Comparison of three stool-processing methods for detection of Salmonella serogroups B, C2, and D by PCR.

Three different stool sample-processing methods (centrifugation, immunomagnetic separation, and selective enrichment cultivation) for the identification of Salmonella serogroups by PCR were studied. The corresponding sensitivities in an ethidium bromide stained-agarose gel were 10(5), 10(3), and 10 bacteria, respectively. The PCR assay with overnight enrichment performed as well as, or even better than, the conventional culture technique. Of 485 clinical stool samples, PCR correctly identified all 230 culture-positive samples as well as mixed Salmonella infections in four cases

Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of Salmonella major serogroups (A, B, C2, and D).

Many parts of the Salmonella rfb gene clusters which are responsible for biosynthesis of the oligosaccharide-repeating units of the O-antigenic lipopolysaccharide have recently been cloned and sequenced. On the basis of this knowledge, three sets of nucleotide primers were selected to target defined regions of the abequose and paratose synthase genes: rfbJ of Salmonella serogroup B, rfbJ of Salmonella serogroup C2, and rfbS of Salmonella serogroup D (also present in serogroup A). For good differentiation among these major serogroups, the primers were designed not only to give precise specificity in priming but also to give DNA products with different sizes in polymerase chain reactions (product sizes, approximately 720 bp for both serogroups A and D, approximately 820 bp for serogroup C2, and approximately 882 bp for serogroup B). In a polymerase chain reaction assay utilizing these rfb-specific primers, all of the 40 salmonellae belonging to serogroups B, C2, and D plus A were accurately identified among a total of 123 clinical isolates tested (including 55 salmonellae from 36 different serotypes and 68 strains from 10 other members of the family Enterobacteriaceae). No false-positive reactions were detected. The selected rfb gene sequences were proved for the first time to be useful DNA-based markers for identification of and differentiation among Salmonella serogroups A, B, C2, and D

An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible