Journal: Journal of Clinical Microbiology

Article Title: Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

doi: 10.1128/jcm.00327-09

Figure Lengend Snippet: FIG. 1. Schematic diagram of the genome organization of the loci used to design primers. The loci were from the three Salmonella strains S. Typhi CT18 (STY CT18), S. Paratyphi A (SPA), and S. Typhi Ty2 (STY Ty2). The expected size of the PCR product is shown at the top of the figure. Solid black arrows depict the position and direction of primer binding sites of the two sets of primers. The genome organizations of the three serovars are depicted as solid arrows, each representing a gene with its GenBank locus tag number given below. Broken lines represent genes that are absent in a particular serovar.

Article Snippet: The complete genomes of Salmonella enterica subsp. enterica serovar Typhi CT18, (accession no. NC_003198), S. enterica subsp. enterica serovar Typhi Ty2 (accession no. NC_004631), S. enterica subsp. enterica serovar Paratyphi A ATCC 9150 (accession no. NC_006511), and Salmonella serovar Typhimurium LT2 (accession no. NC_003197) have been sequenced and deposited in the GenBank database.

Techniques: Binding Assay

Journal: Journal of Clinical Microbiology

Article Title: Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

doi: 10.1128/jcm.00327-09

Figure Lengend Snippet: FIG. 2. The PCR detection method is specific for Salmonella and differentiates various typhoid-causing serovars. Colony PCR was performed for different serovars of Salmonella and other pathogenic and nonpathogenic bacteria. Salmonella enterica subsp. arizonae, many Salmonella enterica serovars, and Salmonella bonogori were used. The other pathogenic and nonpathogenic bacteria included Staphylococcus aureus, Esche- richia coli, uropathogenic E. coli (UPEC), Shigella flexneri, Yersinia enterocolitica, Hafnia alvei, Vibrio cholerae, Citrobacter freundii, Proteus vulgaris, and Pseudomonas syringae. PCR amplification was performed with the two sets of primers for 30 cycles, and the product was run on a 1.5% agarose gel. The samples have been loaded on the gel in the same order as in Table 1. The 384-bp band is the amplification product of the STY0312 gene of S. Typhi CT18 and its homolog in S. Paratyphi A and is found in both the serovars. The 1,043-bp band is the amplification product of the locus spanning the genes STY0313 to STY0316 and their homologs in S. Typhi Ty2. This locus is absent in S. Paratyphi A. Both these bands are absent in all the other bacterial strains tested.

Article Snippet: The complete genomes of Salmonella enterica subsp. enterica serovar Typhi CT18, (accession no. NC_003198), S. enterica subsp. enterica serovar Typhi Ty2 (accession no. NC_004631), S. enterica subsp. enterica serovar Paratyphi A ATCC 9150 (accession no. NC_006511), and Salmonella serovar Typhimurium LT2 (accession no. NC_003197) have been sequenced and deposited in the GenBank database.

Techniques: Bacteria, Agarose Gel Electrophoresis

Journal: Journal of Clinical Microbiology

Article Title: Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

doi: 10.1128/jcm.00327-09

Figure Lengend Snippet: FIG. 3. The genomic locus used as the diagnostic marker is poten- tially stable. Twenty clinical isolates from Pondicherry (southern India) and 12 clinical isolates from Nagpur (central India) were analyzed by colony PCR to show that the region is potentially stable. The amplified products were run on a 0.8% agarose gel. (A) The 20 clinical isolates (lanes 1 to 20) showed two bands, characteristic of S. Typhi CT18, which has also been confirmed through serotyping. (B) This represen- tative gel shows 7 samples out of 12 samples obtained from patients in Nagpur, India. Among seven samples, three samples showed only the lower 384-bp band of S. Paratyphi A (lanes 2, 3, and 5), and four samples showed two bands of S. Typhi CT18 (lanes 1, 4, 6, and 7). Lanes 8, 9, and 10 contain controls with isolated colonies of S. Para- typhi A, S. Typhi CT18, and S. Typhi Ty2, respectively. M lanes contain molecular size markers.

Article Snippet: The complete genomes of Salmonella enterica subsp. enterica serovar Typhi CT18, (accession no. NC_003198), S. enterica subsp. enterica serovar Typhi Ty2 (accession no. NC_004631), S. enterica subsp. enterica serovar Paratyphi A ATCC 9150 (accession no. NC_006511), and Salmonella serovar Typhimurium LT2 (accession no. NC_003197) have been sequenced and deposited in the GenBank database.

Techniques: Diagnostic Assay, Marker, Agarose Gel Electrophoresis, Isolation

Journal: Journal of Clinical Microbiology

Article Title: Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

doi: 10.1128/jcm.00327-09

Figure Lengend Snippet: FIG. 4. The PCR-based assay can detect as few as four bacteria/ml. The minimum number of bacteria that can be detected by this method was determined by diluting a culture which contains 1.5 108 bacteria and adjusting it to an OD600 of 0.3 with various dilutions in PBS and sterile blood. The diluted culture samples were then subjected to one round of PCR amplification for 35 cycles; the products were then visualized by electrophoresis on a 0.8% agarose gel. The corre- sponding number of bacteria/ml is given at the top of each lane. (A) Different numbers of S. Typhi CT18 diluted in PBS, showing that one bacterium/ml can produce a visible band. (B) Different numbers of S. Typhi CT18 diluted in blood, showing that the proce- dure can detect as few as four bacteria/ml of blood. (C) Different numbers of S. Paratyphi A diluted in PBS, showing a sensitivity of detection of four bacteria/ml. (D) Different numbers of S. Paratyphi A diluted in blood, showing a lower detection limit of one bacterium/ml of blood. M lanes contain molecular size markers.

Article Snippet: The complete genomes of Salmonella enterica subsp. enterica serovar Typhi CT18, (accession no. NC_003198), S. enterica subsp. enterica serovar Typhi Ty2 (accession no. NC_004631), S. enterica subsp. enterica serovar Paratyphi A ATCC 9150 (accession no. NC_006511), and Salmonella serovar Typhimurium LT2 (accession no. NC_003197) have been sequenced and deposited in the GenBank database.

Techniques: Bacteria, Sterility, Electrophoresis, Agarose Gel Electrophoresis

Journal: Journal of Clinical Microbiology

Article Title: Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

doi: 10.1128/jcm.00327-09

Figure Lengend Snippet: FIG. 5. The PCR assay is more sensitive than the Widal test is. Two representative gel pictures showing the PCR products amplified from blood samples from patients. The corresponding Widal test result (Wi) (, positive; , negative) is given above each lane. (A) Lanes 1, 2, and 3 contain samples positive for S. Paratyphi A. Lanes 4 to 13 con- tain samples positive for S. Typhi. (B) Lanes 1, 4, 6, 7, 8, and 9 contain samples positive for S. Typhi CT18. Lanes 2, 3, 10, and 11 contain PCR-negative samples. Lane 5 contains a sample positive for S. Typhi Ty2 by PCR. Lane C contains a positive control for S. Typhi CT18. The PCR assay is 40% more sensitive than the Widal test in this given partial sample data. M lanes contain molecular size markers.

Article Snippet: The complete genomes of Salmonella enterica subsp. enterica serovar Typhi CT18, (accession no. NC_003198), S. enterica subsp. enterica serovar Typhi Ty2 (accession no. NC_004631), S. enterica subsp. enterica serovar Paratyphi A ATCC 9150 (accession no. NC_006511), and Salmonella serovar Typhimurium LT2 (accession no. NC_003197) have been sequenced and deposited in the GenBank database.

Techniques: Positive Control