J、K亚群禽白血病病毒血液核酸快速筛查技术的建立

doi:10.11843/j.issn.0366-6964.2025.12.034

Abstract

Abstract: This study aimed to shorten the detection cycle for subgroup J/K avian leukosis virus (ALV-J/K) purification and accelerate the avian leukosis (AL) purification process, this study established a rapid blood nucleic acid screening technique (ALV-J/K blood quantitative polymerase chain reaction, ALV-J/K-B-qPCR) suitable for low-prevalence scenarios of ALV-J/K. By selecting conserved regions of ALV-J and K to design specific primers and TaqMan probes, and optimizing the reaction conditions, a TaqMan qPCR detection method for ALV-J/K was established. Using blood samples identified as ALV-J/K positive through cell culture virus isolation as experimental materials, anticoagulated whole blood DNA, blood cell DNA, and plasma cDNA were prepared and subjected to ALV-J/K TaqMan qPCR detection to identify the optimal detection template. Nucleic acids were extracted using a blood room-temperature lysis kit and a universal column-based genomic DNA extraction kit to screen for the optimal nucleic acid extraction method. The effect of varying dilution factors of positive samples on detection results was investigated to assess the feasibility of mixed sample detection. A simulated blood nucleic acid screening test with an expected prevalence rate of 1% to 2% was conducted using ALV-J/K-B-qPCR, and the results were compared with those obtained by the virus isolation method. The specificity, sensitivity, and repeatability tests of ALV-J/K TaqMan qPCR demonstrated that this method specifically amplifies only ALV-J and ALV-K, with detection limits of 8.95×10¹ copies·μL^-1, 8.16×10¹ copies·μL^-1 for positive standards, respectively. The sensitivity was 100 times higher than that of conventional PCR, and both intra- and inter-assay coefficients of variation <2%. The detection results of 96 clinical samples revealed that the ALV-J/K TaqMan qPCR had a detection rate of 21.9%, which was higher than that of conventional PCR (15.6%) and ALV p27 ELISA (18.8%). In the detection template screening test, anticoagulated whole blood DNA was identified as the optimal detection template. In the nucleic acid extraction method screening test, the universal column-based genomic DNA extraction kit yielded the best nucleic acid detection results. In the feasibility assessment test for mixed sample detection, the method established in this study was capable of detecting samples with low viral loads when the number of mixed samples <10. The simulated nucleic acid screening test for blood with an expected prevalence rate of 1% to 2% showed that the ALV-J/K-B-qPCR had a detection rate of 4%, which was 2.5% higher than that of the virus isolation method. The ALV-J/K-B-qPCR technology established in this study offers the advantages of simultaneous detection and differentiation of ALV-J and ALV-K, high sensitivity, strong specificity, high detection efficiency, and cost-effectiveness, aiming to provide a novel technique for AL purification in breeding poultry farms.

Key words: subgroup J avian leukosis virus, subgroup K avian leukosis virus, qPCR, nucleic acid screening, pooling sample assay

CLC Number:

S852.659.3

WANG Hongmei, ZHOU Wanyi, LUO Xinjie, CHEN Huilin, CHEN Qinxi, SHU Xueli, ZHANG Jinyu, SU Tong, LIAO Ming, CAO Weisheng. Establishment of Blood Nucleic Acid Screening Technology for Subgroup J/K Avian Leukosis Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6316-6325.

