J亚群禽白血病病毒血液核酸筛查技术的建立

doi:10.11843/j.issn.0366-6964.2024.03.024

摘要/Abstract

摘要： 为缩短J亚群禽白血病病毒（subgroup J avian leukosis virus，ALV-J）检测周期，进一步加快禽白血病净化进程，本研究结合SYBR Green Ⅰ qPCR和混样检测，建立了一种适用于低ALV-J流行率场景下的快速筛检ALV-J的血液核酸筛查技术（ALV-J blood quantitative polymerase chain reaction，ALV-J-B-qPCR）。根据GenBank中ALV-J pol及env基因序列，设计ALV-J的特异性引物，并优化反应条件，建立了ALV-J SYBR Green Ⅰ qPCR检测方法。以ALV-J病毒分离为阳性的鸡抗凝血为实验材料，分别制备抗凝血DNA、血细胞DNA、外周血淋巴细胞DNA、外周血淋巴细胞cDNA和血浆cDNA 5类血液检测模板进行ALV-J的PCR检测，筛选最佳检测模板；进一步比较蒸馏水破裂红细胞法提取混合血液DNA的混样方法，血液混样总体积为200 μL的混样方法，血液混样总体积为10 μL的混样方法共3种混样方法的检测准确性，筛选最佳混样方法。综合上述qPCR方法、检测模板与混样方法，成功建立了ALV-J-B-qPCR。运用ALV-J-B-qPCR分别进行预期流行率为1%~2%、4%~5%场景下的模拟筛查试验，并与病毒分离法比较。qPCR方法的特异性、灵敏性和重复性试验显示，该方法仅特异性扩增ALV-J，对标准质粒的最低检测限度为1×10² copies·μL^-1，批内与批间变异系数均<1％。对90份临床送检血液样品的检测结果显示，该qPCR方法对ALV-J的检出率（15.6%）高于p27抗原ELISA和普通PCR的检出率（12.2%）。检测模板筛选试验中，抗凝血DNA最符合检测准确度高、操作复杂度和成本低的要求，为最佳检测模板。混样方法摸索试验中，混样总体积为10 μL（混样规模<12份）的混样方法检测准确性最高，为最佳混样方法。在样本数为400份，预期流行率为4%~5%场景的模拟筛查中，ALV-J-B-qPCR检出率为6.25%，比病毒分离法高2.00%；在样本数为400份，预期流行率为1%~2%场景的模拟筛查中，ALV-J-B-qPCR检出率为2.25%，比病毒分离法高0.75%。本研究建立的ALV-J-B-qPCR技术具有灵敏性高、检测快速和节约成本的优势，为种禽场加快ALV净化进程提供了新的思路和方法。

