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

Abstract

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

CLC Number:

S852.618

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.

Figures/Tables 5

Table 1

Fig. 1

Fig. 2

Table 2

Table 3

References 36

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.
	LIUM D,ZHANGH B,QIF,et al.Research progress on the etiology, prevention and control of bovine paratuberculosis[J].Chinese Journal of Veterinary Medicine,2024,60(1):102-107.
2	CHENGZ,LIUM,WANGP,et al.Characteristics and epidemiological investigation of paratuberculosis in dairy cattle in Tai 'an, China[J].BioMed Res Int,2020,2020,e3896754. doi: 10.1155/2020/3896754
3	MCALOONC G,ROCHES,RITTERC,et al.A review of paratuberculosis in dairy herds — Part 1: Epidemiology[J].Vet J,2019,246,59-65. doi: 10.1016/j.tvjl.2019.01.010
4	MCALOONC G,ROCHES,RITTERC,et al.A review of paratuberculosis in dairy herds — Part 2: On-farm control[J].Vet J,2019,246,54-58. doi: 10.1016/j.tvjl.2019.01.009
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-EWERTT,TICHYA,MADERC,et al.Management and housing factors associated with paratuberculosis-positive herds in small structured alpine cattle husbandry[J].Prev Vet Med,2023,218,105999. doi: 10.1016/j.prevetmed.2023.105999
9	RUSSOS,GIORGIOG,LEOS,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. doi: 10.1016/j.prevetmed.2022.105732
10	DANEH,STEWARTL D,GRANTI R.Culture of Mycobacterium avium subsp. paratuberculosis: challenges, limitations and future prospects[J].J Appl Microbiol,2023,134(1):lxac017. doi: 10.1093/jambio/lxac017
11	XIAZ,JOHANSSONM L,GAOY,et al.Conventional versus real-time quantitative PCR for rare species detection[J].Ecol Evol,2018,8(23):11799-11807. doi: 10.1002/ece3.4636
12	ZEMTSOVAG E,MONTGOMERYM,LEVINM 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. doi: 10.1371/journal.pone.0116658
13	ARSENAULTJ,SINGH SOHALJ,LEBOEUFA,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. doi: 10.1177/1040638718810744
14	CONDEC,PRICE-CARTERM,COCHARDT,et al.Whole-genome analysis of Mycobacterium avium subsp. paratuberculosis IS900 insertions reveals strain type-specific modalities[J].Front Microbiol,2021,12,660002. doi: 10.3389/fmicb.2021.660002
15	ENGLUNDS,BÖLSKEG,JOHANSSONK 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. doi: 10.1111/j.1574-6968.2002.tb11142.x
16	COUSINSD V,WHITTINGTONR,MARSHI,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. doi: 10.1006/mcpr.1999.0275
17	STROMMENGERB,STEVENSONK,GERLACHG 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. doi: 10.1111/j.1574-6968.2001.tb10536.x
18	STABELJ R,BANNANTINEJ 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. doi: 10.1128/JCM.43.9.4744-4750.2005
19	HODGEMANR,LIUY,ROCHFORTS,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. doi: 10.1093/jambio/lxae107
20	RICCHIM,DE CICCOC,KRALIKP,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. doi: 10.1111/1574-6968.12480
21	RAJEEVS,ZHANGY,SREEVATSANS,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. doi: 10.1016/j.vetmic.2004.10.018
22	BANNANTINEJ P,BAECHLERE,ZHANGQ,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. doi: 10.1128/JCM.40.4.1303-1310.2002
23	IMIRZALIOGLUC,DAHMENH,HAINT,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. doi: 10.1128/JCM.01492-10
24	HUNGJ H,WENGZ.Designing polymerase chain reaction primers using Primer3Plus[J].Cold Spring Harb Protoc,2016,2016(9):pdb.prot093096. doi: 10.1101/pdb.prot093096
25	BROWNS S,CHENY W,WANGM,et al.PrimerPooler: automated primer pooling to prepare library for targeted sequencing[J].Biol Meth Protoc,2017,2(1):bpx006. doi: 10.1093/biomethods/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	WHITTINGTONR J,MARSHI B,SAUNDERSV,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. doi: 10.1128/JCM.00210-11
28	LIUT,LINH,ZHUL,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. doi: 10.1016/j.prevetmed.2025.106436
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	RUSSOS,CORTIMIGLIAC,FILIPPIA,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. doi: 10.1016/j.mimet.2023.106825
31	JURADO-MARTOSF,CARDOSO-TOSETF,TARRADASC,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. doi: 10.1016/j.rvsc.2023.06.007
32	HODGEMANR,MANNR,DJITRON,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. doi: 10.1186/s12864-023-09752-0
33	ELLINGSONJ L E,BOLINC A,STABELJ 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. doi: 10.1006/mcpr.1998.0167
34	ELLINGSONJ,KOZICZKOWSKIJ,ANDERSONJ.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. doi: 10.4315/0362-028X-67.10.2310
35	LUN,NIUY L,SONGY,et al.Prevalence of paratuberculosis in cattle in China: A systematic review and meta-analysis[J].Prev Vet Med,2023,220,106043. doi: 10.1016/j.prevetmed.2023.106043
36	IRENGEL M,WALRAVENSK,GOVAERTSM,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.

