副结核分枝杆菌三重TaqMan qPCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.09.051

摘要/Abstract

摘要： 副结核分枝杆菌(Mycobacterium avium subsp. paratuberculosis, MAP)是造成反刍动物约翰病的病原，每年给全球畜牧业带来难以估计的经济损失。由于治疗困难，目前对患畜的及时诊断和尽早淘汰是副结核防控的主要手段。MAP的金标准培养检测周期长，而qPCR技术直接检测样本核酸能够快速确定病原，目前广泛应用。本研究旨在针对MAP非插入序列(insertion sequence, IS)靶标建立一种检测MAP的三重TaqMan qPCR方法，能够提高检测的敏感性和特异性。针对MAP基因组中MAP2765c、F57和hspX基因设计引物探针；以本实验室分离的MAP菌株为对照，建立三重qPCR检测体系；临床采集奶肉牛样品181份，和国标(GB/T 27637—2011)进行符合性试验。结果显示，本研究建立的三重qPCR方法灵敏度能达到10 copies·μL^-1；重复结果变异系数均小于5%；特异性试验中仅MAP核酸出现扩增曲线；标准曲线R²均大于0.999且扩增效率为97%~100%；三重qPCR方法检测临床样品的阳性率为22.65%(41/181)，比国标方法高(15.47%)，两种方法的检测符合率为76.28%(138/181)；根据牧场检测需求对样品进行分类分析：国标方法和三重qPCR方法在“高MAP风险样品”的样品中检出率分别为25%(17/68)和44.12%(30/68)，而两种方法检测较低MAP风险样品的阳性率均为9.73%(11/113)。本研究设计的三重qPCR方法，灵敏度、特异性和重复性良好，线性和定量准确性高，且能比国标方法更灵敏地在高MAP风险样品中检出MAP。研究结果为奶牛和肉牛养殖场开展副结核病的监测、预警、控制和净化提供优选方案。

关键词: 约翰病, 副结核分枝杆菌, qPCR, 三重TaqMan

Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen causing Johne’s disease in ruminants and brings inestimable economic losses to the global livestock industry every year. Due to the difficulty in treatment, timely diagnosis and early culling of diseased animals are currently the main means of MAP prevention and control. The gold-standard culture detection cycle for MAP is long, while the qPCR technology, which directly detects nucleic acids in samples, can quickly identify the pathogen and is currently widely used. This study aims to establish a triplex TaqMan qPCR method for MAP detection targeting non-insertion sequence (IS) of MAP, which can improve the sensitivity and specificity of detection. Design primer probes for the MAP2765c, F57 and hspX genes in the MAP genome. Taking the MAP strains isolated in this laboratory as a control, a triple qPCR detection system was established. A total of 181 samples of dairy and beef cattle were clinically collected, and a compliance test was carried out using the triple qPCR method and the national standard method (GB/T 27637—2011). The results showed that the sensitivity of the triple qPCR method established in this study could reach 10 copies·μL^-1; the coefficient of variation of the repeated results was all less than 5%; in the specificity experiment, only MAP nucleic acid showed an amplification curve; the R² of the standard curves were all greater than 0.999 and the amplification efficiency was between 97% and 100%; the positive rate of clinical samples detected by the triple qPCR method was 22.65% (41/181), which was higher than that of the national standard method (15.47%), and the detection coincidence rate of the two methods was 76.28% (138/181); according to the detection requirements of the ranch, the samples were classified and analyzed: the detection rates of the national standard method and the triple qPCR method in "high-MAP-risk samples" were 25% (17/68) and 44.12% (30/68) respectively, while the positive rates of the two methods for detecting samples with lower MAP risk were both 9.73% (11/113). The triple qPCR method designed in this study has good sensitivity, specificity and repeatability, high linearity and quantitative accuracy, and can detect MAP more sensitively in high-MAP-risk samples than the national standard method. The research results provide an optimal solution for dairy and beef cattle farms to carry out monitoring, early warning, control and purification of paratuberculosis.

Key words: Johne’s disease, Mycobacterium avium subsp. paratuberculosis, qPCR, Triple TaqMan

中图分类号:

S852.618

刘添, 吴雨伦, 姚火春, 潘子豪. 副结核分枝杆菌三重TaqMan qPCR检测方法的建立[J]. 畜牧兽医学报, 2025, 56(9): 4750-4758.

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.

