Identification Of Salmonella Enterica Subspecies Enterica Serovar Typhi-Specific Genes For The Development Of Dna-Based And Antibody-Based Diagnostics For Typhoid Fever
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Date
2018-02
Authors
Goay, Yuan Xin
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Typhoid fever is caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). It is an acute systemic disease which remains a major public health burden worldwide. A lack of specific and sensitive diagnostic markers for detection of S. Typhi at single-gene target resolution prevents effective diagnosis and therefore efficient control of the disease. In the first phase of this study, genome level comparison of S. Typhi with other enteric pathogens was performed using BLASTn. Six genes, i.e. STY0201, STY0307, STY0322, STY0326, STY2020 and STY2021 were found to be specific and sensitive in silico. Six individual single-gene target PCR assays with the incorporation of an internal amplification control (IAC) were developed and optimised using Taguchi method. The analytical specificities of the optimised PCR assays were determined using purified DNA from 39 S. Typhi, 62 non-Typhi Salmonella, and 10 non-Salmonella clinical strains. The analytical sensitivities of the PCR assays were assessed using 5-fold dilutions of genomic DNA and 10-fold dilutions of bacterial culture. Diagnostic specificity and diagnostic sensitivity evaluation tests were further assessed for one of these highly sensitive and specific assays. Of the 6 candidate genes, 5 genes i.e. STY0307, STY0322, STY0326, STY2020 and STY2021 demonstrated 100% analytical specificity (detection of 39/39 bacteria strains). Gene STY0201 demonstrated 97.2% analytical specificity (detection of 70/72 bacteria strains). The sensitivities of the 6 PCR assays by genomic DNA were; 32 pg for STY0322, 6.4 pg for STY0201, STY0326, STY2020 and STY2021, and 1.28 pg for STY0307. The sensitivities of these PCR assays by bacteria counts were as follows; 1.5 × 105 cfu/mL for STY0307 and 1.5 × 106 cfu/mL for STY0201, STY0322, STY0326, STY2020 and STY2021. Since the STY0307 PCR assay demonstrated the highest analytical sensitivity, it was selected for further sensitivity evaluation using spiked stool samples. It was found that with 18 hr enrichment, the sensitivity of STY0307 PCR assay was 1.5 × 104 cfu/mL. The STY0307 PCR assay demonstrated 100% diagnostic specificity and sensitivity when 130 clinical samples were blind screened.
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Keywords
Specific genes for the development of dna-based , and antibody-based diagnostics for typhoid fever