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Updated: Jun 12, 2026
Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples
Published on: October 25, 2018
The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens
Tran Vu Thieu Nga1, Abhilasha Karkey, Sabina Dongol
1Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
Background:
PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive.
Methods:
We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients.
Results:
The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75) and sensitivity of 53.9% (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles.
Conclusions:
Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.
Insights
A novel real-time PCR assay shows high specificity for detecting Salmonella Typhi and Paratyphi A. However, its sensitivity is limited in blood samples, suggesting it may not be ideal for routine enteric fever diagnosis directly from blood.
Area of Science:
- Molecular diagnostics
- Infectious disease detection
- Microbiology
Background:
- Polymerase Chain Reaction (PCR) is a rapid diagnostic tool for pathogens.
- Invasive Salmonella serovars cause enteric fever but are difficult to detect in blood.
- Current blood culture methods for Salmonella are slow and may lack sensitivity.
Purpose of the Study:
- To develop and optimize a multiplex real-time PCR assay.
- To detect Salmonella serovars Typhi and Paratyphi A.
- To evaluate the assay's performance on clinical samples.
Main Methods:
- Developed a novel multiplex three-color real-time PCR assay.
- Targeted specific genomic sequences of Salmonella Typhi and Paratyphi A.
- Tested DNA from blood and bone marrow of enteric fever patients.
Main Results:
- Assay showed high specificity (100%) and reproducibility.
- Bone marrow samples were all positive for Salmonella.
- Sensitivity was limited in blood samples (53.9%).
Conclusions:
- Bacterial load of S. Typhi in peripheral blood is often below PCR detection limits.
- PCR directly on blood may be unsuitable for routine enteric fever diagnosis.
- The assay may be useful for environmental or other biological samples.