引物探针名称 Primer/Probe	引物/探针序列(5′ → 3′) Primer/Probe sequence
GB-F	GTCAGCGGCGGTCCAG
GB-P	FAM-ACGAGCACGCAGGCATTCCAAGTC-BHQ1
GB-R	GCAGCTCCAGATCGTCATTC
F57-F	TTGTCGATCCGCTTAGTTCG
F57-P	FAM-TGAATGGTCGTCTGTGCCAGCCGCCCACT-BHQ1
F57-R	ACGCCCATTTCATCGATACC
251-F	TGTTGTTCGCTGAGTTACGC
251-P	VIC-TCCAGTTCCGTGACGACGATGTGCGCGACG-BHQ1
251-R	CACGCTTGCCTGATATCTCTAC
hspX-F	GAAGATCATGACTGACGTGTGG
hspX-P	Cy5-ACGACGTCTACCAGTCACGAACCGACCGCG-BHQ2
hspX-R	GTGAGTACAACGACGGGAATG

靶标 Target	标准拷贝数/(copies·μL^-1) Concentration of standard plasmi	标准差 SD	平均值 Mean	变异系数 CV%
F57	10⁷	0.30	17.91	1.70
	10⁵	0.80	25.74	3.12
	10³	0.62	31.80	1.96
251	10⁷	0.65	18.02	3.61
	10⁵	0.74	26.00	2.84
	10³	0.52	32.11	1.61
hspX	10⁷	0.37	18.19	2.04
	10⁵	0.71	25.99	2.74
	10³	0.56	32.17	1.74

检测结果 Testing results		样品数 No. of samples	检测副结核样品数 No. of samples tested for MAP	检测其他消化道病原样品数 No. of samples tested for other pathogens
国标方法 National standard method	三重qPCR方法 Triple qPCR method	样品数 No. of samples	检测副结核样品数 No. of samples tested for MAP	检测其他消化道病原样品数 No. of samples tested for other pathogens
阳性 Positive	Positive	13 (7.18%)	10 (14.71%)	3 (2.65%)
阳性 Positive	Negative	15 (8.29%)	7 (10.29%)	8 (7.08%)
阴性 Negative	Positive	28 (15.47%)	20 (29.41%)	8 (7.08%)
阴性 Negative	Negative	125 (69.06%)	31 (45.59%)	94 (83.19%)
合计 Total		181 (100.00%)	68 (100.00%)	113 (100.00%)

