Establishment of a RPA Method for Detecting <i>Mycoplasma ovipneumoniae</i>

LIN Yu-sheng; JIANG Jin-xiu; ZHANG Jing-peng; YOU Wei; HU Qi-lin

doi:10.19303/j.issn.1008-0384.2019.04.006

Volume 34 Issue 4

Jul. 2019

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Article Navigation > Fujian Journal of Agricultural Sciences > 2019 > 34(4): 416-421

LIN Yu-sheng, JIANG Jin-xiu, ZHANG Jing-peng, YOU Wei, HU Qi-lin. Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae[J]. Fujian Journal of Agricultural Sciences, 2019, 34(4): 416-421. doi: 10.19303/j.issn.1008-0384.2019.04.006

Citation:

LIN Yu-sheng, JIANG Jin-xiu, ZHANG Jing-peng, YOU Wei, HU Qi-lin. Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae[J]. Fujian Journal of Agricultural Sciences, 2019, 34(4): 416-421. doi: 10.19303/j.issn.1008-0384.2019.04.006

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Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae

doi: 10.19303/j.issn.1008-0384.2019.04.006

Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China

Received Date: 2018-12-27
Rev Recd Date: 2019-01-14
Publish Date: 2019-04-28

Abstract

Abstract

Objective To develop a rapid detecting method for Mycoplasma ovipneumoniae (Mo). Method Based on the P80 gene sequence, specific primers were designed using Oligo 7 software. Conditions of the recombinase polymerase amplification (RPA) method were optimized for the application. Result The new assay detected P80 gene in Mo specifically, not any other common pathogens of sheep and goats. The detection sensitivity was 70 fg·μL^-1, which was the same as provided by conventional PCR. The inter-and intra-batch tests showed that the modified method could amplify the bands on Mo-positive samples, not on Mo-negative specimens, indicating an acceptable repeatability of the methodology. Furthermore, the RPA assay and conventional PCR methods were simultaneously used on 186 clinic samples, as well as the Mycoplasma isolation and identification on 40 lung samples, to show that the RPA assay positively identified all 13 Mo-positive samples detected by Mycoplasma isolation and identification and 95 positive samples detected by the conventional PCR. Conclusion The newly developed RPA method had desirable specificity and repeatability on Mo detection. It could be applied for rapid detecting and epidemiological studies on Mo.
- Mycoplasma ovipneumoniae,
- recombinase polymerase amplification,
- isothermal amplification

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References(29)

