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

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

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.

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