Optimization and Validation of a Diagnostic Real-Time PCR for Bovine Brucellosis

Mukherjee, Falguni

doi:10.14737/journal.aavs/2015/3.11.577.587

Cited by 6 publications

(2 citation statements)

References 27 publications

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“…Therefore, TaqMan probe-based protocols have gained increasing acceptance to improve the sensitivity, specificity, and reproducibility of Brucella detection. Recently, several TaqMan probe-based Brucella detection studies have achieved dramatic improvements in terms of assay, with lower limits of detection as low as to 1–2 genomic copies ( Table 4 ) ( 35 , 36 ). Whereas previous studies have shown the good performance and analytical sensitivity of in-house assays, the specificity and sensitivity in subsequent assays using clinical specimens have been less than satisfactory; and in some studies, only a small number of clinical specimens or no clinical studies were included ( Table 4 ).…”

Section: Discussionmentioning

confidence: 99%

A novel, highly sensitive, one-tube nested quantitative real-time PCR for Brucella in human blood samples

Liu,

Li,

Liu

et al. 2023

Microbiol Spectr

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Brucellosis is a zoonotic disease caused by Brucella spp. and is a major threat to human and livestock health. The accurate and rapid detection of Brucella DNA is essential for the diagnosis and treatment of this disease. However, traditional diagnostic methods, such as culture and serological tests, have limitations in terms of sensitivity, specificity, and time consumption. To address these challenges, we developed a highly sensitive one-tube nested quantitative real-time PCR (qPCR) method to detect Brucella DNA using a closed-tube approach with two primers and two probes that sequentially react with encoding a Brucella outer membrane protein. The analytical sensitivity of this one-tube nested qPCR approach was 100 fg/μL, which is 100-fold higher than that of a conventional qPCR. Intra-batch and inter-batch replicates showed low coefficients of variation, both less than 5%. The study included 250 clinical samples, showing that this one-tube nested qPCR method has a specificity of 100% and a sensitivity of 98.6%, exceeding the sensitivity of conventional qPCR (84.1%) at a cycle threshold (CT) cutoff of 38. More importantly, the one-tube nested qPCR reduced the CT values by an average of 6.4 compared to conventional qPCR, significantly improving the detection rate of low-load samples (CT > 35). In conclusion, the bcsp31 one-tube nested qPCR method is a promising tool for the detection of brucellosis, owing to its high sensitivity and operational simplicity. This study highlights the potential of qPCR-based methods to improve the accuracy and speed of brucellosis diagnoses. IMPORTANCE This study developed a highly sensitive and efficient method for the detection of brucellosis by introducing a one-tube nested quantitative real-time PCR (qPCR) approach, representing a remarkable advance in the field. The method demonstrated an impressive analytical sensitivity of 100 fg/μL, surpassing conventional qPCR and enabling the detection of even low levels of Brucella DNA. In addition, the study’s comprehensive evaluation of 250 clinical samples revealed a specificity of 100% and a sensitivity of 98.6%, underscoring its reliability and accuracy. Most importantly, the new method significantly improved the detection rate of low-burden samples, reducing cycle threshold values by an average of 6.4. These results underscore the immense potential of this approach to facilitate rapid and accurate brucellosis diagnosis, which is critical for effective disease management and control.

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Section: Discussionmentioning

confidence: 99%

A novel, highly sensitive, one-tube nested quantitative real-time PCR for Brucella in human blood samples

Liu,

Li,

Liu

et al. 2023

Microbiol Spectr

View full text Add to dashboard Cite

show abstract

“…Apart from the above discussed novel real time PCR assays, a lot of work has been done so far on the validation aspect of these real time PCR assays in different countries (Al Dahouk et al, 2007;Queipo-Ortuno et al, 2008;Amoroso et al, 2011;Doosti and Dehkordi, 2011;Kumar et al, 2015;Mukherjee et al, 2015;Awwad et al, 2016;Kumar et al, 2017;Saini et al, 2017 andPatel et al, 2018). All these studies have found real time PCR as fast, sensitive and reliable tool for early detection of causative organism in the biological samples so that control and eradication programmes can be adopted as early as possible to minimise the losses to animal husbandry sector.…”

Section: Real-time Pcrmentioning

confidence: 99%