DOI: https://doi.org/10.14710/jbtr.v6i2.7120

Optimizing Real-Time PCR methods for detection of ssaN gene Salmonella enterica subsp.enterica in the blood specimen

1University of Pembangunan Nasional Veteran Jakarta, Indonesia

2University of Indonesia, Indonesia

Received: 22 Jan 2020; Revised: 28 Jun 2020; Accepted: 8 Jul 2020; Available online: 31 Aug 2020; Published: 31 Aug 2020.
Open Access Copyright (c) 2020 Journal of Biomedicine and Translational Research

Citation Format:
Abstract

Background

Typhoid fever caused by Salmonella typhi is a common acute infection of the reticuloendothelial system, intestinal lymphoid tissue, and gall bladder. Detection of Salmonella spp. is still based on cultures and serological methods.Widal test is one of the serological tests that is still widely used, especially in developing countries including Indonesia.Widal tests have low sensitivity and specificity. They often produce false positive or false negative results.

Objective

The aim of this study were i) real time PCR optimization to develop a Salmonella enterica detection system. ii) molecular detection of new target gene (ssaN gene) from blood specimens in typhoid fever patients.

Methods

An experimental laboratory study was performed from March to October 2016. Extraction of Salmonella typhi DNA is used as templates for the optimization of real time PCR reaction.The blood sample was from patients suspected with typhoid fever obtained from the Menteng Sub-district Health Center according to the inclusion criteria.

Results

Specificity test of real time PCR showed that the primers and probes used are not cross-react against other microorganisms. Sensitivity test obtained minimal detection is at least 10 cfu/ml of blood specimen. In blood clinical specimens, real time PCR could detect 19 (38%) positive samples of 50 blood specimen from suspected typhoid fever patients. Eleven samples with negative Widal serology gives positive results in real time PCR.

Conclusion

Real time PCR used in this study can increase the level of rate of positive testing by 22% of the total specimens.

Keywords : Salmonella enterica subsp.enterica, typhoid fever, ssaN gene, real time PCR

Fulltext View|Download
Keywords: Salmonella enterica subsp.enterica; typhoid fever; ssaN gene; real time PCR

Article Metrics:

Article Info
Section: Original Research Articles
Language : EN
Recent articles
Optimizing Real-Time PCR methods for detection of ssaN gene Salmonella enterica subsp.enterica in the blood specimen Synergistic Potential of the Leaves of Aspillia africana (Compositae) and Psidium guajava (Myrtaceae) Against Some Selected Bacterial and Fungal Isolates In silico Identification of Characteristics Spike Glycoprotein of SARS-CoV-2 in the Development Novel Candidates for COVID-19 Infectious Diseases A Rare Case of 18-year-old female with Primary Sclerosing Cholangitis, Mixed Connective Tissue Disease, Short Stature Knowing When and How to Fight: COVID-19 Between Viral Clearance and Immune Tolerance More recent articles
  1. Cao, J., Xu, L., Yuan, M., Ke, B., Xiang, D., Ke, C. (2013). TaqMan probe real-time PCR detection of foodborne Salmonella enterica and its six serovars. Int.J.Curr.Microbiol.App.Sci, 2(11), 1-12
  2. Rakhman, A., Humardewayanti, R., Pramono, D. (2009). Faktor-faktor risiko yang berpengaruh terhadap kejadian demam tifoid pada orang dewasa. Berita Kedokteran Masyarakat, 25(4),167-175
  3. Amarantini, C., Asmara, W., Kushadiwijaya, H. (2009). Seleksi bakteri Salmonella typhi dari kultur darah penderita demam tifoid. Prosiding Seminar Nasional Penelitian, Pendidikan dan Penerapan MIPA
  4. World Health Organization. (2011) Guidelines for the management of typhoid fever
  5. Crump, J.A., Luby, S.P., Mintz, E.D . (2004) The Global burden of typhoid fever. Bulletin of the World Health Organization, 82,346-353
  6. Crump, J.A., & Mintz, E.D . (2010) Global trends in typhoid and paratyphoid Fever. Emerging Infections. Clinical Infectious Disease, 50, 241-246
  7. Guerrant, R.L., Walke,r D.H., Weller, P.F. (Eds).(2011). Tropical Infectious Disease (3rd ed). Philadelphia : Saunders Elsevier
  8. Ranjbar, R., Naghoni, A., Farshad, S. (2014) Use of TaqMan® real time PCR For rapid detection of Salmonella enterica serovar typhi. Acta Microbiologica et Immunologica Hungarica,61(2),121-130
  9. Zhou, L., & Pollard, A.J. (2010). A Fast and highly sensitive blood culture PCR method for clinical detection of Salmonella enterica serovar typhi. Ann Clin Microbiol Antimicrob,9:14
  10. Alam, A. (2011) Pola Resistensi Salmonella enterica serotype typhi di Departemen IKA RSHS 2006-2010. Sari Pediatri,12(5),296-301
  11. Devrim, I., Ergünay, K., Kara, A. (2008) The comparison of cultures, widal agglutination test and polymerase chain reaction as a diagnostic tool in typhoid fever. Cent. Eur. J. Med,3(4),470-474
  12. Moore, M.M. , Feist, M.D. (2006) Real-time PCR method for Salmonella spp. targeting the stn gene. Journal of Applied Microbiology,102,516-530
  13. Chen, J., Zhang, L., Paoli, G.C . (2010) A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis. International Journal of Food Microbiology,168-174
  14. Kaur, J., & Jain, S.K. (2011) Role of antigens and virulence factors of Salmonella enterica serovar typhi in its pathogenesis. Microbiol Res,167,199-210
  15. Yoshida, Y., Miki, T., Ono, S. (2014) Functional characterization of the type III secretion ATPase SsaN encoded by Salmonella pathogenicity island 2. PLoS ONE,9(4),1-12
  16. Somily, A.M, Adam,M.H, Gad El Rab,M.O. (2011) Detection of Salmonella typhi agglutinins in sera of patients with other febrile illnesses and healthy individuals. Annals of African Medicine,10(1),41-44
  17. Abutiheen,A.A, Altumma,M.A ,Heniwa,R.A. (2015) Prevalence of Positive Widal Test among Healthy Personnel in Kerbala. Karbala J. Med, 8(2),2265-71
  18. Zorgani, A., Ziglam, H. (2014) Typhoid fever: misuse of Widal test in Libya. J Infect Dev Ctries, 8(6), 680-687
  19. Parry, C.M, Wijedoru, L., Arjyal, A., Baker, S. (2011) The utility of diagnostic tests for enteric fever in endemic locations. Expert Rev. Anti Infect Ther, 9(6), 711-25
  20. Nolan, T., Hugget, J., Sanchez, E. Good practice guide for the application of quantitative PCR (qPCR). First Edition 2013

Last update:

No citation recorded.

Last update:

No citation recorded.