Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis
Yıl: 2020 Cilt: 26 Sayı: 5 Sayfa Aralığı: 665 - 670 Metin Dili: İngilizce DOI: 10.9775/kvfd.2020.24110 İndeks Tarihi: 16-10-2020
Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis
Öz: 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.
Anahtar Kelime: Actinobacillus pleuropneumoniae ve Haemophilus parasuis’in Eşzamanlı Saptanması Amacıyla Erime Eğrisi Analizi İle SYBR Green Real-Time PCR Testinin Geliştirilmesi
Öz: 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.
Anahtar Kelime: Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık
- 1. Gajda A, Bladek T, Jablonski A, Posyniak A: The infl uence of Actinobacillus pleuropneumoniae infection on tulathromycin pharmacokinetics and lung tissue disposition in pigs. J Vet Pharmacol Ther, 39 (2): 176-182, 2016. DOI: 10.1111/jvp.12259
- 2. Gottschalk M: The challenge of detecting herds sub-clinically infected with Actinobacillus pleuropneumoniae. Vet J, 206 (1): 30-38, 2015. DOI: 10.1016/j.tvjl.2015.06.016
- 3. Hricinova M, Holoda E, Mudronova D, Ondrasovicova S: Multiplex PCR assay for detection of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis in lungs of pigs from a slaughterhouse. Folia Microbiol, 55 (6): 635-640, 2010. DOI: 10.1007/ s12223-010-0103-9
- 4. Li R, Wang J, Liu L, Zhang R, Hao X, Han Q, Wang J, Yuan W: Direct detection of Actinobacillus pleuropneumoniae in swine lungs and tonsils by real-time recombinase polymerase amplification assay. Mol Cell Probes, 45, 14-18, 2019. DOI: 10.1016/j.mcp.2019.03.007
- 5. Marois-Crehan C, Lacouture S, Jacques M, Fittipaldi N, Kobisch M, Gottschalk M: Development of two real-time polymerase chain reaction assays to detect Actinobacillus pleuropneumoniae serovars 1-9- 11 and serovar 2. J Vet Diagn Invest, 26 (1): 146-149, 2014. DOI: 10.1177/ 1040638713519090
- 6. Zhang B, Tang C, Liao M, Yue H: Update on the pathogenesis of Haemophilus parasuis infection and virulence factors. Vet Microbiol, 168 (1): 1-7, 2014. DOI: 10.1016/j.vetmic.2013.07.027
- 7. Van CN, Thanh TVT, Zou G, Jia M, Wang Q, Zhang L, Ding W, Huang Q, Zhou R: Characterization of serotypes and virulence genes of Haemophilus parasuis isolates from Central Vietnam. Vet Microbiol, 230, 117-122, 2019. DOI: 10.1016/j.vetmic.2019.02.008
- 8. McDowall R, Slavic D, MacInnes JI, Cai HY: Evaluation of a real-time polymerase chain reaction assay of the outer membrane protein P2 gene for the detection of Haemophilus parasuis in clinical samples. Can J Vet Res, 78 (2): 150-152, 2014.
- 9. Turni C, Pyke M, Blackall PJ: Validation of a real-time PCR for Haemophilus parasuis. J Appl Microbiol, 108 (4): 1323-1331, 2010. DOI: 10.1111/j.1365-2672.2009.04526.x
- 10. MacInnes JI, Gottschalk M, Lone AG, Metcalf DS, Ojha S, Rosendal T, Watson SB, Friendship RM: Prevalence of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella multocida, and Streptococcus suis in representative Ontario swine herds. Can J Vet Res, 72 (3): 242-248, 2008.
