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Specific detection of Brucella spp. from slaughter-aged livestock using a dual priming oligonucleotide system

BMC Microbiology volume 25, Article number: 110 (2025) Cite this article

Abstract

Background

Brucella spp. are Gram-negative bacteria causing brucellosis, a major zoonotic disease affecting animals and humans. Annually, over 500,000 human cases are reported globally, with many undiagnosed due to nonspecific symptoms and diagnostic challenges. Current methods for Brucella detection, such as culture and serology, are time-consuming and lack specificity, hindering effective disease control. This study aims to develop a novel dual priming oligonucleotide (DPO) system-based PCR method for the specific detection of Brucella spp. in slaughter-aged livestock. This approach provides a rapid, sensitive, and field-deployable tool to improve early diagnosis and control of brucellosis.

Methods

We developed a DPO system-based PCR assay for the specific detection of Brucella spp. in slaughter-aged livestock. The method utilizes two sets of primers designed to specifically target unique regions of the Brucella genome. The assay was validated for specificity using a panel of 15 non-target bacterial species commonly found in livestock, including Escherichia coli, Salmonella spp., and Campylobacter spp. Sensitivity was evaluated using DNA extracted from a range of Brucella strains, with detection limits assessed using serially diluted samples. The assay’s performance was further tested on 500 samples from slaughter-aged sheep to assess its applicability in field conditions.

Results

The DPO-based PCR assay demonstrated excellent specificity, with no cross-reactivity observed in any of the 15 non-target bacterial species tested. The assay was able to detect Brucella spp. at low DNA concentrations, with a sensitivity limit of approximately 5.3 × 101 CFU/mL of the Brucella per reaction. In the field validation, 500 samples from slaughter-aged sheep were tested, and the assay successfully identified Brucella infections in animals with no false positives or negatives. When compared to conventional PCR, the DPO-PCR method exhibited improved specificity and faster results, with a significantly reduced time to diagnosis.

Conclusions

The DPO-based PCR assay provides a highly specific, rapid, and cost-effective tool for the detection of Brucella spp. in slaughter-aged livestock. This method is suitable for routine surveillance in slaughterhouses, offering a promising solution for early detection and control of brucellosis in both livestock and public health contexts. The assay’s simplicity and robustness make it an ideal candidate for field deployment, particularly in resource-limited settings where timely disease control is crucial.

Clinical trial number

Not applicable

Peer Review reports

Introduction

Brucella species, non-motile, aerobic, Gram-negative cocco-bacilli, are the causative agents of brucellosis, a zoonotic infection with significant implications for both human and animal health [1, 2]. This highly contagious disease is a global public health concern, with the World Health Organization estimating 500,000 new cases annually [3]. Human infections typically occur through direct contact with infected tissues, inhalation of aerosols, or consumption of unpasteurized dairy products [4,5,6]. Given the serious threat posed by brucellosis, the development of an accurate and specific diagnostic assay for Brucella spp. is essential.

Traditional laboratory diagnosis of brucellosis has been based on culture and isolation of the bacteria or serological tests for anti-Brucella antibodies [7, 8]. While culture-based assays are considered the “gold standard,” they are laborious and time-consuming [9]. Serological tests, on the other hand, struggle with differentiating between Brucella spp [10]. To address these limitations, molecular diagnostic techniques, especially PCR-based assays, have been increasingly adopted [11,12,13,14]. For instance, the AMOS-PCR assay has shown promise in detecting Brucella spp. from impure culture isolates, although it has its own set of limitations, such as the complexity of multiple PCR reactions, risk of contamination, and potential for false positives due to non-specific amplification [15,16,17].

