Qiyan XIONG, Hassan Z. A. ISHAG, Zhixin FENG, Lizhong HUA, Beibei LIU, Zhenzhen ZHANG, Hafizah Yousuf CHENIA, Lei ZHANG, Haiyan WANG, Guoqing SHAO, Yuzi WU, Yanna WEI

Mycoplasma hyopneumoniae ve Mycoplasma hyorhinis’in Aynı Anda Tespitinde Gerçek Zamanlı, Dubleks PCR Metodunun Uygulanması

Bu çalışmanın amacı, Mycoplasma hyopneumoniae ve Mycoplasma hyorhinis’in aynı anda tespitinde TaqMan prob temelli, hassas, spesifik dubleks gerçek zamanlı PCR yönteminin geliştirilmesidir. FAM ve Teksas Kırmızısı ile işaretli spesifik primer ve problar M. hyopneumoniae p97 geni ile M. hyorhinis p37 geninin amplifikasyonu amacıyla dizayn edildi. Dubleks gerçek zamanlı PCR reaksiyon karışımları oluşturularak optimize edildi ve yöntemin hassasiyetliği, özgüllüğü ve tekrarlanabilirliği hesaplandı. Dubleks gerçek zamanlı PCR’ın hassasiyetliği hem M. hyopneumoniae hem de M. hyorhinis için 10 kopya/μL olarak bulundu. Diğer yaygın viral ve bakteriyel patojenler ile çapraz reaksiyon yoktu. Ct değerlerinin varyasyonlarının standart katsayısının konsantrasyonu %5’ten az olup iyi bir tekrarlanabilirliğe işaret etmekteydi. Bronkoalveoler lavaj sıvısı, nazal svablar, dokular ve hücre kültürü süpernatantlarını içeren klinik örnekler (n = 937) dubleks gerçek zamanlı PCR ile test edildi. Mycoplasma hyopneumoniae ve Mycoplasma hyorhinis’in aynı anda tespitinde dubleks gerçek zamanlı PCR oldukça yüksek hassasiyetliğe sahip olup klinik örneklerde tanı amacıyla kullanılabilir. Yöntem kısa zamanda uygulanabilmesi ve ekonomik olması sebebiyle hem M. hyopneumoniae hem de M. hyorhinis’in kontrolünde yeni bir yaklaşım olarak kullanılabilir.

Establishment and Application of a Real-time, Duplex PCR Method for Simultaneous Detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis

The objective of this study was to develop a TaqMan probe-based, sensitive, specific duplex real-time PCR assay for simultaneous detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. The specific primers and probes, labeled with FAM and Texas Red, respectively, were designed to amplify the p97 gene of M. hyopneumoniae and p37gene of M. hyorhinis. The duplex real-time PCR reaction mixtures were established and optimized and the sensitivity, specificity and reproducibility of the assay were assessed. The sensitivity of the duplex real-time PCR was found to be 10 copies/μL for both M. hyopneumoniae and M. hyorhinis, respectively. There was no cross reaction with other common viral and bacterial pathogens. The concentration of standard coefficient of variation of Ct values was less than 5%, indicating a good reproducibility. Clinical samples (n = 937) were tested by the duplex real-time PCR assay, including broncho-alveolar lavage flids, nasal swabs, tissues and cell culture supernatant. Duplex real-time PCR for simultaneous detection of M. hyopneumoniae and M. hyorhinis was highly sensitive and can be utilized for diagnosing clinical samples. It is timeefficient and economic, thereby providing a new approach to control both M. hyopneumoniae and M. hyorhinis

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Holst S, Yeske P, Pieters M: Elimination of Mycoplasma hyopneumoniae from breed-to-wean farms: A review of current protocols with emphasis on herd closure and medication. J Swine Health Prod, 23 (6): 321-330, 2015.
Otake S, Dee S, Corzo C, Oliveira S, Deen J: Long-distance airborne transport of infectious PRRSV and Mycoplasma hyopneumoniae from a swine population infected with multiple viral variants. Vet Microbiol, 145 (3-4): 198-208, 2010. DOI: 10.1016/j.vetmic.2010.03.028
Scudamore JM, Harris DM: Control of foot and mouth disease: lessons from the experience of the outbreak in Great Britain in 2001. Rev Sci Tech, 21 (3): 699-710, 2002.
