绵羊肺炎支原体和山羊伪结核棒状杆菌双重PCR检测方法的建立及应用

黄丽丽; 林裕胜; 肖义军; 胡奇林

doi:10.19303/j.issn.1008-0384.2020.01.007

绵羊肺炎支原体和山羊伪结核棒状杆菌双重PCR检测方法的建立及应用

doi: 10.19303/j.issn.1008-0384.2020.01.007

黄丽丽^{1, 2,},
林裕胜²,
肖义军¹,
胡奇林^2, ,

1.
福建师范大学生命科学学院，福建　福州　350108
2.
福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 国家重点研发计划项目（2016YFD0500906）；福建省农业科学院科技创新团队建设项目（STIT2017-1-5）；福建省科技计划公益类专项（2018R1023-1）

详细信息

作者简介:
黄丽丽（1991−），女，硕士研究生，主要从事动物传染病研究（E-mail：huanglilixj2012@163.com）

通讯作者:
胡奇林（1963−），男，研究员，主要从事动物传染病学和免疫学研究（E-mail：hql562713@163.com）

中图分类号: S 852.62
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出版历程
- 收稿日期: 2019-10-15
- 修回日期: 2019-12-25
- 刊出日期: 2020-01-01

A New Multiplex PCR Assay for Simultaneously Detecting Mycoplasma ovipneumoniae and Corynebacterium pseudotuberculosis in Diseased Goats and Sheep

HUANG Li-li^{1, 2
,},
LIN Yu-sheng²,
XIAO Yi-jun¹,
HU Qi-lin^{2
, ,}

1.
College of Life Sciences, Fujian Normal University, Fuzhou, Fujian　350108, China
2.
Institute of Animal Husbandry and Veterinary, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的为实现临床上对绵羊肺炎支原体（Mycoplasma ovipneumoniae, Mo）和山羊伪结核棒状杆菌（Corynebacterium pseudotuberculosis, CP）两种病原混合感染的同时检测，建立Mo和CP的双重PCR检测方法。方法应用基于Mo的P80基因和CP的PLD基因设计两对特异性引物，对反应体系和条件进行优化，验证方法的特异性、灵敏性和重复性；应用该方法和单一PCR方法对临床收集的70份样品进行检测，比较检测结果。结果建立的Mo和CP的双重PCR检测方法能够同时扩增出Mo 700 bp和CP 290 bp左右的片段，而对其他羊病常见病原的DNA扩增均为阴性。Mo和CP的检测下限分别为1 530 pg·μL⁻¹和3 500 pg·μL⁻¹。该方法具有良好的重复性。临床样品检测结果显示：70份临床样品中Mo阳性率为25.71%，CP阳性率为18.57%，同时感染Mo和CP阳性率为7.14%，与单一PCR检测结果符合率为100%。结论本研究建立的双重PCR检测方法具有特异性强、灵敏性较高、重复性好等特点，为临床上Mo和CP混合感染的快速检测以及流行病学调查提供了实用方法。
- 绵羊肺炎支原体 /
- 伪结核棒状杆菌 /
- 双重PCR
Abstract: Objective To establish a multiplex PCR method for simultaneous clinic detection of Mycoplasma ovipneumoniae (Mo), one of the main pathogens causing Mycoplasma pneumonia of goats and sheep(MPGS), and Corynebacterium pseudotuberculosis (CP), the pathogen of goat pseudotuberculosis, in the livestock animals infected by both pathogens at a same time. Method Two pairs of specific primers were designed based on the P80 gene of Mo and the PLD gene of CP for the PCR. After optimizing the detection system and reaction conditions, the specificity, sensitivity, and repeatability of the new method were verified for methodology validation. The results obtained using two separate PCR and the multiplex PCR on 70 clinical specimens were comparatively analyzed. Result The newly developed multiplex PCR method amplified a 700 bp fragment from Mo and a 290 bp fragment from CP, with none from other common pathogens of goats and sheep. The method had a detection limit of 1 530 pg·μL⁻¹ on Mo and 3 500 pg·μL⁻¹ on CP with a high repeatability. A test on 70 clinical specimens using the new method yielded a 25.71% positive rate on Mo, 18.57% on CP, and 7.14% on Mo/CP infections. Its coincidence rate with the two single PCR results was 100%. Conclusion The newly established multiplex PCR detection methodology exhibited high specificity, sensitivity, and reproducibility. It appeared to be applicable for rapid and simultaneous clinic detection on Mo and CP as well as epidemiological studies on the disease infected by the pathogens.
- Mycoplasma ovipneumoniae /
- Corynebacterium pseudotuberculosis /
- multiplex PCR

HTML全文

图 1 Mo和CP单一和双重PCR扩增

注：M: 2 000 DNA Ladder；1: Mo和CP；2：Mo；3: CP；4: 阴性对照。

Figure 1. Single and multiplex PCR amplifications of Mo and CP

Note：M: 2 000 DNA Ladder; 1: Mo and CP; 2: Mo; 3: CP; 4: negative control.

