2014.ISDS.Abstracts.Final.pdf


ISDS Annual Conference Proceedings 2014. This is an Open Access article distributed under the terms of the Creative Commons Attribution-
Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, 
and reproduction in any medium, provided the original work is properly cited.

ISDS 2014 Conference Abstracts

Validation of multiplex PCR for detection and 
differentiation of Salmonellas
Prof. Borys Stegniy, Dr Iryna Gerilovych, Prof. Andrii Zavgorodniy, Anton Gerilovych*, 
Prof. Vasyl’ Vlizlo and Vasyl’ Arefiev

NSC IECVM, Kharkiv, Ukraine

Objective
This study aimed to perform interlaboratory testing and clarification 

of the PCR-based test for its implementation in Ukraine.

Introduction
Salmonellosis is the zoonotic disease caused by Salmonella 

bacteria. These are food-borne pathogens, which require improvement 
of diagnostics and surveillance measures. Prior to implementation 
of a PCR-based system for monitoring Salmonella, presence and 
differentiation of the agent was validated under Office International 
Epizootical (O.I.E.) requirements.

Methods
The PCR-based detection technique for five Salmonella species was 

tested under O.I.E. requirements, to determine specificity, sensitivity, 
and repeatability. Primers were calculated by OLIGO software and 
produced with HPLC purification. Amplification was conducted by 
a T3000 Biometra PCR-machine. Testing was performed on a panel 
of five basic agents: Salmonella enterica ser. Enteritidis, Salmonella 
ser. Typhimurium, Salmonella ser. Typhi, Salmonella ser. Dublin, 
Salmonella ser. Gallinarum-Pullorum

Results
The testing panel included positive samples as controls, containing 

virus at a concentration of 10-1-107 PFU/ml (n = 5 for each species). 
Artificially contaminated clinical materials were also used as positive 
controls (n = 5). Uninfected clinical materials (n = 20) were used as 
negative controls. Samples contaminated with E.coli, Mycobacteria 
spp. were used to test the platform’s ability to differentiate between 
bacterial genus. Testing determined the overall PCR protocol as 89 
% sensitive, 98 % specific (artificial reactions were observed) and 
repeatable. These results allowed for the recommendation that the 
testing technique be implemented in laboratory practice in Ukraine. 
Furthermore, specific guidelines for performing the test have been 
developed

Conclusions
We have validated the PCR-based protocol for indication of 

Salmonella genus agents and identification of its basic agents S. 
Enteritidis, S. Typhimurium, S. Typhi, S. Dublin, S. Gallinarum-
Pullorumon that recognized it to be sensitive, specific, and repeatable 
and able to be implemented in the laboratory practice for food safety 
control.

Keywords
multiplex PCR; Salmonella; detection

*Anton Gerilovych
E-mail: antger@rambler.ru    

Online Journal of Public Health Informatics * ISSN 1947-2579 * http://ojphi.org * 7(1):e162, 2015