Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis

doi:10.3969/j.issn.1004-1524.20230922

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (8): 1811-1819.DOI: 10.3969/j.issn.1004-1524.20230922

• Animal Science • Previous Articles Next Articles

Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis

WANG Jianlin¹(), TIAN Xingmiao¹, WANG Jingsong¹^,², DAI Shasha¹, GUO Yanan², HE Shenghu¹, LI Jidong¹^,^*()

1. College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China
2. Institute of Animal Science, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750002, China

Received:2023-07-28 Online:2024-08-25 Published:2024-09-06
Contact: LI Jidong

Abstract

Abstract:

The aim was to establish an efficient and rapid visual recombinase polymerase amplification (LFD-RPA) clinical diagnostic method for Mycoplasma bovis. With the uvrc gene sequence of Mycoplasma bovis as the target gene, specific primers and probes are designed and validated by sensitivity, specificity, repeatability and clinical sample detection test through screening of primers and probes and optimizing the reaction conditions. The results showed that the optimal primers for LFD-RPA of Mycoplasma bovis established in this test were F2/R2, and the optimal reaction condition was 39 ℃ for 25 min; the detection sensitivity was up to 2.08 copies·μL^-1, 100 times of common PCR; there was no cross reaction with Mycoplasma synoviae, Salmonella, Mycoplasma ovipneumoniae, Pasteurella multocida, Staphylococcus aureus, Streptococcus agalactiae and Clostridium perfringens; the repeatability was stable; 50 nasal swab samples were detected, the positive rate was 26%, and the coincidence rate with domestic industry standard PCR detection methods was 89.6%. In this study, the LFD-RPA detection method for Mycoplasma bovis was successfully established, which has the advantages of easy operation, rapidity, high efficiency, and sensitivity, and provides technical support for the rapid clinical diagnosis of Mycoplasma bovis.

Key words: Mycoplasma bovis, recombinase polymerase amplification assays, visualization, test method

CLC Number:

S852.62

WANG Jianlin, TIAN Xingmiao, WANG Jingsong, DAI Shasha, GUO Yanan, HE Shenghu, LI Jidong. Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1811-1819.

