Shouping ZHANG, Bin HU, Yanhua XU, Zhichen WANG, Qiuxuan REN, Jingfei XU, Yongjun DONG, Lirong WANG

Actinobacillus pleuropneumoniae ve Haemophilus parasuis’in Eşzamanlı Saptanması Amacıyla Erime Eğrisi Analizi İle SYBR Green Real-Time PCR Testinin Geliştirilmesi

Bu çalışmada, bir erime eğrisi analizi yoluyla tek bir reaksiyonda Actinobacillus pleuropneumoniae ve Haemophilus parasuis enfeksiyonunu tanımlamak için bir dubleks SYBR Green gerçek zamanlı PCR analizi geliştirildi. Bu yöntemde, A. pleuropneumoniae Apx IV ve H. parasuis omp P2 geninin yüksek oranda korunmuş bölgelerinin amplifikasyonuna izin veren iki çift spesifik primer kullanıldı. Analizin duyarlılğını belirlemek için PMD - 19T plazmid kullanılarak sulandırma deneyleri gerçekleştirildi. Sonuçlar, A. pleuropneumoniae ve H. parasuis’in erime eğrilerinin Tm değerlerinin sırasıyla bu iki patojeni doğru şekilde ayırt edebilen 83.36±0.09ºC ve 76.48±0.17ºC olduğunu gösterdi. Diğer solunum sistemi patojenleri ile aralarında çapraz reaksiyon gözlenmedi, bu da iki primerin yüksek özgüllüğünü ortaya koydu. Testin saptama hassasiyeti, sıradan PCR yöntemlerinden daha yüksek olan 127 ve 96 kopya/μL idi. Bu hızlı teknik, A. pleuropneumoniae ve H. parasuis’in eşzamanlı tespiti için basit ve kullanışlı bir seçenek sunabilir;i söz konusu seçenek klinik örneklerin teşhisi ve epidemiyolojik araştırmalar için uygulanabilir ve cazip olacaktır.

Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis

and Haemophilus parasuis infection in one reaction, through a melting curve analysis. This method utilized two pairs of specific primersthat allowed the amplification of highly conserved regions of A. pleuropneumoniae Apx IV and H. parasuis omp P2 gene. Reconstitutionexperiments were conducted by using PMD - 19T plasmid in order to determine the sensitivity of the assay. The results showed that the Tmvalues of the melting curves of A. pleuropneumoniae and H. parasuis were 83.36±0.09ºC and 76.48±0.17ºC, respectively that could accuratelydistinguish these two pathogens. And no cross reaction were observed between other respiratory pathogens, which suggested a highspecificity of two primers. The detection sensitivity of the assay was 127 and 96 copies/μL which was higher than that of the ordinary PCRdetection methods. This rapid technique may present a simple, useful option for simultaneous detection of A. pleuropneumoniae and H.parasuis, which would be feasible and attractive for clinical samples diagnosis and epidemiological investigations.

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