Development and validation of multiplex PCR based molecular serotyping of Salmonella serovars associated with poultry in India

• Published in: Journal of microbiological methods Vol. 207; p. 106710
• Main Authors: Mohanapriya, K., Agri, Himani, Anbazhagan, Subbaiyan, Khawaskar, Damini, Jayakumar, Varsha, Lalrinzuala, Michael V., K.M., Himani, I., Sophia, Mariappan, Asok K., Abhishek, Nagaleekar, Viswas Konasagara, Sinha, Dharmendra K., Chaudhuri, Pallab, Chaturvedi, Vinod K., Singh, Bhoj R., Thomas, Prasad
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2023
• Subjects: Animals; Humans; India; Molecular serotyping; Multiplex Polymerase Chain Reaction - methods; PCR; Poultry; Salmonella; Salmonella - genetics; Salmonella enterica - genetics; Serogroup; Serotyping; Serovars; Pullorum disease; Salmonella; Nontyphoidal Salmonella; Poultry; Fowl typhoid; Serovars; Molecular serotyping; White Kauffman Le-Minor scheme; PCR; India
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2023.106710

Salmonella species are Gram-negative bacteria with more than 2600 serovars. Among these serovars, many are associated with various diseases in livestock and humans. White Kauffman Le-Minor (WKL) serotyping scheme applies specific serum to determine the serovars of Salmonella. Recent studies have applied molecular methods for serovar predictions. These methods include PCR, hybridization and sequence data to detect/predict serovar-specific genetic elements. Among these, PCR is a robust method if the unique genetic element is already known. Within this context, also involving novel primers, two multiplex PCR assays were standardized to detect six important Salmonella serovars viz. Typhimurium, Enteritidis, Kentucky, Infantis, Virchow and Gallinarum associated with poultry in India. The developed PCR assays showed targeted serovar specificity. Serial dilution experiments of both kit-based and crude lysate DNA preparations indicated similar applicability of both methods for testing from pure cultures. Further the developed assays were validated with 25 recent field isolates to confirm the applicability in routine diagnosis. The PCR assay could predict all the targeted serovars (17/25) with 100% specificity (CI-95%; 0.63–1). Molecular serotyping can reduce the number of serum used in comparison to the conventional serotyping which involves more random application of serum. [Display omitted] •Two multiplex PCR assays were developed to detect six important Salmonella serovars associated with poultry in India.•Developed molecular serotyping PCR assays showed high specificity for the targeted Salmonella serovars.•Detection limits were comparable for both kit-based and crude lysate DNA preparations during multiplexing.•PCR assays could detect targeted serovars when applied to recent field strains indicating applicability in routine diagnosis.