References

[1] 姜增,郭妍妍,李锦群,等.广东省规模化种鸡场禽白血病流行病学调查[J].中国家禽,2022,44(6):88-93.
JIANG Z, GUO Y Y, LI J Q, et al. Epidemiological investigation of avian leukosis in large-scale breeding chicken farms in Guangdong Province [J]. China Poultry, 2022, 44(6):88-93. (in Chinese)
[2] 窦俊峰,汪最,李丽,等.A、B、J亚群禽白血病病毒混合感染的鉴定及gp85基因序列分析[J].中国畜牧兽医, 2024,51(1):302-311.
DOU J F, WANG Z, LI L, et al. Identification of mixed infections with subgroups A, B, and J avian leukosis viruses and sequence analysis of the gp85 gene [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(1): 302-311. (in Chinese)
[3] LI H, WANG P, LIN L, et al. The emergence of the infection of subgroup J avian leucosis virus escalated the tumour incidence in commercial Yellow chickens in Southern China in recent years[J]. Transbound Emerg Dis, 2019, 66(1):312-316.
[4] 杨凤,孙洪磊,倪伟,等.山东不同品系蛋鸡禽白血病流行病学调查[J].畜牧兽医学报,2011,42(5):698-703.
YANG F, SUN H L, NI W, et al. Epidemiological investigation of avian leukosis in different strains of layer hens in Shandong [J]. Acta Veterinaria et Zootechnica Sinica, 2011, 42(5): 698-703. (in Chinese)
[5] SU Q, CUI Z, ZHANG Z, et al. Whole-genome analysis of an emerging recombinant avian leukosis virus in yellow chickens, south China[J]. Transbound Emerg Dis, 2020, 67(5):2254-2258.
[6] SHAO H, WANG L, SANG J, et al. Novel avian leukosis viruses from domestic chicken breeds in mainland China[J]. Arch Virol, 2017, 162(7):2073-2076.
[7] LIANG X, GU Y, CHEN X, et al. Identification and characterization of a novel natural recombinant avian leucosis virus from Chinese indigenous chicken flock[J]. Virus Gen, 2019, 55(5):726-733.
[8] LI X, YU Y, MA M, et al. Molecular characteristic and pathogenicity analysis of a novel multiple recombinant ALV-K strain[J]. Vet Microbiol, 2021, 260:109184.
[9] 邢林林,李玉杰,徐聪,等.山东省种鸡场禽白血病流行病学调查[J].中国畜牧业, 2020(15):58-59.
XING L L, LI Y J, XU C, et al. Epidemiological investigation of avian leukosis in breeding chicken farms in Shandong Province [J]. China Animal Husbandry, 2020(15):58-59. (in Chinese)
[10] 李海琴,张帆帆,林翠,等.江西地方鸡禽白血病流行与净化现状[J].中国畜禽种业,2024,20(2):62-68.
LI H Q, ZHANG F F, LIN C, et al. Prevalence and purification status of avian leukosis in local chickens in Jiangxi [J]. China Livestock and Poultry Breeding, 2024, 20(2):62-68. (in Chinese)
[11] 曹利利,董航,郭衍冰,等.禽白血病病毒ELISA检测方法的建立与初步应用[J].中国动物传染病学报, 2020, 28(1):16-21.
CAO L L, DONG H, GUO Y B, et al. Establishment and preliminary application of an ELISA detection method for avian leukosis virus [J]. Chinese Journal of Animal Infectious Diseases, 2020, 28(1):16-21.
[12] 郭慧君,李中明,李宏梅,等.3种ELISA试剂盒检测不同亚型外源性鸡白血病病毒的比较[J]. 畜牧兽医学报, 2010, 41(3):310-314.
GUO H J, LI Z M, LI H M, et al. Comparison of three ELISA kits for detecting different subtypes of exogenous avian leukosis virus [J]. Acta Veterinaria et Zootechnica Sinica, 2010, 41(3): 310-314. (in Chinese)
[13] 王瑞明,都玉印,相英杰,等. K亚群禽白血病病毒实时荧光定量PCR检测方法的建立及应用[J].中国兽医学报, 2020, 40(3):524-529.
WANG R M, DU Y Y, XIANG Y J, et al. Establishment and application of a real-time fluorescent quantitative PCR detection method for subgroup K avian leukosis virus [J]. Chinese Journal of Veterinary Science, 2020, 40(3):524-529. (in Chinese)
[14] DAI M M, FENG M, LIU D, et al. Development and application of SYBR Green I real-time PCR assay for the separate detection of subgroup J avian leukosis virus and multiplex detection of avian leukosis virus subgroups A and B[J]. Virol J, 2015(12): 52.