参考文献

[1] 刘蒙达, 张皓博, 亓菲, 等. 牛副结核病的病原学和防控研究进展[J]. 中国兽医杂志, 2024, 60(1): 102-107.
LIU M D, ZHANG H B, QI F, et al. Research progress on the etiology，prevention and control of bovine paratuberculosis[J]. Chinese Journal of Veterinary Medicine, 2024, 60(1): 102-107.(in Chinese)
[2] CHENG Z, LIU M, WANG P, et al. Characteristics and epidemiological investigation of paratuberculosis in dairy cattle in Tai’an, China[J]. BioMed Res Int, 2020, 2020: e3896754.
[3] MCALOON C G, ROCHE S, RITTER C, et al. A review of paratuberculosis in dairy herds — Part 1: Epidemiology[J]. Vet J, 2019, 246: 59-65.
[4] MCALOON C G, ROCHE S, RITTER C, et al. A review of paratuberculosis in dairy herds — Part 2: On-farm control[J]. Vet J, 2019, 246: 54-58.
[5] IMADA J, KELTON D F, BARKEMA H W. Epidemiology, global prevalence and economics of infection[M]//BEHR M A, STEVENSON K, KAPUR V. Paratuberculosis: organism, disease, control. 2nd ed. Wallingford, Oxfordshire, UK: CABI, 2020: 1-13.
[6] SULLIVAN J R, BEHR M A. Drug susceptibility testing and antimicrobial resistance in Mycobacterium avium subsp. paratuberculosis[M]//BEHR M A, STEVENSON K, KAPUR V. Paratuberculosis: organism, disease, control. 2nd ed. Wallingford, Oxfordshire, UK: CABI, 2020: 139-148.
[7] JUSTE R A, GARRIDO J M, ELGUEZABAL N, et al. Paratuberculosis vaccines and vaccination[M]//BEHR M A, STEVENSON K, KAPUR V. Paratuberculosis: organism, disease, control. 2nd ed. Wallingford, Oxfordshire, UK: CABI, 2020: 365-379.
[8] LIENING-EWERT T, TICHY A, MADER C, et al. Management and housing factors associated with paratuberculosis-positive herds in small structured alpine cattle husbandry[J]. Prev Vet Med, 2023, 218: 105999.
[9] RUSSO S, GIORGIO G, LEO S, et al. Validation of IS900- qPCR assay to assess the presence of Mycobacterium avium subs. paratuberculosis in faecal samples according to the OIE procedure.[J]. Prev Vet Med, 2022, 208: 105732.
[10] DANE H, STEWART L D, GRANT I R. Culture of Mycobacterium avium subsp. paratuberculosis: challenges, limitations and future prospects[J]. J Appl Microbiol, 2023, 134(1): lxac017.
[11] XIA Z, JOHANSSON M L, GAO Y, et al. Conventional versus real-time quantitative PCR for rare species detection[J]. Ecol Evol, 2018, 8(23): 11799-11807.
[12] ZEMTSOVA G E, MONTGOMERY M, LEVIN M L. Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals[J]. PLoS One, 2015, 10(1): e0116658.
[13] ARSENAULT J, SINGH SOHAL J, LEBOEUF A, et al. Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep[J]. J Vet Diagn Invest, 2019, 31(1): 58-68.
[14] CONDE C, PRICE-CARTER M, COCHARD T, et al. Whole-genome analysis of Mycobacterium avium subsp. paratuberculosis IS900 insertions reveals strain type-specific modalities[J]. Front Microbiol, 2021, 12: 660002.
[15] ENGLUND S, BÖLSKE G, JOHANSSON K E. An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis[J]. FEMS Microbiol Lett, 2002, 209(2): 267-271.
[16] COUSINS D V, WHITTINGTON R, MARSH I, et al. Mycobacteria distenct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable IS900 polymerase chain reaction: implications for diagnosis[J]. Mol Cell Probes, 1999, 13(6): 431-442.
[17] STROMMENGER B, STEVENSON K, GERLACH G F. Isolation and diagnostic potential of ISMav2, a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis[J]. FEMS Microbiol Lett, 2001, 196(1): 31-37.
[18] STABEL J R, BANNANTINE J P. Development of a nested PCR method targeting a unique multicopy element, ISMap02, for detection of Mycobacterium avium subsp. paratuberculosis in fecal samples[J]. J Clin Microbiol, 2005, 43(9): 4744-4750.
[19] HODGEMAN R, LIU Y, ROCHFORT S, et al. Development and evaluation of genomics informed real-time PCR assays for the detection and strain typing of Mycobacterium avium subsp. paratuberculosis[J]. J Appl Microbiol, 2024, 135(5): lxae107.