The Dual Priming Oligonucleotide (DPO) system represents a novel approach to PCR primer design, offering a simpler and more efficient alternative to traditional methods. The DPO primer is characterized by a longer 5’-segment, a shorter 3’-segment, and a polydeoxyinosine (poly I) linker, which together enhance specificity and allow for a broader range of annealing temperatures. This system has been successfully applied to detect a wide range of foodborne pathogens in various studies. For example, a DPO-based multiplex PCR assay was developed for the simultaneous detection of Salmonella spp., Listeria monocytogenes, Shigella spp., Staphylococcus aureus, Campylobacter jejuni, and Yersinia enterocolitica from food samples [18]. The assay demonstrated high specificity, amplifying only the target genes in the presence of 238 target and 83 non-target bacterial strains, with an analytical detection limit of 102-103 CFU/mL. The successful application of the DPO-PCR system to detect multiple pathogens in food matrices underscores the versatility and reliability of this system for diagnostic purposes. Given its success in detecting various pathogens, the DPO-PCR system is ideally suited for Brucella spp. detection due to its high specificity, reduced cross-reactivity, and ability to handle complex sample matrices, such as those encountered in livestock and food samples. The economic and health implications of misdiagnosis or delayed diagnosis of Brucella spp. are significant. Misdiagnosis or delayed detection can lead to the spread of the disease among livestock populations, resulting in costly outbreaks that affect animal health, food safety, and the economy. Brucellosis also has serious public health implications, as it is a zoonotic disease that can cause long-term chronic illness in humans, leading to prolonged treatment and increased healthcare costs. Early and accurate diagnosis is essential not only for controlling the disease within animal populations but also for preventing human infections. In particular, the use of a reliable and rapid diagnostic tool can reduce the time to diagnosis, limit disease transmission, and mitigate the broader socio-economic impact.

Similarly, this study introduces a DPO-based PCR assay for the specific detection of Brucella spp. in slaughter-aged livestock, targeting the Brucella Omp25 gene, with the aim of overcoming the limitations of existing diagnostic methods. It is reported that no DPO-PCR method has been employed for the detection of Brucella spp. to date, highlighting the novelty and potential impact of this research.

Methods

Bacterial strains

In the present study, 24 bacterial strains were used to evaluate the specificity of the DPO-based PCR assay (Table 1). Bacterial strains, including Brucella spp., Campylobacter, Vibrio, and Lactobacillus, were cultured under conditions optimized in our previous study, using selective media and temperature-controlled environments specific to each strain [19].

Table 1 The bacterial strains used in the study
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DPO primers

The DPO primers targeting Brucella spp. gene Omp25 were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China), following our established protocol as previously described [19]. Briefly, the primers were optimized for high specificity under varying annealing temperatures, as reported. Details of the DPO primers are summarized in Table 2.

Table 2 DPO primers used in this study
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Development of the DPO-based pcr assay for Brucella spp

For the DPO-based PCR assay, the optimal reaction conditions were determined by testing various concentrations of Mg2+ (0–5.0 µL, 25 mmol/L), rTaq DNA polymerase (0.02–0.2 µL, 5 U/µL), dNTP (0.25-4 µL, 2.5 mmol/L), and DPO primer pair (0.2–1.4 µL, 10 µmol/µL) in a total volume of 25 µL. PCR was performed under the following conditions: 95℃ for 5 min; 35 cycles of 94℃ for 30 s, 50–70℃for 30 s, and 72℃ for 30 s; and a final extension at 72℃ for 10 min. PCR products were analyzed by agarose gel electrophoresis.

Determination of detection limit and specificity of the DPO-based PCR assay

The detection limit of the DPO-based PCR assay for Brucella spp. was assessed in both pure cultures and spiked tissue samples, following our established protocol [19]. Serial 10-fold dilutions of Brucella cultures were prepared in phosphate-buffered saline (PBS, pH 7.4), and genomic DNA was extracted using the TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, Dalian, China). For spiked tissue samples, 100 µL of each dilution was added to 100 mg of Brucella-negative lamb liver, and total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Beijing, China). The detection limit was defined as the last dilution yielding a positive PCR result in triplicate experiments. Bacterial concentrations were confirmed by the standard plate count method. The specificity of the assay was evaluated using the bacterial strains listed in the “Bacterial Strains” section. DNA from each strain was extracted using the TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit and tested by the DPO-based PCR assay. To assess the reproducibility of the DPO-based PCR assay, intra-assay and inter-assay variability were calculated using the coefficient of variation (CV). Each sample was tested in triplicate within the same run (intra-assay variability) and across three independent runs on different days (inter-assay variability).