下载: 全尺寸图片幻灯片

图 2 双重PCR特异性试验

注：M：2 000 DNA Ladder；1：Mo+CP；2–11分别为CP、Mo、Mmc、Mcc、Mccp、AL、Mb、SE、Ec、SA；12: 空白对照。

Figure 2. Specificity of multiplex PCR

Note：M: 2 000 DNA Ladder; 1: Mo and CP; 2–11: CP, Mo, Mmc, Mcc, Mccp, AL, Mb, SE, Ec, and SA, respectively; 12: blank control.

下载: 全尺寸图片幻灯片

图 3 双重PCR灵敏性试验

注：M: 2 000 DNA Ladder；1: Mo＋CP阳性对照；2–9: 分别为10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸倍稀释的含Mo和CP的阳性样本；10: 阴性对照。

Figure 3. Sensitivity of multiplex PCR

Note：M: 2 000 DNA Ladder; 1: Mo and CP positive control; 2–9: positive specimens containing Mo and CP diluted 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ times, respectively; 10: negative control.

下载: 全尺寸图片幻灯片

图 4 单一PCR灵敏性试验

注：A中，M: 2 000 DNA Ladder；1: Mo阳性对照；2–9: 分别为10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸倍稀释的含Mo的阳性样本；10: 阴性对照；B中，M: 2 000 DNA Ladder；1: CP 阳性对照；2–9分别为10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸倍稀释的含CP 的阳性样本；10: 阴性对照。

Figure 4. Sensitivity of single PCR

Note：A：M: 2 000 DNA Ladder; 1: Mo positive control; 2–9: positive specimens containing Mo diluted 10¹，10²，10³，10⁴，10⁵，10⁶，10⁷，10⁸ times, respectively; 10: negative control; B: M: 2 000 DNA Ladder; 1: CP positive control; 2–9: positive specimens containing CP diluted 10¹，10²，10³，10⁴，10⁵，10⁶，10⁷，10⁸ times, respectively; 10: negative control.

下载: 全尺寸图片幻灯片

图 5 双重PCR扩增重复性试验

注：M: 2 000 DNA Ladder；1: 阳性对照；2–5: Mo；6–10: CP；11–16分别为Mccp、Mmc、AL、Ec、SA和SE；17: 空白对照。

Figure 5. Repeatability of multiplex PCR

Note：M: 2 000 DNA Ladder; 1: positive control; 2–5: Mo; 6–10: CP; 11–16: Mccp, Mmc, AL, Ec, SA, and SE, respectively; 17: blank control.

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

靶基因 Target gene	引物名称 Primer name	引物序列（5′→ 3′） Primer sequence（5′→ 3′）	扩增片段大小 Amplified fragment size/bp
P80基因	F1	GCCTTGGGGTTGGAATTCCTTTGTCTTATTC	705
P80基因	R1	CATTTGATGCTGAGGTCGGATTTGGACTAAC	705
PLD基因	F2	GTGAGAAGAACCCCGGTATAAG	291
PLD基因	R2	TACCGCACTTATTCTGACACTG	291

下载: 导出CSV

表 2 临床样品检测结果

Table 2. Detection of pathogens on clinical specimens

样品名称 Sample name	Mo阳性率 Mo positive rate/%	CP阳性率 CP positive rate/%	Mo + CP阳性率 Mo + CP positive rate/%
鼻拭子 Nasal cotton swab	18.60	11.63	4.65
肺组织 Lung tissue	37.04	29.63	11.11
所有样品 All samples	25.71	18.57	7.14

下载: 导出CSV

参考文献(41)

