Reliable means of diagnosis and serovar determination of blood-borne Salmonella strains: quick PCR amplification of unique genomic loci by novel primer sets

Abstract

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

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.

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, Escherichia 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.

FIG. 3.

The genomic locus used as the diagnostic marker is potentially 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 representative 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. Paratyphi A, S. Typhi CT18, and S. Typhi Ty2, respectively. M lanes contain molecular size markers.

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 corresponding 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 procedure 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.

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 contain 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.