Figures/Tables 10

References 31

[1]	NICHOLAS R A J, AYLING R D. Mycoplasma bovis: disease, diagnosis, and control[J]. Research in Veterinary Science, 2003, 74(2): 105-112.
[2]	KANDA T, TANAKA S, SUWANRUENGSRI M, et al. Bovine endocarditis associated with Mycoplasma bovis[J]. Journal of Comparative Pathology, 2019, 171: 53-58.
[3]	HAAPALA V, POHJANVIRTA T, VÄHÄNIKKILÄ N, et al. Semen as a source of Mycoplasma bovis mastitis in dairy herds[J]. Veterinary Microbiology, 2018, 216: 60-66.
[4]	GILLE L, EVRARD J, CALLENS J, et al. The presence of Mycoplasma bovis in colostrum[J]. Veterinary Research, 2020, 51(1): 54.
[5]	ASKAR H, CHEN S L, HAO H F, et al. Immune evasion of Mycoplasma bovis[J]. Pathogens, 2021, 10(3): 297.
[6]	季文恒, 储岳峰, 赵萍, 等. 牛支原体逃避宿主免疫的研究进展[J]. 畜牧兽医学报, 2017, 48(3): 393-402.
	JI W H, CHU Y F, ZHAO P, et al. Advances in the research of immune evasion by Mycoplasma bovis[J]. Chinese Journal of Animal and Veterinary Sciences, 2017, 48(3): 393-402. (in Chinese with English abstract)
[7]	NICHOLAS R A J. Bovine mycoplasmosis: silent and deadly[J]. The Veterinary Record, 2011, 168(17): 459-462.
[8]	HALE H H, HELMBOLDT C F, PLASTRIDGE W N, et al. Bovine mastitis caused by a Mycoplasma species[J]. The Cornell Veterinarian, 1962, 52: 582-591.
[9]	温靖, 李丹, 郭婷, 等. 中国奶牛牛支原体流行病学调查分析[J]. 中国农业大学学报, 2022, 27(3): 75-82.
	WEN J, LI D, GUO T, et al. Epidemiological investigation and analysis of Mycoplasma bovis in different dairy farming areas in China[J]. Journal of China Agricultural University, 2022, 27(3): 75-82. (in Chinese with English abstract)
[10]	PIEPENBURG O, WILLIAMS C H, STEMPLE D L, et al. DNA detection using recombination proteins[J]. PLoS Biology, 2006, 4(7): e204.
[11]	DAHER R K, STEWART G, BOISSINOT M, et al. Recombinase polymerase amplification for diagnostic applications[J]. Clinical Chemistry, 2016, 62(7): 947-958.
[12]	LIU Y L, LIU L B, WANG J F, et al. Rapid detection of bovine rotavirus a by isothermal reverse transcription recombinase polymerase amplification assays[J]. BMC Veterinary Research, 2022, 18(1): 339.
[13]	LIU L, LI R, ZHANG R, et al. Rapid and sensitive detection of Mycoplasma hyopneumoniae by recombinase polymerase amplification assay[J]. Journal of Microbiological Methods, 2019, 159: 56-61.
[14]	DUDEK K, NICHOLAS R A J, SZACAWA E, et al. Mycoplasma bovis infections-occurrence, diagnosis and control[J]. Pathogens, 2020, 9(8): 640.
[15]	THOMAS A, DIZIER I, LINDEN A, et al. Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis[J]. Veterinary Journal, 2004, 168(1): 100-102.
[16]	BOONYAYATRA S, FOX L K, BESSER T E, et al. A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis[J]. Journal of Dairy Science, 2012, 95(1): 196-205.
[17]	BEHERA S, RANA R, GUPTA P K, et al. Development of real-time PCR assay for the detection of Mycoplasma bovis[J]. Tropical Animal Health and Production, 2018, 50(4): 875-882.
[18]	ASHRAF A, IMRAN M, YAQUB T, et al. Development and validation of a loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis in mastitic milk[J]. Folia Microbiologica, 2018, 63(3): 373-380.
[19]	NIELSEN P K, PETERSEN M B, NIELSEN L R, et al. Latent class analysis of bulk tank milk PCR and ELISA testing for herd level diagnosis of Mycoplasma bovis[J]. Preventive Veterinary Medicine, 2015, 121(3/4): 338-342.
[20]	SALGADU A, FIRESTONE S M, WATT A, et al. Evaluation of the MilA ELISA for the diagnosis of herd infection with Mycoplasma bovis using bulk tank milk and estimation of the prevalence of M. bovis in Australia[J]. Veterinary Microbiology, 2022, 270: 109454.
[21]	DUDEK K, SZACAWA E, NICHOLAS R A J. Recent developments in vaccines for bovine mycoplasmoses caused by Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides[J]. Vaccines, 2021, 9(6): 549.
[22]	CLOTHIER K A, JORDAN D M, THOMPSON C J, et al. Mycoplasma bovis real-time polymerase chain reaction assay validation and diagnostic performance[J]. Journal of Veterinary Diagnostic Investigation: Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 2010, 22(6): 956-960.
[23]	PARKER A M, SHEEHY P A, HAZELTON M S, et al. A review of mycoplasma diagnostics in cattle[J]. Journal of Veterinary Internal Medicine, 2018, 32(3): 1241-1252.
[24]	DEVI K R, LEE L J, YAN L T, et al. Occupational exposure and challenges in tackling M. bovis at human-animal interface: a narrative review[J]. International Archives of Occupational and Environmental Health, 2021, 94(6): 1147-1171.
[25]	于新友, 李天芝, 李书光, 等. 牛病毒性腹泻病毒和牛支原体SYBR GreenⅠ双重荧光PCR检测方法的建立[J]. 中国兽医科学, 2021, 51(10): 1227-1232.
	YU X Y, LI T Z, LI S G, et al. Establishment of a duplex SYBR GreenⅠ real-time RT-PCR assay for simultaneous detection of emerging BVDV and Mycoplasma bovis[J]. Chinese Veterinary Science, 2021, 51(10): 1227-1232. (in Chinese with English abstract)
[26]	刘立兵, 史云鹏, 高雅欣, 等. 牛支原体微滴式数字PCR检测方法的建立及应用[J]. 中国预防兽医学报, 2022, 44(5): 502-507.
	LIU L B, SHI Y P, GAO Y X, et al. Development and application of droplet digital PCR for detection of Mycoplasma bovis[J]. Chinese Journal of Preventive Veterinary Medicine, 2022, 44(5): 502-507. (in Chinese with English abstract)
[27]	ZHANG H, WANG Z, CAO X D, et al. Loop-mediated isothermal amplification assay targeting the mpb70 gene for rapid differential detection of Mycobacterium bovis[J]. Archives of Microbiology, 2016, 198(9): 905-911.
[28]	HIGGINS M, RAVENHALL M, WARD D, et al. PrimedRPA: primer design for recombinase polymerase amplification assays[J]. Bioinformatics, 2019, 35(4): 682-684.
[29]	LI R W, WANG J F, SUN X X, et al. Direct and rapid detection of Mycoplasma bovis in bovine milk samples by recombinase polymerase amplification assays[J]. Frontiers in Cellular and Infection Microbiology, 2021, 11: 639083.
[30]	ZHAO G M, HOU P L, HUAN Y J, et al. Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis[J]. BMC Veterinary Research, 2018, 14(1): 412.
[31]	孙魁. 基于LFD-RPA的核酸可视化检测技术的建立及应用[D]. 北京: 中国人民解放军军事医学科学院, 2017.
	SUN K. Establishment of nucleic acid visual detection based on LFD-RPA and its application[D]. Beijing: Academy of Military Medical Sciences, 2017. (in Chinese with English abstract)