[1]	邓守全, 霍晓伟. 一株绵羊肺炎支原体的分离及鉴定 [J]. 中国生物制品学杂志, 2017, 30(11):1153−1156. DENG S Q, HUO X W. Separation and identification of a strain of sheep Mycoplasma pneumonia [J]. Chinese Journal of Biologicals, 2017, 30(11): 1153−1156.(in Chinese)
[2]	崔康康, 姚亚军, 肖虎锁, 等. 一起羊支原体性肺炎的诊疗 [J]. 山东畜牧兽医, 2017, 38(10):38. doi: 10.3969/j.issn.1007-1733.2017.10.026 CUI K K, YAO Y J, XIAO H S, et al. A diagnosis and treatment of Mycoplasma pneumoniae of sheep [J]. Shandong Journal of Animal Science and Veterinary Medicine, 2017, 38(10): 38.(in Chinese) doi: 10.3969/j.issn.1007-1733.2017.10.026
[3]	陆震, 吴翠兰, 李军, 等. 绵羊肺炎支原体病的诊断与防制 [J]. 今日畜牧兽医, 2019, 35(6):80−81. doi: 10.3969/j.issn.1673-4092.2019.06.075 LU Z, WU C L, LI J, et al. Research Progress of Mycoplasma Pneumonia in Sheep [J]. Today Animal Husbandry and Veterinary Medicine, 2019, 35(6): 80−81.(in Chinese) doi: 10.3969/j.issn.1673-4092.2019.06.075
[4]	BESSER T E, CASSIRER E F, POTTER K A, et al. Association of Mycoplasma ovipneumoniae infection with population limiting respiratory disease in free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) [J]. Journal of Clinical Microbiology, 2008, 46(2): 423−430. doi: 10.1128/JCM.01931-07
[5]	DASSANAYAKE R P, SHANTHALINGAM S, HERNDON C N, et al. Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia [J]. Veterinary Microbiology, 2010, 145(3-4): 354−359. doi: 10.1016/j.vetmic.2010.04.011
[6]	RIFATBEGOVIC M, MAKSIMOVIC Z, HULAJ B. Mycoplasma ovipneumoniae associated with severe respiratory disease in goats [J]. Veterinary Record, 2011, 168(21): 565.
[7]	YANG F, DAO X, RODRIGUEZ- PALACIOS A, et al. A Real-time PCR for detection and quantification of Mycoplasma ovipneumoniae [J]. Journal of Veterinary Medical Science, 2014, 76(12): 1631−1634. doi: 10.1292/jvms.14-0094
[8]	储岳峰. 我国山羊(接触)传染性胸膜肺炎病原学、流行病学研究及灭活疫苗的研制[D]. 北京: 中国农业科学院研究生院兰州兽医研究所, 2011. CHU Y F. An etiological and epidemiological study on contagious caprine pleuropneumonia in China and development of a bacterin against it[D]. Beijing: Lanzhou Veterinary Research Institute, Graduate School of Chinese Academy of Agricultural Sciences, 2011. (in Chinese)
[9]	李文杨, 刘远, 张晓佩, 等. 山羊伪结核棒状杆菌的分离鉴定 [J]. 中国草食动物科学, 2014, 34(5):49−51. doi: 10.3969/j.issn.2095-3887.2014.05.015 LI W Y, LIU Y, ZHANG X P, et al. Isolation and Identification of Corynebacterium pseudotuberculosis [J]. China Herbivore Science, 2014, 34(5): 49−51.(in Chinese) doi: 10.3969/j.issn.2095-3887.2014.05.015
[10]	BAIRD G J, FONTAINE M C. Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis [J]. Journal of Comparative Pathology, 2007, 137(4): 179−210. doi: 10.1016/j.jcpa.2007.07.002
[11]	周作勇, 李和贤, 杨浩钺, 等. 伪结核棒状杆菌毒力因子的研究进展 [J]. 中国人兽共患病学报, 2017, 33(12):1115−1119. doi: 10.3969/j.issn.1002-2694.2017.12.012 ZHOU Z Y, LI H X, YANG H Y, et al. Research advances on the virulence factors of Corynebacterium pseudotuberculosis [J]. Chinese Journal of Zoonoses, 2017, 33(12): 1115−1119.(in Chinese) doi: 10.3969/j.issn.1002-2694.2017.12.012
