]	张新建，马向红，陈威.禽痘的流行诊断和防控［J］.饲料博览，2020（5）：69.
	ZHANG X J，MA X H，CHEN W.Epidemiology，diagnosis and prevention of avian pox［J］.Feed Review，2020（5）：69.（in Chinese）
[40]	王鉴波.禽痘的流行和诊治［J］.现代畜牧科技，2016（4）：109.
	WANG J B.The prevalence and diagnosis of fowlpox［J］.Modern Animal Husbandry Science & Technology，2016（4）：109.（in Chinese）

引物/探针 Primer/probe	序列（5→3'） Sequence	位置 Position	长度/bp Product
FWPV-F	AGAAAACGAAGCGTCGCAATAGC	88 824—88 846	132
FWPV-R	ATTGTGGCCGCGCTTAACGGTG	88 715—88 736	132
FWPV-P	TTCACGCAAATAGCTGTCAAACTGG	88 797—88 821	/
FWPV-1	CAGCAGGTGCTAAACAACAA	201 978—201 997	558
FWPV-2	CGGTAGCTTAACGCCGAATA	201 420—201 439	558
FWPV-3	ACGACCTATGCGTCTTC	201 962—201 978	419
FWPV-4	ACGCTTGATATCTGGATG	201 560—201 577	419

质粒浓度/（拷贝·反应^-1） Plasmids concentration（copies per reaction）	微滴式数字PCR ddPCR
质粒浓度/（拷贝·反应^-1） Plasmids concentration（copies per reaction）	检测值（x±s）/（拷贝·反应^-1） Detected values （x±s）/（copies per reaction）	阳性/样本数 Positive/Reactions	相对标准偏差 RSD
10³	1 003.50 ±61.43	30/30	0.88%
10²	103.73±7.37	30/30	1.52%
10	9.80±1.53	30/30	6.80%
7.5	8.32±2.11	30/30	12.23%
5	5.83±1.37	30/30	14.02%
2.5	ND	29/30	/
1	ND	22/30	/

结果 Results	方法 Method
结果 Results	PCR （SN/T 1226—2015）	微滴式数字PCR ddPCR
阳性 Positive	18	72
阴性 Negative	62	8
总数 Total	80	80
符合率/% Compliance rate	25	100

检测部位 Detection site	检出阳性数 Number of positive
	攻毒组（n=3） Challenge group		阴性组（n=2） Negative group
	ddPCR	PCR	ddPCR	PCR
阳性检出率/% Positive detection rate	100（15/15）	33.3（5/15）	0（0/10）	0（0/10）
鸡冠 Cockscomb	3/3	3/3	0/2	0/2
鸡爪 Foot	3/3	2/3	0/2	0/2
肺 Lung	3/3	0/3	0/2	0/2
肾脏 Kidney	3/3	0/3	0/2	0/2
血液 Blood	3/3	0/3	0/2	0/2

Establishment and Application of a Droplet Digital PCR Assay for Fowlpox Virus

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 10

References 40

Related Articles 12

Recommended Articles

Metrics

Comments

[1]	CHEN Bingbing, CAI Weiyou, LIU Yutong, WANG Xiuwu, SUN Shouhu, HE Dongsheng. Establishment and Application of Droplet Digital PCR Method for the Detection of Porcine Rotavirus A [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 4095-4100.
[2]	MEI Li, WANG Yingchao, CHENG Rujia, YU Guoji, FAN Xuezheng, GAO Xiaolong, GAO Min, QIN Yuming, LI Xiaoying, LI Qiaoling, ZHU Liangquan, FENG Xiaoyu. A Droplet Digital PCR Method for Detection of Brucella [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1753-1759.
[3]	MEI Li, WANG Yingchao, WU Di, LI Yue, SONG Yanjun, WEI Haitao, WANG Lin, GAO Xiaolong, FENG Xiaoyu. A Droplet Digital PCR Method for Detection of Newcastle Disease Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3187-3192.
[4]	CHEN Ya-na, WANG Jing, ZI Zhan-chao, KANG Wen-hua, WANG Bao-yue, NI Jian-qiang, YUAN Lin. Development of Droplet Digital PCR for the Detection of Pseudorabies Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(9): 1705-1710.
[5]	CHEN Sujuan#;DING Yanhong#;QIAN Cheng;CHAI Mao;PENG Daxin;LIU Xiufan. Antibody Dynamics and Protective Efficacy of Recombinant Fowlpox Virus Coexpressing Cytokine IL6 Gene and HA Gene of H5 Subtype Avian Influenza Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(4): 596-601.
[6]	WANG Yanhong;CHEN Sujuan;LIU Wujie;QIU Xusheng;DONG Li;PENG Daxin;LIU Xiufan. Comparison of Immune Efficacy of Three Recombinant Fowlpox Virus Expressing HA Gene of H5 AIV [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2010, 41(1): 60-64.
[7]	SHI Xing-ming;WANG Yun-feng;WANG Mei;LAN De-song;REN Gang;LI Ji-song;ZENG Wei-wei;SUN Yan;LIU Sheng-wang;TONG Guang-zhi. Safety Evaluation and Minimum Immune-doses Analysis of the Recombinant Fowl Pox Virus Expressing S1 Gene of Infectious Bronchitis Virus and IFN-γ Gene [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(5): 712-716.
[8]	LIU Wu-jie;SUN Lei;CHEN Su-juan;XU Zhong-lin;LIU Jin-biao;LIU Xiu-fan. Construction and Protective Efficacies of Two Recombinant Fowlpox Viruses Expressing the HA Gene of H9 Subtype AIV [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2008, 39(3): 327-332.
[9]	WANG Yun-feng;SHI Xing-ming;WANG Mei;LIU Sheng-wang;TONG Guang-zhi;CUI Hong-yu;MU Gu-li;XU Ling-long;HE Lai;HE Sun;GE Jun-wei;LI Yi-jing. Partial Biological Character Detection of Fowlpox Virus RecombinantCo-expressing the IBV-S1 Gene and the Chicken IFNγ Gene [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2007, 38(10): 1077-1082.
[10]	TIAN Zhan-cheng;SUN Yong-ke;WANG Yun-feng;ZHI Hai-dong;LIU Sheng-wang;SUN Hui-ling;WANG Mei;TONG Guang-zhi. cnThe Immunological Efficacies of Recombinant Fowlpox Virus Expressing the S1 Gene of LX4 Strain of Infectious Bronchitis Virus in Specific-pathogen-free (SPF) Chickens [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2006, 37(6): 580-586.
[11]	ZHI Hai-dong;WANG Yun-feng;ZHANG Jing;WANG Mei;WANG Feng-xue;TONG Guang-zhi. Efficiency of the Recombinant Fowlpox Virus Expressing gB Gene of Larngotracheitis Virus and Fusion Gene of Newcastle Disease Viru [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2005, 36(8): 807-811.
[12]	SUN Yong-ke;TIAN Zhan-cheng;WANG Yun-feng;TONG Guang-zhi;ZHI Hai-dong;LIU Sheng-wang;WANG Mei;YANG Zeng-qi. Co-expression of S1 Gene of Infectious Bronchitis Virus and Chicken Type II Interferon Gene in Recombinant Fowlpox Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2005, 36(8): 800-806.