Jiang Y, Shang H, Xu H, Zhu L, Chen W, Zhao L, Fang L: Simultaneous detection of porcine circovirus type 2, classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction. Vet J, 183 (2): 172-175, 2010. DOI: 10.1016/j.tvjl.2008.11.016
Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE: Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev, 13 (4): 559-570, 2000. DOI: 10.1128/CMR.13.4.559
Pomeranz LE, Reynolds AE, Hengartner CJ: Molecular biology of pseudorabies virus: Impact on neurovirology and veterinary medicine. Microbiol Mol Biol R, 69 (3): 462-500, 2005. DOI: 10.1128/MMBR.69.3.462- 500.2005
Yang H, Qu L, Ma H, Chen L, Liu W, Liu C, Meng L, Wu J, Shou C: Mycoplasma hyorhinis infection in gastric carcinoma and its effcts on the malignant phenotypes of gastric cancer cells. BMC Gastroenterol, 10:132, 2010. DOI: 10.1186/1471-230X-10-132
Bongtae K, Kichan L, Kiwon H, Duyeol K, Yooncheol H, Chung HK, Yeonsu O, Ikjae K, Jeehoon L, Chanhee C: Development of in situ hybridization for the detection of Mycoplasma hyorhinis in formalin-fixed paraffin-embedded tissues from naturally infected pigs with polyserositis. J Vet Med Sci, 72 (9): 1225-1227, 2010. DOI: 10.1292/jvms.10-0062
Chen DJ, Wei YW, Huang LP, Wang YP, Sun JH, Du WJ, Wu HL, Liu CM: Synergistic pathogenicity in sequential coinfection with Mycoplasma hyorhinis and porcine circovirus type 2. Vet Microbiol, 182, 123-130, 2016. DOI: 10.1016/j.vetmic.2015.11.003
Palzer A, Haedke K, Heinritzi K, Zoels S, Ladinig A, Ritzmann M: Associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome virus infections in pigs with polyserositis. Can Vet J, 56 (3): 285-287, 2015.
Du GM, Liu MJ, Wu YZ, Xiong QY, Bai FF, Feng ZX, Shao GQ: Development of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma hyorhinis. Clin Lab, 59 (11-12): 1363-1371, 2013. DOI: 10.7754/Clin.Lab.2013.121223
Kang I, Kim D, Han K, Seo HW, Oh Y, Park C, Lee J, Gottschalk M, Chae C: Optimized protocol for multiplex nested polymerase chain reaction to detect and diffrentiate Haemophilus parasuis, Streptococcus Suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded tissues from pigs with polyserositis. Can J Vet Res, 76 (3): 195-200, 2012.
Liu MJ, Du GM, Bai FF, Wu YZ, Xiong QY, Feng ZX, Li B, Shao GQ: A rapid and sensitive loop-mediated isothermal amplification procedure (LAMP) for Mycoplasma hyopneumoniae detection based on the p36 gene. Genet Mol Res, 14 (2): 4677-4686, 2015. DOI: 10.4238/2015. May.4.27
Tocqueville V, Ferre S, Nguyen NHP, Kempf I, Marois-Créhan C: Multilocus sequence typing of Mycoplasma hyorhinis strains identified by a real-time Taq Man PCR assay. J Clin Microbiol, 52 (5): 1664-1671, 2014. DOI: 10.1128/JCM.03437-13
Wu YZ, Zhang X, Bai FF, Liu MJ, Hua LZ, Du GM, Shao GQ: Evaluation on clinical application of three nucleic acid amplification assays for the detection of Mycoplasma hyorhinis. Chinese J Prev Vet Med, 35, 985-988, 2013.