关键词: J亚群禽白血病病毒, 实时荧光定量PCR, 混样检测, 筛查技术

Abstract: To shorten the detection cycle of subgroup J avian leukosis virus (ALV-J) and further accelerate the process of avian leukosis decontamination, this study combined SYBR Green Ⅰ qPCR and pooling sample assay to establish a blood nucleic acid screening technique (ALV-J-B-qPCR) for rapid screening of ALV-J in low prevalence. Based on the sequences of ALV-J pol and env genes in GenBank, the specific primers for ALV-J were designed and the reaction conditions were optimized to establish the ALV-J SYBR Green Ⅰ qPCR assay. Anticoagulant DNA, blood cell DNA, peripheral blood lymphocyte DNA, peripheral blood lymphocyte cDNA and plasma cDNA were respectively prepared to perform PCR detection of ALV-J, and the best assay templates were screened. Further, the accuracy of the three mixing methods of extracting DNA from mixed blood by red blood cell cracking method, the mixed method with 200 μL total volume of blood mixed sample and the mixed method with 10 μL total volume of blood mixed sample were compared, and the best mixing method was selected. The ALV-J-B-qPCR was established by combining the above qPCR method, assay template and mixing method. Simulated screening tests with expected prevalence scenarios of 1%-2% and 4%-5% were performed using ALV-J-B-qPCR respectively, and compared with the virus isolation method. Specificity, sensitivity and reproducibility tests of the qPCR method showed that the method specifically amplifies only ALV-J and has a minimum detection limit of 1×10² copies·μL^-1 for standard plasmids, with intra-and inter-batch coefficients of variation <1%. Results on 90 clinical samples showed that the detection rate of ALV-J by this qPCR method (15.6%) was higher than that of p27 antigen ELISA and PCR (12.2%). In the assay template screening test, anticoagulated blood DNA best met the requirements of high assay accuracy, operational complexity and low cost, and was the best assay template. In the mixing method screening test, the mixing method with a total mixing volume of 10 μL (mixing size <12) had the highest detection accuracy and was the best mixing method. In a simulated screening with a sample size of 400 and an expected prevalence of 4%-5%, the detection rate of ALV-J-B-qPCR was 6.25%, which was 2.00% higher than that of the virus isolation method; in a simulated screening with a sample size of 400 and an expected prevalence of 1%-2%, the detection rate of ALV-J-B-qPCR was 2.25%, which was 0.75% higher than that of the virus isolation method. The ALV-J-B-qPCR technique established in this study has the advantages of high sensitivity, rapid detection and cost saving, which provides a new idea and method to accelerate the ALV purification process in breeding poultry farms.

Key words: ALV-J, qPCR, pooling sample assay, screening technique

中图分类号:

S855.3

董欣怡, 李锦群, 陈钦玺, 廖明, 曹伟胜. 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 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.

