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Establishment of a SYBR Green Ⅰ qRT-PCR for Rapid Detection of Mycoplasma mycoides subsp. capri |
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Abstract Mycoplasma mycoides subsp. capri (Mmc) causes Mycoplasma pneumonia of goats and sheep (MPGS). The existing methods for Mmc detection are time-consuming and unquantifiable, but not suitable for accurate and rapid detection of Mmc. In order to establish a SYBR Green I real-time PCR assay for detecting Mmc. A pair of primers was designed based on the hypothetical protein gene (MLC_1770) gene of Mmc. The recombinant plasmid of Mmc-1770 was constructed as positive control for standard curve development, and the specificity, sensitivity and reproducibility of the assay were evaluated. The result showed that the correlation coefficient (R2) of standard curve was 0.999 and the amplification efficiency (E) was 101.3%. There was no cross reactions with Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma ovipenumoniae (Mo), Pasteurella multocida (Pm), Escherichia coli (Ec), Staphylococcus aureus (SA) and Orf virus (ORFV), indicating a good specificity. The sensitivity of this assay was 226 copies/μL which was 100 times higher than that of the conventional PCR. The coefficients of variation between the intra-groups assay were 0.54%~0.79%, and the inter-groups assay was 0.70%~0.96%. The 95 clinical samples were tested with this assay, the Mmc positive rate was 11.6% (11/95). The assay could be used to detect Mmc rapidly in clinical samples with strong specificity, high sensitivity and reliable reproducibility.
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Received: 21 April 2017
Published: 01 November 2017
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[1]Kama-Kama F, Midiwo J, Nganga J, et al.Selected enthno-medicinal plants from Kenya with in vitro activity against major African livestock pathogens belonging to the “Mycoplasma mycoides cluster”[J]., 2016, 192:524-534
[2]Tatay-Dualde J, Prats-ban der Ham M, de la Fe C, et al..Multilocus sequence typing of Mycoplasma mycoides subsp. capri to assess its genetic variability in a contagious agalactia endemic area[J]., 2016, 191:30-64
[3]Cillara G, Manca MG, Longheu C, et al.Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR[J]., 2015, 205(3):421-423
[4]Corona L, Cillara G, Tola S, et al. Proteomic approach for identification of immunogenic proteins of Mycoplasma mycoides subsp. capri. [J]., 2013, 167(3-4):434-439
[5]Xu C G, Hao Y Q, Zhang L, et al.Molecular cloning and immune response analysis of putative variable lipoproteins from Mycoplasma mycoides subsp capri.[J]., 2014, 13(1):1527-1539
[6]Algire MA, Montague M G, Vashee S, et al.A Type III restriction-modification system in Mycoplasma mycoedes subsp. capri.[J]., 2012, 2(10):115-120
[7]D’Angelo A R, DiProvvido A, Di Francesco G, et al.Experimental infection of goats with an unusual strain of Mycoplasma mycoides subsp. capri. Isolated in Jordan: comparison of different diagnostic methods[J].Veterinaria Italiana, 2010, 46(2):189-207
[8]王慧, 周碧君, 张双翔, 等. 丝状支原体山羊亚种贵州株的分离与鉴定[J].畜牧与兽医, 2013, 45(12):18-23
[9]杜永凤, 文心田, 曹三杰, 等.山羊传染性胸膜肺炎病原分离与鉴定[J]., 2006, 28(6):618-621
[10]韩赟, 郭晗, 逯忠新.青海地区丝状支原体山羊亚种的流行病学调查[J].中国动物检疫, 2010, 27(5):41-42
[11]陶立, 李军, 王仲挺, 等.隆林黑山羊肺炎病原的分离鉴定[J].畜牧与兽医, 2015, 47(2):67-69
[12]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
[13]贺英, 赵萍, 储岳峰, 等.由丝状霉形体山羊亚种引起的羊传染性胸膜肺炎诊断方法的研究进展[J]., 2009, 30(2):169-170
[14]万一元, 万晴娇, 杨何, 等.山羊传染性胸膜肺炎垂直感染试验[J].中国兽医杂志, 2000, 26(11):19-19
[15]万春和, 朱海侠, 施少华, 等.鸭坦布苏病毒SYBR Green I 实时荧光定量检测方法的建立[J]., 2013, 33(7):973-978
[16]程振涛, 张双翔, 王慧, 等.绵羊肺炎支原体实时荧光定量PCR检测方法的建立[J].西北农业学报, 2013, 22(2):7-12
[17]Balamurugan V, Sen A, Venkatesan G, et al.A rapid and sensitive one step-SYBR Green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples [J]., 2012, 27(1):1-9
[18]Wang Y, Yang K K, Bai C X, et al.Development of a SYBR Green I real-time PCR for the detection of the orf virus[J]., 2017, 7(1):21-26
[19]秦子禹, 孙建设, 王娜, 等.一种高效的苹果茎沟病毒(ASGV)TaqMan探针实时荧光定量(qRT-PCR)检测方法[J]., 2015, 23(6):816-822
[20]朱彤, 赵贵民, 沈付娆, 等.牛肠道病毒SYBR Green I实时荧光定量PCR检测方法的建立及初步应用[J]., 2015, 31(5):488-493
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