Charlebois A, Marois-Créhan C, Hélie P, Gagnon CA, Gottschalk M, Archambault M: Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs. Vet Microbiol, 168 (2-4): 348-356, 2014. DOI: 10.1016/j.vetmic.2013.11.006
Hillen S, Von Berg S, Köhler K, Reinacher M, Willems H, Reiner G: Occurrence and severity of lung lesions in slaughter pigs vaccinated against Mycoplasma hyopneumoniae, with diffrent strategies. Prev Vet Med, 113 (4): 580-588, 2014. DOI: 10.1016/j.prevetmed.2013.12.012
Marois C, Dory D, Fablet C, Madec F, Kobisch M: Development of a quantitative Real-Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae, strain 116 required to induce pneumonia in SPF pigs. J Appl Microbiol, 108 (5): 1523-1533, 2010. DOI: 10.1111/j.1365-2672.2009.04556.x
Zhang X, Bai FF, Wu YZ, Liu MJ, Feng ZX, Xiong QY, Zhang Y, Shao GQ: Research advances on the detection of Mycoplasma hyopneumoniae by PCR. Biotechnol Bullet, 5, 54-60, 2012.
Cho JG, Dee SA, Deen J, Guedes A, Trincado C, Fano E, Jiang Y, Faaberg K, Collins JE, Murtaugh MP, Joo HS: Evaluation of the effcts of animal age, concurrent bacterial infection, and pathogenicity of porcine reproductive and respiratory syndrome virus on virus concentration in pigs. Am J Vet Res, 67 (3): 489-493, 2006. DOI: 10.2460/ajvr.67.3.489
Hua LZ, Wu YZ, Bai FF, William KK, Feng ZX, Liu MJ, Yao JT, Zhang X, Shao GQ: Comparative analysis of mucosal immunity to Mycoplasma hyopneumoniae in Jiangquhai porcine lean strain and DLY piglets. Genet Mol Res, 13 (3): 5199-5206, 2014. DOI: 10.4238/2014.July.7.13
Nathues H, Woeste H, Doehring S, Fahrion AS, Doherr MG, Beilage E: Herd specific risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning. Acta Vet Scand, 55:30, 2013. DOI: 10.1186/1751-0147-55-30
Pieters M, Cline GS, Payne BJ, Prado C, Ertl JR, Rendahl AK: Intra-farm risk factors for Mycoplasma hyopneumoniae colonization at weaning age. Vet Microbiol, 172 (3-4): 575-580, 2014. DOI: 10.1016/j. vetmic.2014.05.027
Ginzinger DG: Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Exp Hematol, 30 (6): 503- 512, 2002. DOI: 10.1016/S0301-472X(02)00806-8
Hu WX, Zhou WY, Zhu XL, Wen Z, Wu LH, Wu XM, Wei HP, Wang WD, He D, Xiang Q, Hu GZ: Anti-interleukin-1 beta/tumor necrosis factoralpha IgY antibodies reduce pathological allergic responses in guinea pigs with allergic rhinitis. Mediat Inflmm, 2016:3128182, 2016.
Fourour S, Fablet C, Tocqueville V, Dorenlor V, Eono F, Eveno E, Kempf I, Marois-Créhan C: A new multiplex real-time TaqMan® PCR for quantification of Mycoplasma hyopneumoniae, M. hyorhinis and M. flcculare: Exploratory epidemiological investigations to research mycoplasmal association in enzootic pneumonia-like lesions in slaughtered pigs. J Appl Microbiol, 125 (2): 345-355, 2018. DOI: 10.1111/jam.13770
Rebaque F, Camacho P, Parada J, Lucchesi P, Ambrogi A, Tamiozzo P: Persistence of the same genetic type of Mycoplasma hyopneumoniae in a closed herd for at least two years. Rev Argent Microbiol, 50 (2): 147-150, 2018. DOI: 10.1016/j.ram.2017.05.002
Strait EL, Madsen ML, Minion FC, Christopher-Hennings J, Dammen M, Jones KR, Thacker EL: Real-time PCR assays to address genetic diversity among strains of Mycoplasma hyopneumoniae. J Clin Microbiol, 46 (8): 2491-2498, 2008. DOI: 10.1128/JCM.02366-07
Bai FF, Wu YZ, Liu MJ, Feng ZX, Xiong QY, Wei YN, Ma QH, Shao GQ: Development of TaqMan MGB probe real-time PCR for detection of Mycoplasma hyorhinis. Chinese J Prev Vet Med, 35, 833-836, 2013.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaf lW, Shipley GL, Vandesompele J, Wittwer CT: The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 55 (4): 611-622, 2009. DOI: 10.1373/clinchem.2008.112797
Lung O, Ohene-Adjei S, Buchanan C, Joseph T, King R, Erickson A, Detmer S, Ambagala A: Multiplex PCR and microarray for detection of swine respiratory pathogens. Transbound Emerg Dis, 64 (3):834-848, 2017. DOI: 10.1111/tbed.12449
Wu JB, Nan WJ, Huang JQ, Hu HH, Peng GL: Establishment and application of duplex PCR assay for detection of Haemophilus parasuis and Mycoplasma hyorhinis. Chinese Vet Sci, 9, 1094-1101, 2016.