参考文献

[1] LI H J, WANG P K, LIN L 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.
[2] LIU P, LI L Z, JIANG Z, et al. Molecular characteristics of subgroup J avian leukosis virus isolated from yellow breeder chickens in Guangdong, China, during 2016-2019[J]. Infect Genet Evol, 2021, 89:104721.
[3] MENG F F, LI Q C, ZHANG Y B, et al. Isolation and characterization of subgroup J avian leukosis virus associated with hemangioma in commercial Hy-line chickens[J]. Poult Sci, 2018, 97(8):2667-2674.
[4] ZHOU D F, XUE J W, ZHANG Y, et al. Outbreak of myelocytomatosis caused by mutational avian leukosis virus subgroup J in China, 2018[J]. Transbound Emerg Dis, 2019, 66(2):622-626.
[5] 郭雨欣, 游广炬, 李晓齐, 等. 2株J亚群禽白血病病毒毒株的分离鉴定及全基因组序列分析[J]. 中国兽医杂志, 2021, 57(6):7-12, 18.
GUO Y X, YOU G J, LI X Q, et al. Isolation, Identification and genome sequence analysis of two avian leukemia virus strains of subgroup J[J]. Chinese Journal of Veterinary Medicine, 2021, 57(6):7-12, 18. (in Chinese)
[6] 马美哥, 于蒙蒙, 许传田, 等. 进口白羽肉种鸡J亚群禽白血病病毒的全基因组序列分析[J]. 中国预防兽医学报, 2019, 41(7):750-754.
MA M G, YU M M, XU C T, et al. Full-length genome sequence analysis of subgroup J avian leukosis viruses isolated from imported white feather broiler breeders[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(7):750-754. (in Chinese)
[7] WU L, LI Y J, CHEN X Y, et al. Isolation and characterization of avian leukosis virus subgroup J associated with hemangioma and myelocytoma in layer chickens in China[J]. Front Vet Sci, 2022, 9:970818.
[8] PAYNE L N, NAIR V. The long view:40 years of avian leukosis research[J]. Avian Pathol, 2012, 41(1):11-19.
[9] 崔治中, 孙淑红, 赵鹏, 等. 对种鸡场禽白血病净化方案的建议[J]. 中国家禽, 2014, 36(1):3-6.
CUI Z Z, SUN S H, ZHAO P, et al. Suggestions on the purification plan for avian leukemia in poultry farms[J]. China Poultry, 2014, 36(1):3-6. (in Chinese)
[10] 叶建强, 秦爱建, 邵红霞, 等. J亚群禽白血病病毒(ALV-J)ELISA检测方法的建立[J]. 中国兽医学报, 2006, 26(3):235-237.
YE J Q, QIN A J, SHAO H X, et al. Establishment of ELISA for diagnosis of subgroup J of avian leukosis virus(ALV-J)[J]. Chinese Journal of Veterinary Science, 2006, 26(3):235-237. (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. Development and preliminary application of ELISA method for detection of avian leukosis virus[J]. Chinese Journal of Animal Infectious Diseases, 2020, 28(1):16-21. (in Chinese)
[12] GAO Q, YUN B L, WANG Q, et al. Development and application of a multiplex PCR method for rapid differential detection of subgroup A, B, and J avian leukosis viruses[J]. J Clin Microbiol, 2014, 52(1):37-44.
[13] CHEN J, ZHAO Z J, CHEN Y Y J, et al. Development and application of a SYBR green real-time PCR for detection of the emerging avian leukosis virus subgroup K[J]. Poult Sci, 2018, 97(7):2568-2574.
[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] 张自豪, 程菲, 刘双, 等. 多种比例混合样本对新型冠状病毒核酸检测结果的影响[J]. 中华医院感染学杂志, 2021, 31(19):2886-2890.
ZHANG Z H, CHENG F, LIU S, et al. Impact of mixed samples in various proportions on the detection of novel coronavirus nucleic acids[J]. Chinese Journal of Nosocomiology, 2021, 31(19):2886-2890. (in Chinese)
[16] 黄中钰, 赵秀英. 新型冠状病毒核酸混合样本检测的研究进展[J]. 北京医学, 2021, 43(3):246-249.
HUANG Z Y, ZHAO X Y. Research progress in detection of novel coronavirus nucleic acid mixed samples[J]. Beijing Medical Journal, 2021, 43(3):246-249. (in Chinese)
[17] SUN X, DENG L, ZHAN X, et al. Pooled sampling is an efficient and economical strategy for SARS-CoV-2 detection in low-prevalence areas[J]. Clin Lab, 2022, 68(5):2390-2395.
[18] PHAN T, TRAN N Y K, GOTTLIEB T, et al. Evaluation of the influenza and respiratory syncytial virus (RSV) targets in the AusDiagnostics SARS-CoV-2, influenza and RSV 8-well assay:sample pooling increases testing throughput[J]. Pathology, 2022, 54(4):466-471.