[12]	徐志豪, 黄诗琴, 李乡城, 等. 四川省山羊皮下脓肿的流行病学调查及病原体分析 [J]. 中国兽医科学, 2016, 46(8):991−996. XU Z H, HUANG S Q, LI X C, et al. Epidemiological survey and etiological analysis in goat with subcutaneous abscess in Sichuan Province [J]. Chinese Veterinary Science, 2016, 46(8): 991−996.(in Chinese)
[13]	马玉馨, 李文贵, 郑国英, 等. 山羊伪结核棒状杆菌TaqMan荧光定量PCR检测方法的建立和初步应用 [J]. 畜牧与兽医, 2018, 50(10):82−87. MA Y X, LI W G, ZHENG G Y, et al. Establishment and preliminary application of TaqMan real-time PCR for detection of Corynebacterium pseudotuberculosis in goats [J]. Animal Husbandry & Veterinary Medicine, 2018, 50(10): 82−87.(in Chinese)
[14]	赵宏坤, 范伟兴, 胡敬东, 等. 羊伪结核病研究进展 [J]. 中国预防兽医学报, 2000, 22(3):77−78. ZHAO H K, FAN W X, HU J D, et al. Research progress of pseudotuberculosis in sheep [J]. Chinese Journal of Preventive Veterinary Medicine, 2000, 22(3): 77−78.(in Chinese)
[15]	王全溪, 李国平. 波尔山羊伪结核棒状杆菌的分离鉴定 [J]. 中国兽医杂志, 2004, 40(9):53−54. doi: 10.3969/j.issn.0529-6005.2004.09.023 WANG Q X, LI G P. Isolation and Identification of pseudotuberculosis corynebacterium on Boer Goat [J]. Chinese Journal of Veterinary Medicine, 2004, 40(9): 53−54.(in Chinese) doi: 10.3969/j.issn.0529-6005.2004.09.023
[16]	赵宏坤, 周维武, 色音巴亚尔, 等. 由溶解的山羊胎儿分离出一株羊伪结核棒状杆菌 [J]. 中国畜禽传染病, 1994, 16(2):31−32. ZHAO H K, ZHOU W W, SAYINBAYAR, et al. A strain of Corynebacterium pseudotuberculosis isolated from a dissolved goat fetus [J]. Chinese Journal of Animal and Poultry Infectious Diseases, 1994, 16(2): 31−32.(in Chinese)
[17]	韩瑞鑫, 申捷, 申之义. 绵羊肺炎支原体TaqMan实时荧光定量PCR检测方法的建立与应用 [J]. 中国兽医科学, 2018, 48(8):965−970. HAN R X, SHEN J, SHEN Z Y. Development and application of TaqMan real-time fluorescent quantitation PCR for the detection of Mycoplasma ovipneumoniae [J]. Chinese Veterinary Science, 2018, 48(8): 965−970.(in Chinese)
[18]	林裕胜, 江锦秀, 张靖鹏, 等. 绵羊肺炎支原体RPA检测技术的建立 [J]. 福建农业学报, 2019, 34(4):416−421. LIN Y S, JIANG J X, ZHANG J P, et al. Establishment of a RPA Method for Detecting Mycoplasma pneumoniae [J]. Fujian Journal of Agricultural Sciences, 2019, 34(4): 416−421.(in Chinese)
[19]	王娟, 刀筱芳, 刘海燕, 等. 绵羊肺炎支原体恒温热隔绝式PCR方法的建立 [J]. 中国预防兽医学报, 2019, 41(4):380−384. WANG J, DAO X F, LIU H Y, et al. Development of an insulated isothermal PCR for detection of Mycoplasma pneumoniae [J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(4): 380−384.(in Chinese)
[20]	张双翔, 周碧君, 程振涛, 等. 绵羊肺炎支原体LAMP检测方法的建立及初步应用 [J]. 中国兽医学报, 2013, 33(3):362−366. ZHANG S X, ZHOU B J, CHENG Z T, et al. Development and application of a loop-mediated isothermal amplification assay for detection of Mycoplasma ovipneumoniae [J]. Chinese Journal of Veterinary Science, 2013, 33(3): 362−366.(in Chinese)
[21]	赵萍, 储岳峰, 高鹏程, 等. 绵羊肺炎支原体间接ELISA检测方法的建立及应用 [J]. 福建农林大学学报(自然科学版), 2008, 37(5):510−513. ZHAO P, CHU Y F, GAO P C, et al. Establishment and application of indirect ELISA method for Mycoplasma ovipneumoniae [J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2008, 37(5): 510−513.(in Chinese)
