A METHOD FOR DETECTING AND TYPING OF SALMONELLA BY MULTIPLEX PCR

Today in Ukraine’s market is increasing the volume of trade with livestock products. Also the number of catering services and grocery shops selling ready-made food is growing throughout the country. Th e veterinary service should have time to check the quality of all of these products. Only traditional bacteriological methods of isolation and identifi cation of pathogens of toxicoinfection, which is not enough in terms of increasing turnover of products, are used today. Th e one of the most dangerous toxicoinfections is salmonellosis. Typing diff erent Salmonella species gives an answer about the source of infection. Th e aim of our work was to develop a system of identifi cation of Salmonella and typing among them fi ve serovars based on the polymerase chain reaction (multiplex PCR). We performed analysis of the nucleotide sequences of the fi ve members of the genus Salmonella, on the basis of which a primer designed for the identifi cation of any member of the genus Salmonella with simultaneous typing Salmonella enteriса ser. Enteritidis, Salmonella enteriса ser. Typhimurium, Salmonella enterica ser. Тyphi, Salmonella enterica ser. Dublin, Salmonella enterica ser. GallinarumPullorum by multiplex PCR. Th e protocol of multiplex PCR was optimization with simples positive DNA matrix.


INTRODUCTION
Salmonellosis -one of the most dangerous diseases that is caused by serotypes of bacteria of the genus Salmonella, which have mechanisms for habitat and parasitism in the gastrointestinal tract (Althouse et al., 2003;Chiu et al., 2010).
According to the current classifi cation, S. enterica is divided into six subspecies: Salmonella enterica subspecies enterica, Salmonella enterica subspecies salamae, Salmonella enterica subspecies arizonae, Salmonella enterica subspecies diarizonae, Salmonella enterica subspecies houtenae and Salmonella enterica subspecies indica, which diff erentiate by the biochemical activity and represent the number of subtypes I, II, IIIa, IIIb, IV , and VI , respectively. Salmonella enterica subspecies enterica is mostly isolated in the majority of cases of Salmonella infection from animal and human (Althouse et al., 2003;Battistuzzi et al., 2004).
Salmonella contamination occurs through the consumption of contaminated food: eggs and egg products, milk and dairy products, meat birds and other animals. Another way of infection is the transfer of infections through tap water, in addition, the sources of infection can be the open water (Bailey, 1998). According to the FAO, 20% of poultry products in the world are contaminated with Salmonella, and they can persist for a long time in the animal facilities because they can form a surface fi lm (Vestby et al., 2009; http://www. fao.org/docrep/012/i1133e/i1133e00.htm). Annually on the planet are registered 21 million cases of typhoid fever, and about 216 thousand cases (Zhou and Pollard, 2010).
Worldwide, the monitoring of the incidence of salmonellosis in which tracked various options for its manifestation. As well as a comparison of Salmonella strains isolated from humans and animals (Chiu et  Analysis of antigen alleles H1 (i, g, m, r or z10) allowed fast typed serological variants enteritidis, hadar, heidelberg and typhimurium (Hong et al., 2008).
To date, Ukraine has not yet widespread methods of rapid diagnosis of salmonellosis. Typing of the pathogen is an essential component of diagnosis, because it can give an answer about the alleged source of infection. For this reason, the aim of our work was the development of the national test system based on the polymerase chain reaction, which would like to identify and typed some key members of the genus Salmonella (Gerylovich, 2011).

METHODS AND MATERIALS
Th e objects of our study were Salmonella spp., Salmonella enterica ser. Enteritidis, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Typhi, Salmonella enterica ser. Dublin, Salmonella enterica ser. Gallinarum-Pullorum. For the construction of genus-and species-specifi c primers electronic databases of sequences of essential genes in Salmonella contained in the international database GenBank (http://www.ncbi.nlm.nih.gov/genbank/) were analyzed.
Multiple alignment of selected sequences, and their subsequent analysis to select PCR primers was performed using the computer program Bio Edit (v.7.2.4).
Th e protocol of polymerase chain reaction has been developed on the basis of the primer systems with a certain temperature, the selection of components for the formulation of the multiplex PCR and the identifi cation of the genus Salmonella spp. and typing of the fi ve listed above serotypes (Elnifro, 2000;Kaderali, 2007).
One-day-old cultures of Salmonella from the museum sector study mycoplasmoses and salmonellosis are grown for meat -peptone medium were used as the source of positive DNA-matrix.
Extraction of total nucleic acid was carried out using micro columns. To 450 μl of Extraction buff er was added 100 μl of Salmonella culture. Aft er lysis of the containments from the tubes were transferred to microcolumns and centrifuged. Th is was followed by washing with ethanol followed by extraction of total nucleic acid of TE-buff er.
DNA concentration was calculated by spectrophotometery at 260 nm.

RESULTS AND DISCUSSIONS
Th e nucleotide sequences of the major genes were analyzed. Th e greatest breadth of sample homogeneity and sequenced portions of the gene was detected in invA for all members of the genus Salmonella. In the computer analysis of the gene sequences invA was selected 22 pairs of oligonucleotides -potential pairs of primers for PCR. Th e PCR product limited by size of 387 bp in length, and olygonucleotides were called Salm3_4.
For Salmonella enterica ser. Enteritidis specifi c motifs were found in the gene SefA. Sequence analysis of this gene allowed to establish the potential 6 primer pairs. Th e primers fl anking portion length 299 bp were selected.
Th e gene fl iC demonstrated specifi city for Salmonella enterica ser. Typhimurium. Th e primers fl anking region 420 bp were choosed.
Gene viaB contained specifi c motives for Salmonella enterica ser. Typhi. Accordingly, on this basis was chosen area, which limited the targeted gene fragment length 738 bp.
For the genome of Salmonella enterica ser. Dublin serospecifi c motifs were found in SeD_A1104 gene. When bioinformatics studies were identifi ed primers fl anking the product of 203 bp.
Finally, gene SG0266 was elected by containing specifi c motifs for Salmonella enterica ser. Gallinarum-Pullorum. Specifi c primers fl anking length of 97 bp region were selected in analyzed area. Aft er synthesis of primers, we performed optimization of the PCR protocol. As the positive control for PCR we used DNA extracted from the oneday-old culture of Salmonella which have been stored in the museum NSC " IECVM ".
Th e obtained DNA matrix concentration aft er measuring with a spectrophotometer, we have led to the same concentration and then put PCR.
Th e fi rst stage was carried out testing each primer pair using the standard composition of the reaction mixture at diff erent temperatures.
Multiplex PCR protocol could be applied in the laboratories for identification and typing of Salmonella in the shortest possible time. Also, the system can be convenient for monitoring Salmonella contamination of various objects, while typing their main representatives.

ACKNOWLEDGE
Th e authors thank senior researcher studying pathology of reproduction Bolotin Vitaly I., and Roxana Sanchez-Ingunza, DVM, PhD Research Microbiologist Egg Safety and Quality Research Unit USDA, ARS, RRC 950 College Station Road Athens, GA 30605 USA for assistance in the Science.