[19] 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.
[20] 罗颖, 陈晓辉, 李维, 等. 用于新冠肺炎大规模筛查和监测的混合样本采集策略[J]. 中国国境卫生检疫杂志, 2022, 45(1):34-36.
LUO Y, CHEN X H, LI W, et al. Hybrid sample pooling strategy for large-scale COVID-19 screening and surveillance[J]. Chinese Journal of Frontier Health and Quarantine, 2022, 45(1):34-36. (in Chinese)
[21] PROCOP G W, TUOHY M, RAMSEY C, et al. Asymptomatic patient testing after 10:1 pooling using the Xpert Xpress SARS-CoV-2 Assay[J]. Am J Clin Pathol, 2021, 155(4):522-526.
[22] PANHOTRA B R, JOSHI C S, UL HASSAN Z, et al. Nucleic acid amplification technology for screening of HBV DNA among anti-HBc-positive units of blood and blood donors[J]. Transfus Med, 2006, 16(1):73-74.
[23] LAPERCHE S. Blood safety and nucleic acid testing in Europe[J]. Euro Surveill, 2005, 10(2):3-4.
[24] GRANT P R, BUSCH M P. Nucleic acid amplification technology methods used in blood donor screening[J]. Transfus Med, 2002, 12(4):229-242.
[25] 黄小珍, 曾宾, 刘佳霖. 核酸检测技术在无偿献血者血液筛查中的应用[J]. 海南医学, 2018, 29(10):1465-1467.
HUANG X Z, ZENG B, LIU J L. Application of nucleic acid detection technology in blood screening of voluntary blood donors[J]. Hainan Medical Journal, 2018, 29(10):1465-1467. (in Chinese)
[26] 李榕. 核酸检测背后的数学原理[J]. 中学数学研究:华南师范大学版, 2021(7):46-47.
LI R. The mathematical principles behind nucleic acid testing[J]. Middle School Mathematics Research:South China Normal University Edition, 2021(7):46-47. (in Chinese)
[27] 赵小艳. 基于数学期望的新冠肺炎核酸检测方法[J]. 大学数学, 2020, 36(6):19-22.
ZHAO X Y. COVID-19 testing for nucleic acid based on mathematical expectation[J]. College Mathematics, 2020, 36(6):19-22. (in Chinese)
[28] 陈杨一骏. 惠阳胡须鸡禽白血病病原学调查及受体基因遗传多态性分析[D]. 广州:华南农业大学, 2018.
CHEN Y Y J. Etiological investigation of avian leukosis and analysis on genetic polymorphism of avian leukosis virus receptor in Huiyang bearded chicken[D]. Guangzhou:South China Agricultural University, 2018. (in Chinese)
[29] LEE J, KIM S Y, SUNG H, et al. Challenges and issues of SARS-CoV-2 pool testing[J]. Lancet Infect Dis, 2020, 20(11):1232-1233.
[30] 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 Path, 2023, 32(3):375-381.
[31] 余波, 霍细香, 汪杨, 等. 新型冠状病毒灭活样品混合进行核酸检测效果分析[J]. 公共卫生与预防医学, 2020, 31(5):11-14.
YU B, HUO X X, WANG Y, et al. Impact of pooling of inactivated SARS-CoV-2 samples on nucleic acid detection[J]. Journal of Public Health and Preventive Medicine, 2020, 31(5):11-14. (in Chinese)
[32] 孙微超, 崔志刚, 吉尚志, 等. 核酸血筛试剂6混样和48混样检测在血液筛查中的适用性评价[J]. 检验医学与临床, 2019, 16(19):2803-2806.
SUN W C, CUI Z G, JI S Z, et al. Evaluation of applicability of nucleic acid blood screen reagent 6 mixed samples and 48 mixed samples detection in blood screening[J]. Laboratory Medicine and Clinic, 2019, 16(19):2803-2806. (in Chinese)
[33] 彭昊, 吴元俊, 秦丽莉, 等. 不同品种禽类ALV和REV感染情况调查[J]. 畜牧兽医学报, 2013, 44(12):1989-1993.
PENG H, WU Y J, QIN L L, et al. Investigation on the infections of ALV and REV in different poultry species and breeds[J]. Acta Veterinaria et Zootechnica Sinica, 2013, 44(12):1989-1993. (in Chinese)
[34] 李家俊, 郑潇, 盛杰, 等. 新型冠状病毒及其临床检测方法研究进展[J]. 生物技术通报, 2021, 37(4):282-292.
LI J J, ZHENG X, SHENG J, et al. Novel coronavirus and research progress of related clinical detection methods[J]. Biotechnology Bulletin, 2021, 37(4):282-292. (in Chinese)
[35] 钱榕, 熊丽红, 刘强. 血液标本凝块对核酸检测混合加样的影响[J]. 中国输血杂志, 2012, 25(6):586-587.
QIAN R, XIONG L H, LIU Q. The effect of blood sample clots on mixed sample addition in nucleic acid testing[J]. Chinese Journal of Blood Transfusion, 2012, 25(6):586-587. (in Chinese)
[36] 吴晓晋, 陈宏伟, 黄志翔, 等. 两种血液核酸筛查系统的核酸筛查能力比较研究[J]. 中国医学装备, 2022, 19(9):26-30.
WU X J, CHEN H W, HUANG Z X, et al. Comparative research of screening abilities of blood nucleic acid between two kinds of blood nucleic acid screening systems[J]. China Medical Equipment, 2022, 19(9):26-30. (in Chinese)