引物名称 Primer name	引物序列 Primer sequence (5'-3')	长度 Length/bp
F1	CCTGGTGTTTATCTATGAAAAGATGCTAAA	119
R1	TTAGTTTTATATGAATTAATAGCGCCGTCA
F2	CACAAAACCAAAGCCTTAATTGACCTAGAT	173
R2	CCTTTTATGTTTCTTAGTTTGCCTTCTAGTGA
F3	TTTACTATGGTCCTTTTCCTTCTGGTTATG	207
R3	TGGCTGCTTGATGCATTTTGTTAGTTAGTT
uvrc-LFD-F	CACAAAACCAAAGCCTTAATTGACCTAGAT
uvrc-LFD-R	[5’-biotin]-CCTTTTATGTTTCTTAGTTTGCCTTCTAGTGA
uvrc-LFD-Probe	[5'FAM]-ATGACTTAGAACTATACAACTACTTAGTTCAAAT/idSp/CAAGTTGAAGTTGACC-[3'C3 Spacer]
NY-T-F	TTTTAGCTCTTTTTGAACAAAT	1 900
NY-T-R	GGCTCTCATTAAGAATGTC
uvrc-F	GTGGATGCAATTGAAGCTGAAC	2 098
uvrc-R	AAAGAGCAGAAGAGAGAGAGCT

引物名称 Primer name	引物序列 Primer sequence (5'-3')	长度 Length/bp
F1	CCTGGTGTTTATCTATGAAAAGATGCTAAA	119
R1	TTAGTTTTATATGAATTAATAGCGCCGTCA
F2	CACAAAACCAAAGCCTTAATTGACCTAGAT	173
R2	CCTTTTATGTTTCTTAGTTTGCCTTCTAGTGA
F3	TTTACTATGGTCCTTTTCCTTCTGGTTATG	207
R3	TGGCTGCTTGATGCATTTTGTTAGTTAGTT
uvrc-LFD-F	CACAAAACCAAAGCCTTAATTGACCTAGAT
uvrc-LFD-R	[5’-biotin]-CCTTTTATGTTTCTTAGTTTGCCTTCTAGTGA
uvrc-LFD-Probe	[5'FAM]-ATGACTTAGAACTATACAACTACTTAGTTCAAAT/idSp/CAAGTTGAAGTTGACC-[3'C3 Spacer]
NY-T-F	TTTTAGCTCTTTTTGAACAAAT	1 900
NY-T-R	GGCTCTCATTAAGAATGTC
uvrc-F	GTGGATGCAATTGAAGCTGAAC	2 098
uvrc-R	AAAGAGCAGAAGAGAGAGAGCT

		行业标准PCR Industry standard PCR		总计 Total
		阳性Positive	阴性Negative	总计 Total
LFD-RPA	阳性Positive	12	1	13	92.31% (PPV)
	阴性Negative	0	36	37	97.29% (NPV)
	总计Total	12	38	50
		92.31% (灵敏度) (Sensitivity)	97.30% (特异度) (Specificity)	0.896(Kappa值) (Kappa value)

		行业标准PCR Industry standard PCR		总计 Total
		阳性Positive	阴性Negative	总计 Total
LFD-RPA	阳性Positive	12	1	13	92.31% (PPV)
	阴性Negative	0	36	37	97.29% (NPV)
	总计Total	12	38	50
		92.31% (灵敏度) (Sensitivity)	97.30% (特异度) (Specificity)	0.896(Kappa值) (Kappa value)

Establishment of a visual recombinase polymerase amplification assays for Mycoplasma bovis

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