[1]	WANG Chun, WANG Qing, WU Xiaoqian, HU Ting, CUI Weitao, PEI Jie, SONG Tieping, HU Sishun, ZHANG Wanpo, LI Zili, ZHOU Zutao. Analysis of Mycoplasma synoviae Infection in the Laying Hens in Hubei Province based on Fluorescence Quantitative PCR Detection and in situ Hybridization Technology [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1396-1407.
[2]	Yue NAN, Meizhen LONG, Yuanzhi WANG, Yiduo LIU, Xiangmei ZHOU. Establishment and Preliminary Application of Recombinase-mediated Amplification-sidestream Detection for Detection of Mycobacterium paratuberculosis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3255-3260.
[3]	HU Zeqi, LI Runcheng, TAN Zuming, XIE Xiuyan, WANG Jiangping, QIN Lejuan, LI Rong, GE Meng. Establishment and Preliminary Application of PEDV, PoRVA and PDCoV TaqMan Triple RT-qPCR Assay [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2267-2272.
[4]	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.
[5]	YAN Chao, LIU Yonggang, XIE Hao, PENG Cuiting, ZHANG Caiyong, ZHAO Yulan, QI Lin, CHEN Zhilong, TANG Zhonglin. Detection of Gene Expression in Trace Cells of Early Porcine Embryo by Pre-amplified Quantitative PCR [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5567-5574.
[6]	ZHAI Gang, GU Wenyuan, LIU Tao, LIU Ying, ZHANG Shuai, FAN Jinghui, ZUO Yuzhu. Establishment of TaqMan Detection Method of Porcine Epidemic Diarrhea Virus and Analysis of Genetic Variation based on S Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 847-854.
[7]	ZHOU Shiwei, WU Tingjie, WANG Qian, CAI Bei, HAN Saizheng, WU Yanhu, WANG Xiaolong, CHEN Yulin. Establishment of the Efficient Method to Detect the FecB Mutation and the Construction of the Prolific Tan Sheep Population [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 2920-2929.
[8]	WANG Qin, XIONG Yan, ZI Xiangdong. Effects of PAF Supplementation during in vitro Maturation Medium on Yak Oocyte Development Competence and Gene Expression [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 514-523.
[9]	LIU Haoli, TENG Man, LI Huizhen, YU Zuhua, LIU Jingling, DING Ke, ZHANG Gaiping, LUO Jun. Screening and Identification of Host mRNA Targets for the Viral microRNA miR-M11-5p Encoded by Marek's Disease Virus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1026-1038.
[10]	WANG Yunhe, JIN Xin, ZHANG Man, WEI Fang, WEN Jingyi, ZHAO Feifei, YANG Yinfeng. Effects of Saccharomyces Cerevisiae and Its Inactivated Bacteria on the Expression of β-defensin-1 in Ruminal Explants of Sheep [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 581-591.
[11]	LIAO Bo, CUI Yan, YANG Xue, HE Jun-feng, SONG Liang-li, YANG Xiao-qing, LIU Peng-gang. Distribution and Change Rule of HSP70 in Yak Intestine [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1735-1742.
[12]	LI Chang, KU Xu-gang, WANG Jun-wei, LI Jing, ZHU Ling, HE Qi-gai. Development and Application of a TaqMan-based Real-time Fluorescent PCR for Specific Detection of Porcine Circovirus Type 3 [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1320-1326.
[13]	ZHANG Man, JIN Xin, WANG Yun-he, WEI Fang, WEN Jing-yi, LI Zheng-yi, YANG Yin-feng. Effects of Saccharomyces cerevisiae β-glucan on the Expression of SBD-1 in Ovine Ruminal Epithelial Cells [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2416-2424.
[14]	XU Qian-ru, ZHANG Er-qin, GUO Jun-qing, YANG Ji-fei, LIU Yun-chao, WANG Ai-ping, WANG Li, WAN Bo, WANG Lei, JIANG Da-wei, DENG Rui-guang, ZHANG Gai-ping. Expression and Detection of NDV-F Protein in Rice Endosperm [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1167-1172.
[15]	LIU Jiao, ZHANG Man, JIN Xin, FAN Yan-ru, TIAN Qiao-zhen, YANG Yin-feng. Ghrelin Expression and Cell Location Analysis inside Sika Deer [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(1): 157-165.

Establishment of a Triple TaqMan qPCR Detection Method for Mycobacterium avium subsp. Paratuberculosis

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 5

References 36

Related Articles 15

Recommended Articles

Metrics

Comments