Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars

Abstract

A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rfb , fliC , fljB , and viaB groups that encode the O , H , and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ , wzx , fliC , fljB , wcdB , the sdf-l sequence , and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella O:4 , O:9 , O:7 , O:8 , and O:3 ,10 serogroups. Once the serogroups were identified , the results of a second round of reactions that used primers and probes for the flagellar antigen l genes , 1 ,2; e ,h; g ,m; d; e ,n ,x; and z 10 , and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedure's reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus , our technique is a quick , sensitive, reliable , and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars. (J Mol Diagn 2010, 12:220 -225;

Rapid Genoserotyping Tool for Classification of Salmonella Serovars

“…Blind samples were found to be in agreement with traditional serotyping results as long as the serogroup designation was correct; however, all of the possible serovars based on somatic factors are listed in Table S2 in the supplemental material. This limitation is shared by all other molecular serotyping methods that rely on serogroup detection rather than individual factors (16,49).…”

“…In recent years, molecular typing assays have been developed based upon multiplex real-time PCR (31,49), primer extension (5), microarrays (61,66,69), DNA sequencebased approaches (47), and bead-based suspension arrays (16,41). To date, many of the molecular techniques have been able to type only a very small subset of the thousands of Salmonella serovars.…”

Serotype Distribution and Antimicrobial Susceptibility Profiles of Salmonella Isolates from South Marmara Region, Turkey

“…Serotiplendirme; sürve-yans, gıda kaynaklı salgınların tespiti, kontrolü ve önlenmesi, yeni türlerin ve geçiş mekanizmalarının belirlenmesi ve suşların epidemiyolojik sınıflandırması açısından önem taşımaktadır (5,6) .…”

Determination of Salmonella Serotypes by Conventional and Molecular Methods

“…Salmonella serotiplerinin belirlenmesi, ulusal sürveyansın yönelimi, suşların epidemiyolojik olarak sınıflandırılması, salgınların incelenmesi ve uluslararası bir dil oluşması açı-sından önem taşır 1,2 .…”

“…Özgül Salmonella an-tiserumları ile lam veya tüp aglütinasyon tekniği kullanılarak mikroorganizmanın önce "O" somatik antijenine göre serogrubu, daha sonra "H" kirpik antijeninin tanımlanması ile serotipi belirlenir 1 . Konvansiyonel serotiplendirme; zaman alan, yorumlanması subjektif olabilen ve bu nedenle iyi eğitimli kişilerce çalışılması gereken, yüksek kaliteli çok sayıda antiserum gerektiren, pahalı bir yöntemdir 2,3 .…”