ORIGINAL ARTICLE Rapid detection of Actinobacillus pleuropneumoniae targeting the apxIVA gene for diagnosis of contagious porcine pleuropneumonia in pigs by polymerase spiral reaction R. Sarkar, P. Roychoudhury, S. Kumar, S. Dutta, N. Konwar, P. K. Subudhi and T. K. Dutta Department of Veterinary Microbiology, Central Agricultural University, Aizawl, Mizoram, India Significance and Impact of the Study: Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia associated with serious economic impact on pig hus- bandry worldwide. Diagnosis of the disease by existing techniques including isolation and identification of bacteria followed by serotyping, serological techniques, conventional PCR, real-time PCR and LAMP assays are cumbersome, time-consuming, costly and not suitable for rapid field application. A novel isothermal polymerase spiral reaction (PSR) is developed for the specific detection of A. pleuropneumo- niae in porcine nasal swabs. The technique is highly sensitive, specific, cost-effective and applicable to the field condition for rapid diagnosis of the disease. To the best of our knowledge, this is the first-ever report on the development of PSR for specific detection of A. pleuropneumoniae. Keywords Actinobacillus pleuropneumoniae, diagnosis, India, pig, polymerase chain reaction (PSR). *Correspondence Tapan K. Dutta, Professor & Head, Depart- ment of Veterinary Microbiology, Central Agricultural University, Selesih, Aizawl, Mizo- ram – 796014, India. E-mail: tapandutta@rediffmail.com 2022/LAMICRO-2022-0163.R3: received 20 March 2022, revised 13 May 2022 and accepted 16 May 2022 doi:10.1111/lam.13749 Abstract Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia associated with serious economic impact on pig husbandry worldwide. Diagnosis of the disease by existing techniques including isolation and identification of bacteria followed by serotyping, serological techniques, conventional PCR, real-time PCR and LAMP assays are cumbersome, time-consuming, costly and not suitable for rapid field application. A novel isothermal polymerase chain reaction (PSR) technique is standardized for all the reagents, incubation time and incubation temperature against A. pleuropneumoniae. The sensitivity of the assay was determined against various dilutions of purified DNA and total bacterial count. The specificity of the assay was determined against 11 closely related bacterial isolates. The relative sensitivity and specificity were compared with bacterial isolation, conventional PCR and real-time PCR assays. The PSR assay for specific detection was standardized at 64°C for 30 min of incubation in a water bath. The result was visible by the naked eye after centrifugation of the reaction mixture or after incorporation of SYBR Green dye as yellowish-green fluorescence. The technique was found to be 100% specific and equally sensitive with real-time PCR and 10 times more sensitive than conventional PCR. The PSR assay could be applicable in the detection of the organisms in porcine nasal swabs spiked with A. pleuropneumoniae. This is the first-ever report on the development of PSR for specific detection of A. pleuropneumoniae and can be applied for early diagnosis at the field level. Introduction Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia (Dayao et al., 2016; Watt et al., 2020) associated with serious economic impact on pig husbandry worldwide (Gottschalk and Broes, 2019). Pigs of 12–16 weeks of age are found to be most susceptible to the disease (Makrai et al., 2019), where infection may be contracted by direct contact with diseased animals including asymptomatic Letters in Applied Microbiology 75, 442--449 © 2022 The Society for Applied Microbiology. 442 Letters in Applied Microbiology ISSN 0266-8254 Downloaded from https://academic.oup.com/lambio/article/75/2/442/6989312 by guest on 19 January 2023