[15] QIN L, GAO Y, NI W, et al. Development and application of real-time PCR for detection of subgroup J avian leukosis virus[J]. J Clin Microbiol, 2013, 51(1):149-154.
[16] SOKOL D L, ZHANG X, LU P, et al. Real time detection of DNA.RNA hybridization in living cells[J]. Proc Natl Acad Sci U S A, 1998, 95(20):11538-11543.
[17] NORTON D M, BATT C A. Detection of viable Listeria monocytogenes with a 5' nuclease PCR assay[J]. Appl Environ Microbiol, 1999, 65(5):2122-2127.
[18] VET J A, MAJITHIA A R, MARRAS S A, et al. Multiplex detection of four pathogenic retroviruses using molecular beacons[J]. Proc Natl Acad Sci U S A, 1999, 96(11):6394-6399.
[19] PAN J, XIANG Q, BALL S. Use of a novel real-time quantitative reverse transcription-polymerase chain reaction method to study the effects of cytokines on cytochrome P450 mRNA expression in mouse liver[J]. Drug Metab Dispos, 2000, 28(6):709-713.
[20] MALLAPATY S. The mathematical strategy that could transform coronavirus testing[J]. Nature, 2020,583(7817):504-505.
[21] ABDALHAMID B, BILDER C R, MCCUTCHEN E L, et al. Assessment of specimen pooling to conserve SARS CoV-2 testing resources[J]. Am J Clin Pathol, 2020, 153(6):715-718.
[22] PALMER S, DIJKSTRA M, KET J C, et al. Acute and early HIV infection screening among men who have sex with men, a systematic review and meta-analysis[J]. J Int AIDS Soc, 2020, 23(Suppl 6):e25590.
[23] ZENG J, HUANG H, LIU X, et al. Pooling sputum samples for the Xpert MTB/RIF assay: a practical screening strategy for highly infectious tuberculosis cases[J]. BMC Infect Dis, 2024, 24(1):122.
[24] HOGAN C A, SAHOO M K, PINSKY B A. Sample pooling as a strategy to detect community transmission of SARS-CoV-2[J]. JAMA, 2020, 323(19):1967-1969.
[25] CHONG B S W, TRAN T, DRUCE J, et al. Sample pooling is a viable strategy for SARS-CoV-2 detection in low-prevalence settings[J]. Pathology, 2020, 52(7):796-800.
[26] 袁东升.新冠病毒核酸检测数学建模研究[J].中学数学研究(华南师范大学版), 2022(17):38-41.
YUAN D S. Mathematical modeling research on nucleic acid detection of COVID-19 [J]. Secondary School Mathematics Research (South China Normal University Edition), 2022(17): 38-41. (in Chinese)
[27] SMITH L M, BROWN S R, HOWES K, et al. Development and application of polymerase chain reaction (PCR) tests for the detection of subgroup J avian leukosis virus[J]. Virus Res, 1998, 54(1):87-98.
[28] CHEN J, ZHAO Z, CHEN Y, et al. Development and application of a SYBR green real-time PCR for detection of the emerging avian leukosis virus subgroup K[J]. Poul Sci, 2018, 97(7):2568-2574.
[29] 董欣怡,李锦群,陈钦玺,等.J亚群禽白血病病毒血液核酸筛查技术的建立[J].畜牧兽医学报, 2024, 55(3):1115-1126.
DONG X Y, LI J Q, CHEN Q X, et al. Establishment of a blood nucleic acid screening technology for subgroup J avian leukosis virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3):1115-1126. (in Chinese)
[30] YANG Y, YANG M, YUAN J, et al. Laboratory diagnosis and monitoring the viral shedding of SARS-CoV-2 infection[J]. Innovation (Camb), 2020, 1(3):100061.
[31] 杨晓雯,刘佳音,张宇,等.不同方法提取血培养瓶培养的抗凝血中布鲁氏菌核酸的比较[J].疾病监测, 2022, 37(7):964-966.
YANG X W, LIU J Y, ZHANG Y, et al. Comparison of different methods for extracting Brucella nucleic acid from anticoagulated blood cultured in blood culture bottles [J]. Disease Surveillance, 2022, 37(7):964-966. (in Chinese)
[32] ABDELRAZIK A M, SAID M N E, ABDELAZIZ H M. Evaluation of pooling strategy of SARS-CoV-2 RT-PCR in limited resources setting in Egypt at low prevalence[J]. Comp Clin Pathol, 2023, 32(3):375-381.