[22]	许国洋, 付利芝, 龙小飞, 等. 山羊伪结核棒状杆菌PMA-PCR检测方法的建立 [J]. 现代畜牧兽医, 2018(11):9−13. XU G Y, FU L Z, LONG X F, et al. Establishment of PMA-PCR detection method for Corynebacterium pseudotuberculosis in Goat [J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2018(11): 9−13.(in Chinese)
[23]	郑敏, 胡杰, 欧绍毅, 等. 广西黑山羊伪结核棒状杆菌的分离与鉴定 [J]. 广西农业科学, 2010, 41(10):1128−1130. ZHENG M, HU J, OU S Y, et al. Isolation and Identification of Corynebacterium pseudotuberculosis from black goat in Guangxi [J]. Guangxi Agricultural Sciences, 2010, 41(10): 1128−1130.(in Chinese)
[24]	韦志锋, 李常挺, 陶立, 等. 山羊干酪性淋巴结炎病原的分离和鉴定 [J]. 中国动物检疫, 2011, 28(7):54−55, 59. doi: 10.3969/j.issn.1005-944X.2011.07.028 WEI Z F, LI C T, TAO L, et al. Isolation and identification of goat caseinous lymphadenitis [J]. China Animal Health Inspection, 2011, 28(7): 54−55, 59.(in Chinese) doi: 10.3969/j.issn.1005-944X.2011.07.028
[25]	朱伟英, 猴银行, 霍宁宁, 等. 一株奶山羊伪结核棒状杆菌的分离与鉴定 [J]. 动物医学进展, 2017, 38(5):127−132. doi: 10.3969/j.issn.1007-5038.2017.05.029 ZHU W Y, HOU Y H, HUO N N, et al. Isolation and identification of corynebacterium pseudotuberculosis from goats [J]. Progress in Veterinary Medicine, 2017, 38(5): 127−132.(in Chinese) doi: 10.3969/j.issn.1007-5038.2017.05.029
[26]	王韡, 张彦明, 邢福珊, 等. 羊伪结核棒状杆菌的分离鉴定及ELISA检测方法的建立 [J]. 中国兽医学报, 2005, 25(1):34−36. doi: 10.3969/j.issn.1005-4545.2005.01.012 WANG W, ZHANG Y M, XING F S, et al. Isolation, identification of Corynebacterium pseudotuberculosis and establishment of ELISA detection method [J]. Chinese Journal of Veterinary, 2005, 25(1): 34−36.(in Chinese) doi: 10.3969/j.issn.1005-4545.2005.01.012
[27]	黄丽丽, 林裕胜, 江锦秀, 等. 山羊伪结核棒状杆菌的分离鉴定及耐药性分析 [J]. 福建农业学报, 2019, 34(8):925−932. HUANG L L, LIN Y S, JIANG J X, et al. Isolation, identification and antibiotics resistance of corynebacterium pseudotuberculosis from goats [J]. Fujian Journal of Agricultural Sciences, 2019, 34(8): 925−932.(in Chinese)
[28]	江锦秀, 林甦, 林裕胜, 等. 绵羊肺炎支原体FJ01-CL株的分离和鉴定 [J]. 福建农业学报, 2015, 30(5):430−434. doi: 10.3969/j.issn.1008-0384.2015.05.002 JIANG J X, LIN S, LIN Y S, et al. Isolation and identification of Mycoplasma ovipneumoniae FJ01-CL [J]. Fujian Journal of Agricultural Sciences, 2015, 30(5): 430−434.(in Chinese) doi: 10.3969/j.issn.1008-0384.2015.05.002
[29]	江锦秀, 林裕胜, 游伟, 等. 丝状支原体山羊亚种FJ-GT株的分离和鉴定 [J]. 中国农学通报, 2016, 32(29):11−16. doi: 10.11924/j.issn.1000-6850.casb16030148 JIANG J X, LIN Y S, YOU W, et al. Isolation and identification of Mycoplasma mycoides subsp. capri FJ-GT strain [J]. Chinese Agricultural Science Bulletin, 2016, 32(29): 11−16.(in Chinese) doi: 10.11924/j.issn.1000-6850.casb16030148
[30]	江锦秀, 林裕胜, 游伟, 等. 山羊莱氏无胆甾原体FJ-NP株的分离鉴定 [J]. 中国畜牧兽医, 2016, 43(12):3329−3335. JIANG J X, LIN Y S, YOU W, et al. Isolation and identification of acholeplasma laidlawii FJ-NP strains in goat [J]. China Animal Husbandry & Veterinary Medicine, 2016, 43(12): 3329−3335.(in Chinese)