References

[1]	BESSER T E, FRANCES CASSIRER E, HIGHLAND M A, et al. Bighorn sheep pneumonia:sorting out the cause of a polymicrobial disease[J]. Preventive Veterinary Medicine, 2013, 108(2-3):85-93. doi: 10.1016/j.prevetmed.2012.11.018
[2]	DASSANAYAKE R P, SHANTHALINGAM S, HEMDON 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
[3]	NIANG M, ROSENBUSCH R F, ANDREWS J J, et al. Demonstration of a capsule on Mycoplasma ovipneumoniae[J]. American Journal of Veterinary Research, 1998, 59(5):557-562.
[4]	NIANG M, ROSENBUSCH R F, DEBEY M C, et al. Field isolates of Mycoplasma ovipneumoniae exhibit distinct cytopathic effects in ovine tracheal organ cultures[J]. Zentralbl Veterinarmed A, 1998, 45(1):29-40. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=0459530fa80250620db67a0991b47fbf
[5]	ETTORRE C, SACCHINI F, SCACCHIA M, et al. Pneumonia of lambs in the Abruzzo region of Italy:anatomopathological and histopathological studies and localisation of Mycoplasma ovipneumoniae[J].Veterinaria Italiana, 2007, 43(1):149-155.
[6]	RIFATBEGOVIC M, MAKSIMOVIC Z, HULAJ B, et al. Mycoplasma ovipneumoniae associated with severe respiratory disease in goats[J]. Veterinary Record, 2011, 168(21):565.
[7]	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 (Oviscanadensiscanadensis)[J]. Journal of Clinical Microbiology, 2018, 46(2):423-430.
[8]	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
[9]	林裕胜, 江锦秀, 江斌, 等.绵羊肺炎支原体、丝状支原体山羊亚种和山羊支原体山羊肺炎亚种多重PCR检测方法的建立[J].中国预防兽医学报, 2018, 40(9):812-817. http://d.old.wanfangdata.com.cn/Periodical/zgyfsyxb201809009 LIN Y S, JIANG J X, JIANG B, et al. Development of a triplex PCR assay for detection of Mycoplasma ovipneumoniae, M.mycoides subsp. capri and M.capricolum subsp. capripneumoniae[J]. Chinese Journal of Preventive and Veterinary Medicine, 2018, 40(9):812-817.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgyfsyxb201809009
[10]	LI M, MA C J, LIU X M, et al. Molecular cloning of HSP70 in Mycoplasma ovipneumoniae and comparison with that of other mycoplasmas[J]. Genetics and Molecular Research, 2011, 10(2):834-848. doi: 10.4238/vol10-2gmr1193
[11]	YANG F, TANG C, WANG Y, et al. Genome sequence of Mycoplasma ovipneumoniae strain SC01[J]. Journal of Bacteriology, 2011, 193(18):5018. doi: 10.1128/JB.05363-11
[12]	RONG G, ZHAO J M, HOU G Y, et al. Seroprevalence and molecular detection of Mycoplasma ovipneumoniae in goats in tropical China[J]. Tropical Animal Health and Production, 2014, 46(8):1491-1495. doi: 10.1007/s11250-014-0645-y
[13]	赵军, 王川庆, 吴艳阳, 等.一种用于检测胞内劳森菌的RPA引物及其检测方法: CN201710652790.5[P]. 2018-08-24. ZHAO J, WANG C Q, Wu Y Y, et al. A RPA primer for detection of intracellular Lawson bacteria and its detection method: CN201710652790.5[P]. 2018-08-24.(in Chinese)
[14]	EULER M, WANG Y J, OTTO P, et al. Recombinase polymerase amplification assay for rapid detection of Francisella tularensis[J]. Journal of Clinical Microbiology, 2012, 50(7):2234-2238. doi: 10.1128/JCM.06504-11
[15]	ABD EI W A, EI-DEDB A, EI-THOLOTH M, et al. A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot and mouth disease virus[J]. PLoS One, 2013, 8(8):e71642. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_3748043
[16]	王建昌, 王金凤, 刘立兵, 等.非洲猪瘟病毒RPA等温检测方法的建立[J].中国动物检疫, 2016, 33(7):78-81, 94. doi: 10.3969/j.issn.1005-944X.2016.07.024 WANG J C, WANG J F, LIU L B, et al. Rapid and sensitive detection of African swine fever virus by recombinase polymerase amplification[J]. China Animal Health Inspection, 2016, 33(7):78-81, 94.(in Chinese) doi: 10.3969/j.issn.1005-944X.2016.07.024
[17]	LILJANDER A, YU M, O'BRIEN E, et al. Field-applicable recombinase polymerase amplification assay for rapid detection of Mycoplasma capricolum subsp. capripneumoniae[J]. Journal of Clinical Microbiology, 2015, 3(9):2810-2815. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=b57bfe26fbdaed22ed395d7b5097fd61
[18]	费媛媛, 李兆才, 娄忠子, 等.动物衣原体RPA检测方法的建立[J].中国兽医科学, 2018, 48(2):142-147. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgsykj201802002 FEI Y Y, LI Z C, LOU Z Z, et al. Detection of Chlamydia in livestock by recombinase polymerase amplification[J]. Chinese Veterinary Science, 2018, 48(2):142-147.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgsykj201802002
[19]	BOYLE D S, MCNEMEY R, TENG L H, et al. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification[J].PLoS One, 2014, 9(8):103091. doi: 10.1371/journal.pone.0103091
[20]	KERSTING S, RAUSCH V, BIER F F, et al. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis[J].Malar Journal, 2014(13):99.
[21]	林裕胜, 江锦秀, 张靖鹏, 等.绵羊肺炎支原体TaqMan荧光定量PCR检测方法的建立[J].中国预防兽医学报, 2018, 40(4):316-320. http://d.old.wanfangdata.com.cn/Periodical/zgyfsyxb201804009 LIN Y S, JIANG J X, ZHANG J P, et al. Establishment of a TaqMan real-time fluorescent quantitative PCR assay for detection of Mycoplasma ovipneumoniae[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(4):316-320.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgyfsyxb201804009
[22]	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. http://d.old.wanfangdata.com.cn/Periodical/nyswjsxb201708018 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. http://d.old.wanfangdata.com.cn/Periodical/nyswjsxb201708018
[23]	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
[24]	吴耀东, 徐民俊, 郑文斌, 等.重组聚合酶扩增技术及其在动物病原快速检测中的应用[J].中国兽医学报, 2016, 26(10):1797-1802. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgsyxb201610029 WU Y D, XU M J, ZHENG W B, et al. Development of recombinant polymerase amplification technology and its applications in quick diagnosis of animal pathogen[J]. Chinese Journal of Veterinary Science, 2016, 26(10):1797-1802.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgsyxb201610029
[25]	秦立得, 南文龙, 陈义平, 等.重组聚合酶扩增技术及其在动物病毒检测中的应用[J].中国动物检疫, 2017, 34(5):81-85. doi: 10.3969/j.issn.1005-944X.2017.05.023 QIN L D, NAN W L, CHEN Y P, et al. Recombinase polymerase amplification and its application in detection of animal diseases[J]. China Animal Health Inspection, 2017, 34(5):81-85.(in Chinese) doi: 10.3969/j.issn.1005-944X.2017.05.023
[26]	刘占旭, 刘新生, 方玉珍, 等.猪嵴病毒RPA快速诊断方法的建立和应用[J].中国兽医科学, 2018, 48(9):1073-1079. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgsykj201809001 LIU Z X, LIU X S, FANG Y Z, et al. Development and application of a recombinase polymerase amplification assay for rapid diagnosis of porcine kobuvirus[J]. Chinses Veterinary Science, 2108, 48(9):1073-1079.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgsykj201809001
[27]	WANG J, WANG J, LIU L, et al. Rapid detection of Porcine circovirus-2 by recombinase polymerase amplification[J]. Journal of Veterinary Diagnostic Investigation, 2016, 28(5):1-4. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=10.1177/1040638716654201
[28]	YANG Y, QIN X, WANG G, et al.Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus[J]. Virology Journal, 2015, 12(1):206.
[29]	樊晓旭, 宋翥远, 赵永刚, 等.塞尼卡病毒重组聚合酶扩增-侧流层析试纸条检测方法的建立[J].中国预防兽医学报, 2018, 40(5):406-410. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgyfsyxb201805008 FANG X X, SONG Z Y, ZHAO Y G, et al. Establishment of recombinase polymerase amplification-lateral flow dipstick for the detection of Seneca valley virus[J].Chinese Journal of Preventive Veterinary Medicine, 2018, 40(5):406-410.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgyfsyxb201805008