Molecular detection of Salmonella enterica serovar Typhi by Vi-qPCR

Nik Noorul Shakira Mohamed Shakrin; Siti Noor Adnalizawati Adnan; Asmah Hani Abdul Wahab; R Pusparani Ramasamy; Wan Noraini Wan Yussof; Noorliza Noordin; Khebir Verashahib; Rohani Jahis

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Molecular detection of Salmonella enterica serovar Typhi by Vi-qPCR

Author: Nik Noorul Shakira Mohamed Shakrin ¹ ; Siti Noor Adnalizawati Adnan ² ; Asmah Hani Abdul Wahab ³ ; R. Pusparani Ramasamy ³ ; Wan Noraini Wan Yussof ³ ; Noorliza Noordin ³ ; Khebir Verashahib ³ ; Rohani Jahis ⁴
Author Information

1. Faculty of Medicine and Defence Health, Universiti Pertahanan Nasional Malaysia (UPNM), 57000 Sungai Besi, Kuala Lumpur, Malaysia
2. Faculty of Dentistry, Universiti Sains Islam Malaysia (USIM), Level 15, Tower B, Persiaran MPAJ, Jalan Pandan Utama, 55100 Kuala Lumpur, Malaysia
3. Bacteriology Section, National Public Health Laboratory, Lot 1853, Kampung Melayu, 47000 Sungai Buloh, Selangor, Malaysia
4. Bahagian Kawalan Penyakit, Kementerian Kesihatan Malaysia, Blok E10, Kompleks E, 62590 Putrajaya, Malaysia
Publication Type:Journal Article
Keywords: PCR; diagnostic; pathogen
From:Malaysian Journal of Microbiology 2018;14(6):483-489
CountryMalaysia
Language:English
Abstract: Aims:To develop a real-time polymerase chain reaction system Vi-qPCR in the detection of Salmonella enterica serovar Typhi (S. Typhi), targeting the vexC gene encoding for Vi antigen (capsular polysaccharide antigen) and to evaluate its sensitivity and specificity performance using pure cultures of S. Typhi and other enteric pathogens.
Methodology and results:Microbiological, biochemical and serotyping tests were conducted to determine the phenotypic characteristics of S. Typhi and other enteric pathogens in our collection. Primers were designed using Primer3 software and their in-silico specificity were analysed using Basic Local Alignment System Tool (BLAST). Optimisation of PCR annealing temperature was done prior to assessment of sensitivity and specificity performance against artificial serially diluted seeded stools. The primers were found to be 100% specific in the detection of S. Typhi towards 32 tested clinical strains. Verification of gene amplification by comparing the nucleotide sequences against reference genes in the GenBank database revealed high specificity to S. Typhi. Statistical analysis indicates that this method results in 100% sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Moreover, Vi-qPCR allows the detection of S. Typhi as low as 131.4 CFU/g of stool sample.
Conclusion, significance and impact of study:A rapid and sensitive method for detection of Salmonella enterica serovar Typhi (S. Typhi) is desired as a diagnostic tool to improve typhoid management. The Vi-qPCR represent a promising non-invasive diagnostic tool for medical microbiology laboratories as a method for the detection of S. Typhi in both pure culture and stool specimens especially in chronic asymptomatic carriers where shedding of S. Typhi is intermittent and sometimes occurs in low level.
Full text:20.2018my0480.pdf