In a diagnostic comparative study evaluating rapid detection methods for Brucella species in human clinical blood samples, loop-mediated isothermal amplification identified Brucella DNA in 78.33 percent of tested specimens and demonstrated a predicted clinical sensitivity of 86 percent relative to conventional polymerase chain reaction testing. Published in BMC Microbiology, the study evaluated the isothermal technique alongside blood culture and molecular amplification to determine whether isothermal methods could provide faster diagnosis for patients suspected of having brucellosis.

Brucellosis is a well-known animal disease found across the world that poses a significant threat to individuals in direct contact with livestock. Traditional diagnostic methods rely on bacterial isolation and cultivation from patient specimens, but these conventional approaches can be time-consuming. Because early diagnosis and appropriate treatment of brucellosis are essential, researchers evaluated loop-mediated isothermal amplification as a molecular tool offering the potential for faster diagnosis with high sensitivity and specificity compared to conventional culture-based methods.

To evaluate the utility of loop-mediated isothermal amplification for diagnosing Brucella species in clinical blood samples, the study enrolled 60 patients presenting with clinical signs suggestive of brucellosis and positive initial serological test results. From each patient, ten milliliters of blood were collected under sterile conditions to enable parallel diagnostic evaluations. Five milliliters of blood was designated for blood culture, and five milliliters was collected in EDTA for molecular analyses.

The blood culture samples were inoculated into BACTEC aerobic bottles and, upon instrument alert, subcultured onto Brucella Agar from Merck in Germany with incubation at 37 degrees Celsius for up to one week. Of the 60 blood samples examined using this culture method, 9 samples, or 15 percent, were positive for bacterial growth, whereas 51 samples, or 85 percent, were negative.

For molecular analyses, conventional polymerase chain reaction assays targeting Brucella-specific genes were conducted on DNA extracted from peripheral blood samples. A commercial kit from Bioneer Co. of South Korea was used to extract the DNA and confirm the presence of Brucella DNA. Amplification products were analyzed by gel electrophoresis or real-time detection. Conventional polymerase chain reaction testing identified 38 of the 60 blood samples, or 63.33 percent, as positive for Brucella species.

The loop-mediated isothermal amplification assay was performed on part of the sample using Brucella-specific primers to enable rapid detection at a constant temperature. Requiring only a temperature-controlled water-bath or dry-bath, the technique allows results to be analyzed directly by the naked eye within 60 minutes. Across the 60 examined blood samples, the loop-mediated isothermal amplification assay showed that 47 samples, or 78.33 percent, were positive for Brucella species.

Patient results from culture, loop-mediated isothermal amplification, and conventional polymerase chain reaction were recorded and compared to assess diagnostic concordance, sensitivity, and specificity using SPSS version 17 software. Assuming culture and polymerase chain reaction methods serve as the gold standard, the researchers used Stata/MP version 17.0 software to calculate performance metrics at a 95 percent confidence interval. Compared to conventional polymerase chain reaction, the loop-mediated isothermal amplification method demonstrated a predicted clinical sensitivity of 86 percent and a clinical specificity of 36.36 percent, with a 95 percent confidence interval between 14.49 percent and 58.23 percent.

Statistical analysis further established that loop-mediated isothermal amplification had a positive predictive value of 70.21 percent, with a 95 percent confidence interval from 51.67 percent to 88.75 percent, and a negative predictive value of 61.54 percent, with a 95 percent confidence interval from 33.92 percent to 89.16 percent. The overall accuracy of the loop-mediated isothermal amplification method compared to polymerase chain reaction was 68.33 percent, with a 95 percent confidence interval spanning 56.49 percent to 80.17 percent. The authors concluded that loop-mediated isothermal amplification is a specific and sensitive method for detecting Brucella strains in clinical samples. Given the importance of early diagnosis and appropriate treatment of brucellosis, the authors noted that the technique offers a faster, more sensitive, and more cost-effective way to identify Brucella species in blood samples while saving time and providing remarkable and precise results.

Subject of Research: Diagnostic performance of loop-mediated isothermal amplification versus culture and PCR for Brucella species

Article Title: Comparison of loop-mediated isothermal amplification and conventional methods for rapid diagnosis of Brucella species

Article References: Bakhtiari, S., Arabestani, M. R., Keramat, F., Hashemi, S. H., Alikhani, M. S., Khodavirdipour, A., Hossainpour, H., & Alikhani, M. Y. (2026). Comparison of loop-mediated isothermal amplification and conventional methods for rapid diagnosis of Brucella species. BMC Microbiology. https://doi.org/10.1186/s12866-026-05903-x

Image Credits: AI Generated

DOI: 10.1186/s12866-026-05903-x

Keywords: Brucella, brucellosis, loop-mediated isothermal amplification, LAMP assay, polymerase chain reaction, blood culture, molecular diagnostics, bacterial detection, Comparison, loop-mediated, isothermal, amplification