Establishment of Blood Nucleic Acid Screening Technology for Subgroup J/K Avian Leukosis Virus

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LIU Tian, WU Yulun, YAO Huochun, PAN Zihao. Establishment of a Triple TaqMan qPCR Detection Method for Mycobacterium avium subsp. Paratuberculosis [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4750-4758.
[2]	WANG Chun, WANG Qing, WU Xiaoqian, HU Ting, CUI Weitao, PEI Jie, SONG Tieping, HU Sishun, ZHANG Wanpo, LI Zili, ZHOU Zutao. Analysis of Mycoplasma synoviae Infection in the Laying Hens in Hubei Province based on Fluorescence Quantitative PCR Detection and in situ Hybridization Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1396-1407.
[3]	CHEN Qian, TONG Jiang, PAN Yuheng, MA Jianfeng, SHI Yuqian, CHEN Siyu, LI Jiaxin, ZHOU Ting, LIU Chendong, ZHU Li, SHEN Linyuan, GAN Mailin. The Impact of Gossypol on Spermatogenic Capacity of the Testis and Apoptosis of Sertoli Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5575-5587.
[4]	Li ZHANG, Mengmeng YU, Ying WANG, Suyan WANG, Zhuangzhuang XU, Peng LIU, Yuntong CHEN, Xiaole QI, Liuan LI, Yulong GAO. Analysis of Cellular Receptor chNHE1 Expression in Chicken Tissues Infected with Avian Leukosis Virus Subgroup J [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3631-3639.
[5]	HU Zeqi, LI Runcheng, TAN Zuming, XIE Xiuyan, WANG Jiangping, QIN Lejuan, LI Rong, GE Meng. Establishment and Preliminary Application of PEDV, PoRVA and PDCoV TaqMan Triple RT-qPCR Assay [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2267-2272.
[6]	DONG Xinyi, LI Jinqun, CHEN Qinxi, LIAO Ming, CAO Weisheng. Establishment of Blood Nucleic Acid Screening Technology for Subgroup J Avian Leukosis Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1115-1126.
[7]	YAN Chao, LIU Yonggang, XIE Hao, PENG Cuiting, ZHANG Caiyong, ZHAO Yulan, QI Lin, CHEN Zhilong, TANG Zhonglin. Detection of Gene Expression in Trace Cells of Early Porcine Embryo by Pre-amplified Quantitative PCR [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5567-5574.
[8]	ZHAI Gang, GU Wenyuan, LIU Tao, LIU Ying, ZHANG Shuai, FAN Jinghui, ZUO Yuzhu. Establishment of TaqMan Detection Method of Porcine Epidemic Diarrhea Virus and Analysis of Genetic Variation based on S Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 847-854.
[9]	ZHOU Shiwei, WU Tingjie, WANG Qian, CAI Bei, HAN Saizheng, WU Yanhu, WANG Xiaolong, CHEN Yulin. Establishment of the Efficient Method to Detect the FecB Mutation and the Construction of the Prolific Tan Sheep Population [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 2920-2929.
[10]	WANG Qin, XIONG Yan, ZI Xiangdong. Effects of PAF Supplementation during in vitro Maturation Medium on Yak Oocyte Development Competence and Gene Expression [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 514-523.
[11]	LIU Haoli, TENG Man, LI Huizhen, YU Zuhua, LIU Jingling, DING Ke, ZHANG Gaiping, LUO Jun. Screening and Identification of Host mRNA Targets for the Viral microRNA miR-M11-5p Encoded by Marek's Disease Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1026-1038.
[12]	WANG Yunhe, JIN Xin, ZHANG Man, WEI Fang, WEN Jingyi, ZHAO Feifei, YANG Yinfeng. Effects of Saccharomyces Cerevisiae and Its Inactivated Bacteria on the Expression of β-defensin-1 in Ruminal Explants of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 581-591.
[13]	LIAO Bo, CUI Yan, YANG Xue, HE Jun-feng, SONG Liang-li, YANG Xiao-qing, LIU Peng-gang. Distribution and Change Rule of HSP70 in Yak Intestine [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1735-1742.
[14]	LI Chang, KU Xu-gang, WANG Jun-wei, LI Jing, ZHU Ling, HE Qi-gai. Development and Application of a TaqMan-based Real-time Fluorescent PCR for Specific Detection of Porcine Circovirus Type 3 [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1320-1326.
[15]	ZHANG Man, JIN Xin, WANG Yun-he, WEI Fang, WEN Jing-yi, LI Zheng-yi, YANG Yin-feng. Effects of Saccharomyces cerevisiae β-glucan on the Expression of SBD-1 in Ovine Ruminal Epithelial Cells [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2416-2424.