[31]	江锦秀, 林裕胜, 游伟, 等. 牛支原体FJ-HJ分离株的分离及鉴定 [J]. 中国农学通报, 2017, 33(20):107−112. JIANG J X, LIN Y S, YOU W, et al. Isolation and identification of mycoplasma bovis FJ-HJ strain [J]. Chinese Agricultural Science Bulletin, 2017, 33(20): 107−112.(in Chinese)
[32]	林裕胜, 江锦秀, 江斌, 等. ORFV和Mo双重PCR检测方法的建立及应用 [J]. 农业生物技术学报, 2017, 25(8):1374−1380. LIN Y S, JIANG J X, JIANG B, et al. Establishment and application of a duplex pcr assay for detection of orf virus (ORFV) and Mycoplasma ovipneumoniae (Mo) [J]. Journal of Agricultural Biotechnology, 2017, 25(8): 1374−1380.(in Chinese)
[33]	ANDERSON D E, RINGS D M, KOWALSKI J. Infection with Corynebacterium pseudotuberculosis in five alpacas [J]. Journal of the American Veterinary Medical Association, 2004, 225(11): 1743−1747, 1702. doi: 10.2460/javma.2004.225.1743
[34]	STEHMAN S M. Paratuberculosis in small ruminants, deer, and south american camelids [J]. Veterinary Clinics of North America: Food Animal Practice, 1996, 12(2): 441−455. doi: 10.1016/S0749-0720(15)30416-3
[35]	DASSANAYAKE R P, SHANTHALINGAM S, HERNDON C N, et al. Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia [J]. Veterinary Microbiology, 2010, 145(3/4): 354−359.
[36]	李庆超.多重PCR技术同时高效检测食源性致病菌的研究[D]. 吉林大学, 2017. LI Q C. Study of multiplex PCR technology in the simultaneously and efficiently detection of food-borne pathogenic bacteria[D]. Changchun: Jilin University, 2017. (in Chinese)
[37]	侯宏艳, 惠文巧, 张丹俊, 等. 山羊的绵羊肺炎支原体的分离和鉴定 [J]. 安徽农业科学, 2015, 43(28):159−160. doi: 10.3969/j.issn.0517-6611.2015.28.062 HOU H Y, HUI W Q, ZHANG D J, et al. Isolation and Identification of Mycoplasma ovipneumoniae from goats [J]. Journal of Anhui Agricultural Sciences, 2015, 43(28): 159−160.(in Chinese) doi: 10.3969/j.issn.0517-6611.2015.28.062
[38]	张洁, 曹军军, 祝明松, 等. 基于绵羊肺炎支原体EF-Tu蛋白间接ELISA方法的建立 [J]. 动物医学进展, 2019, 40(11):6−13. ZHANG J, CAO J J, ZHU M S, et al. Establishment of indirect ELISA method based on Mycoplasma pneumoniae EF-Tu protein [J]. Progress in Veterinary Medicine, 2019, 40(11): 6−13.(in Chinese)
[39]	王璇, 潘淑惠, 万一元. 山羊伪结核棒状杆菌病的ELISA诊断 [J]. 上海畜牧兽医通讯, 2009(2):29. doi: 10.3969/j.issn.1000-7725.2009.02.013 WANG X, PAN S H, WAN Y Y. ELISA diagnosis of corynebacterium tuberculosis in goats [J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine, 2009(2): 29.(in Chinese) doi: 10.3969/j.issn.1000-7725.2009.02.013
[40]	BESSER T E, HIGHLAND M, BAKER K, et al. Causes of pneumonia epizootics among Bighorn sheep, western United States, 2008–2010 [J]. Emerging Infectious Diseases, 2012, 18(3): 406−414. doi: 10.3201/eid1803.111554
[41]	许国洋, 付利芝, 杨金龙, 等. 山羊淋巴结炎病原菌的分离鉴定及多重PCR检测方法的建立 [J]. 畜牧兽医学报, 2017, 48(2):324−330. doi: 10.11843/j.issn.0366-6964.2017.02.016 XU G Y, FU L Z, YANG J L, et al. Isolation and identification of pathogenic bacteria of lymphadenitis in goat and establishment of multiplex PCR detection method [J]. Chinese Journal of Animal and Veterinary Sciences, 2017, 48(2): 324−330.(in Chinese) doi: 10.11843/j.issn.0366-6964.2017.02.016