[20] RICCHI M, DE CICCO C, KRALIK P, et al. Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and propidium monoazide qPCR.[J]. FEMS Microbiol Lett, 2014, 356(1): 127-133.
[21] RAJEEV S, ZHANG Y, SREEVATSAN S, et al. Evaluation of multiple genomic targets for identification and confirmation of Mycobacterium avium subsp. paratuberculosis isolates using real-time PCR[J]. Vet Microbiol, 2005, 105(3-4): 215-221.
[22] BANNANTINE J P, BAECHLER E, ZHANG Q, et al. Genome scale comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium reveals potential diagnostic sequences[J]. J Clin Microbiol, 2002, 40(4): 1303-1310.
[23] IMIRZALIOGLU C, DAHMEN H, HAIN T, et al. Highly specific and quick detection of Mycobacterium avium subsp. paratuberculosis in feces and gut tissue of cattle and humans by multiple real-time PCR assays[J]. J Clin Microbiol, 2011, 49(5): 1843-1852.
[24] HUNG J H, WENG Z. Designing polymerase chain reaction primers using Primer3Plus[J]. Cold Spring Harb Protoc, 2016, 2016(9): pdb.prot093096.
[25] BROWN S S, CHEN Y W, WANG M, et al. PrimerPooler: automated primer pooling to prepare library for targeted sequencing[J]. Biol Meth Protoc, 2017, 2(1): bpx006.
[26] 刘中勇, 陈茹, 杨国海, 等.副结核分枝杆菌实时荧光PCR检测方法：GB/T 27637—2011[S]. 中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会, 2011.
LIU Z Y, CHEN R, YANG G H, et al. Real-time PCR method the detection of Mycobacterium avium subsp. paratuberculosis: GB/T 27637—2011[S]. General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China, 2011.(in Chinese)
[27] WHITTINGTON R J, MARSH I B, SAUNDERS V, et al. Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts[J]. J Clin Microbiol, 2011, 49(5): 1822-1830.
[28] LIU T, LIN H, ZHU L, et al. Accuracy of real-time PCR for the detection of paratuberculosis in actual samples: A systematic review and meta-analysis[J]. Prev Vet Med, 2025, 237: 106436.
[29] PLAIN K M, MARSH I, PURDIE A C. Diagnosis of paratuberculosis by PCR[M]//BEHR M A, STEVENSON K, KAPUR V. Paratuberculosis: organism, disease, control. 2nd ed. Wallingford, Oxfordshire, UK: CABI, 2020: 305-332.
[30] RUSSO S, CORTIMIGLIA C, FILIPPI A, et al. Validation of digital PCR assay for the quantification of Mycobacterium avium subsp. paratuberculosis in bovine faeces according to the ISO 20395:2019[J]. J Microbiol Methods, 2023, 213:106825.
[31] JURADO-MARTOS F, CARDOSO-TOSET F, TARRADAS C, et al. Diagnostic performance of faecal and tissue multiplex qPCR IS900/F57 for the detection of Mycobacterium avium subspecies paratuberculosis in cattle.[J]. Res Vet Sci, 2023, 161: 156-162.
[32] HODGEMAN R, MANN R, DJITRO N, et al. The pan-genome of Mycobacterium avium subsp. paratuberculosis (Map) confirms ancestral lineage and reveals gene rearrangements within Map Type S[J]. BMC Genomics, 2023, 24(1): 656.
[33] ELLINGSON J L E, BOLIN C A, STABEL J R. Identification of a gene unique to Mycobacterium avium subspecies paratuberculosis and application to diagnosis of paratuberculosis[J]. Mol Cell Probes, 1998, 12(3): 133-142.
[34] ELLINGSON J, KOZICZKOWSKI J, ANDERSON J. Comparison of PCR prescreening to two cultivation procedures with PCR confirmation for detection of Mycobacterium avium subsp. paratuberculosis in US Department of Agriculture fecal check test samples[J]. J Food Prot, 2004, 67(10): 2310-2314.
[35] LU N, NIU Y L, SONG Y, et al. Prevalence of paratuberculosis in cattle in China: A systematic review and meta-analysis[J]. Prev Vet Med, 2023, 220: 106043.
[36] IRENGE L M, WALRAVENS K, GOVAERTS M, et al. Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples[J]. Vet Microbiol, 2009, 136(1): 166-172.