Wu YZ, Xiong QY, Bai Y, Wei YN, Zhang ZZ, Wang HY, Feng ZX, Chenia HY, Shao GQ: Standardization study of Real-time PCR method for the quantitative detection of Mycoplasma hyopneumoniae Culture. Agric Sci Technol, 18, 2479-2484, 2017.
Markoulatos P, Siafakas N, Moncany M: Multiplex polymerase chain reaction: A practical approach. J Clin Lab Anal, 16 (1): 47-51, 2002. DOI: 10.1002/jcla.2058
Wu YZ, Zhang X, Hua LZ, Liu MJ, Shao GQ: Introduction on Mycoplasma contamination in cell culture. Prog Vet Med, 34, 112-117, 2013.
Jiangsu Province Quality and Technical Supervision Bureau: DB32/ T 1461-2009 Method of PCR for detecting Mycoplasma hyopneumoniae. Agricultural local standard of Jiangsu province, 2009.
Cai HY, Van Dreumel T, Mcewen B, Hornby G, Bell-Rogers P, Mcraild P, McRaild P, Josephson G, Maxie G: Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples. J Vet Diagn Invest, 19 (1): 91-95, 2007. DOI: 10.1177/104063870701900115
Ministry of Agriculture of the People’s Republic of China: NY/T 1186-2017. Diagnostic Criteria for Mycoplasmal Pneumonia of Swine. National Standard of the People’s Republic of China, 2017.
Dubosson CR, Conzelmann C, Miserez R, Boerlin P, Frey J, Zimmermann W, Hani H, Kuhnert P: Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples. Vet Microbiol, 102 (1-2): 55-65, 2004. DOI: 10.1016/j.vetmic.2004.05.007
Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH: Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques, 23 (3): 504-511, 1997. DOI: 10.2144/97233rr01
Wernike K, Hoffann B, Beer M: Single-tube multiplexed molecular detection of endemic porcine viruses in combination with background screening for transboundary diseases. J Clin Microbiol, 51 (3): 938-944, 2013. DOI: 10.1128/JCM.02947-12
Kurth KT, Hsu T, Snook ER, Thacker EL, Thacker BJ, Minion FC: Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine. J Vet Diagn Invest, 14 (6): 463-469, 2002. DOI: 10.1177/104063870201400603
Verdin E, Saillard C, Labbé A, Bové JM, Kobisch M: A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs. Vet Microbiol, 76 (1):31-40, 2000. DOI: 10.1016/S0378-1135(00)00228-5
Friis NF: Mycoplasma suipneumoniae and Mycoplasma flcculare in comparative pathogenicity studies. Acta Vet Scand, 15 (4): 507-518, 1974.
Jin MM: Development and application of the real-time florescent quantitative PCR detection of Mycoplasma hyopneumoniae. MSc Thesis, Nanjing Agriculture University, 2012.
Wang GP, Li JP, Wang YN, Luo G, Hu SF, Li XY: Distribution of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in the respiratory tract and blood of slaughter pigs. Swine Prod, 1, 119-120, 2016.