- 11. Gimenez-Lirola LG, Jiang YH, Sun D, Hoang H, Yoon KJ, Halbur PG, Opriessnig T: Simultaneous detection of antibodies against Apx toxins ApxI, ApxII, ApxIII, and ApxIV in pigs with known and unknown Actinobacillus pleuropneumoniae exposure using a multiplexing liquid array platform. Clin Vaccine Immunol, 21 (1): 85-95, 2014. DOI: 10.1128/ CVI.00451-13
- 12. Li P, Bai J, Li JX, Zhang GL, Song YH, Li YF, Wang XW, Jiang P: Molecular cloning, sequencing, and expression of the outer membrane protein P2 gene of Haemophilus parasuis. Res Vet Sci, 93 (2): 736-742, 2012. DOI: 10.1016/j.rvsc.2011.08.019
- 13. Otaguiri ES, Morguette AEB, Morey AT, Tavares ER, Kerbauy G, de Almeida Torres RSL, Chaves Junior M, Tognim MCB, Goes VM, Krieger MA, Perugini MRE, Yamauchi LM, Yamada-Ogatta SF: Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides. BMC Pregnancy Childbirth, 18:126, 2018. DOI: 10.1186/s12884-018-1774-5
- 14. Chiers K, Van Overbeke I, Donne E, Baele M, Ducatelle R, De Baere T, Haesebrouck F: Detection of Actinobacillus pleuropneumoniae in cultures from nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide sequence of a dsbE-like gene. Vet Microbiol, 83 (2): 147- 159, 2001. DOI: 10.1016/s0378-1135(01)00414-x
- 15. Balboni A, Dondi F, Prosperi S, Battilani M: Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2. J Virol Methods, 222, 34-40, 2015. DOI: 10.1016/j.jviromet.2015.05.009
- 16. Yamaguchi A, Shimizu K, Mishima T, Aoki N, Hattori H, Sato H, Ueda N, Watanabe T, Hino A, Akiyama H, Maitani T: Detection method of genetically modified papaya using duplex PCR. Shokuhin Eiseigaku Zasshi, 47 (4): 146-150, 2006. DOI: 10.3358/shokueishi.47.146
| APA | Hu B, zhang s, Xu Y, WANG Z, ren q, Xu J, Dong Y, WANG L (2020). Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. , 665 - 670. 10.9775/kvfd.2020.24110 |
| Chicago | Hu Bin,zhang shouping,Xu YanHua,WANG ZHICHEN,ren qiuxuan,Xu Jingfei,Dong Yongjun,WANG Lirong Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. (2020): 665 - 670. 10.9775/kvfd.2020.24110 |
| MLA | Hu Bin,zhang shouping,Xu YanHua,WANG ZHICHEN,ren qiuxuan,Xu Jingfei,Dong Yongjun,WANG Lirong Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. , 2020, ss.665 - 670. 10.9775/kvfd.2020.24110 |
| AMA | Hu B,zhang s,Xu Y,WANG Z,ren q,Xu J,Dong Y,WANG L Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. . 2020; 665 - 670. 10.9775/kvfd.2020.24110 |
| Vancouver | Hu B,zhang s,Xu Y,WANG Z,ren q,Xu J,Dong Y,WANG L Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. . 2020; 665 - 670. 10.9775/kvfd.2020.24110 |
| IEEE | Hu B,zhang s,Xu Y,WANG Z,ren q,Xu J,Dong Y,WANG L "Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis." , ss.665 - 670, 2020. 10.9775/kvfd.2020.24110 |
| ISNAD | Hu, Bin vd. "Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis". (2020), 665-670. https://doi.org/10.9775/kvfd.2020.24110 |
| APA | Hu B, zhang s, Xu Y, WANG Z, ren q, Xu J, Dong Y, WANG L (2020). Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 26(5), 665 - 670. 10.9775/kvfd.2020.24110 |
| Chicago | Hu Bin,zhang shouping,Xu YanHua,WANG ZHICHEN,ren qiuxuan,Xu Jingfei,Dong Yongjun,WANG Lirong Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 26, no.5 (2020): 665 - 670. 10.9775/kvfd.2020.24110 |
| MLA | Hu Bin,zhang shouping,Xu YanHua,WANG ZHICHEN,ren qiuxuan,Xu Jingfei,Dong Yongjun,WANG Lirong Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, vol.26, no.5, 2020, ss.665 - 670. 10.9775/kvfd.2020.24110 |
| AMA | Hu B,zhang s,Xu Y,WANG Z,ren q,Xu J,Dong Y,WANG L Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2020; 26(5): 665 - 670. 10.9775/kvfd.2020.24110 |
| Vancouver | Hu B,zhang s,Xu Y,WANG Z,ren q,Xu J,Dong Y,WANG L Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2020; 26(5): 665 - 670. 10.9775/kvfd.2020.24110 |
| IEEE | Hu B,zhang s,Xu Y,WANG Z,ren q,Xu J,Dong Y,WANG L "Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis." Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 26, ss.665 - 670, 2020. 10.9775/kvfd.2020.24110 |
| ISNAD | Hu, Bin vd. "Development of a SYBR Green Real-Time PCR Assay with Melting Curve Analysis for Simultaneous Detection of Actinobacillus pleuropneumoniae and Haemophilus parasuis". Kafkas Üniversitesi Veteriner Fakültesi Dergisi 26/5 (2020), 665-670. https://doi.org/10.9775/kvfd.2020.24110 |