Practical application of the DPO-based PCR assay

A total of 500 samples including 250 liver samples and 250 blood samples were collected from sheep at livestock slaughter plants. Liver samples were aseptically collected during post-mortem examination, and blood samples were drawn from the jugular vein using sterile vacutainers. All samples were transported to the laboratory on ice and processed within 24 h of collection. Liver tissues were homogenized in sterile PBS, and 100 mg of each homogenate was used for DNA extraction. Whole blood samples were centrifuged at 3,000 × g for 10 min to separate plasma and buffy coat. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Beijing, China) according to the manufacturer’s instructions. The extracted DNA from both liver and blood samples was subjected to the DPO-based PCR assay and under the optimized conditions described above. Liver homogenates and whole blood samples were streaked onto Brucella-selective agar plates and incubated at 37℃ under 5% CO2 for 7–10 days. Colonies suspected to be Brucella were identified based on morphology, Gram staining, and biochemical tests. Serum was separated from blood samples by centrifugation at 3,000 × g for 10 min and stored at -20 °C until analysis. The presence of anti-Brucella antibodies was detected using a commercial ELISA kit (Brucella Competitive ELISA Antibody Detection Kit, Shanghai Huzheng Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions.

Results

Establishment of the DPO-based PCR assay

Following the optimization of the reaction system, the DPO-based PCR assay for specific detection of Brucella spp. was successfully developed (Fig. 1). The optimal reaction system of the DPO-based PCR assay in a total volume of 25 µL was as follows: 10×PCR buffer 2.5 µL, Mg2+ (25 mmol/L) 2.5 µL (Fig. 1a), rTaq DNA polymerase (5U/µL) 0.05 µL (Fig. 1b), dNTP (2.5 mmol/L for each) 1 µL (Fig. 1c), DPO primer pair (10 µmol/µL) 0.4 µL (Fig. 1d), DNA template 0.5 µL and deionized water added up to 25 µL. In this DPO-based PCR assay, the longer 5’-segment of DPO initiates stable annealing and its shorter 3’-segment determines target-specific extension, resulting in unparalleled high specificity. The DPO system enables a wide annealing temperature range of 50–70 °C for effective target gene amplification, comparable to conventional PCR. Through optimization, we selected 61 °C as the optimal annealing temperature for our DPO-PCR assay, showcasing the system’s adaptability (Fig. 2) Thus, the DPO system provides a powerful tool for the development of multiplex PCR assay [20, 21].

Fig. 1
figure 1

Development of the DPO-based PCR assay for Brucella spp. (a) Optimization of Mg2+ concentration: Lanes 1–10 represent Mg2+ concentrations of 0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, and 5 mM, respectively. The optimal concentration was determined to be 2.5 mM (lane 6). (b) Optimization of rTaq DNA polymerase: Lanes 1–10 represent rTaq concentrations of 0.1 U, 0.15 U, 0.2 U, 0.25 U, 0.3 U, 0.35 U, 0.4 U, 0.45 U, 0.5 U, and 1 U, respectively. The optimal concentration was determined to be 0.25 U (lane 4). (c) Optimization of dNTP: Lanes 1–10 represent dNTP concentrations of 0.025 mM, 0.05 mM,0.075 mM,0.1 mM,0.125 mM,0.15 mM,0.175 mM,0.2 mM,0.3 mM,0.4 mM, respectively. The optimal concentration was determined to be 0.1 mM (lane 4). (d) Optimization of DPO primer: Lanes 1–7 represent DPO primer concentrations of 0.08 µM, 0.16 µM,0.24 µM,0.32 µM,0.4 µM,0.48 µM,0.56 µM, respectively. The optimal concentration was determined to be 0.16 µM (lane 2). (e) Successful development of the DPO-based PCR assay: This panel illustrates the assay’s performance under optimal conditions, demonstrating efficient and specific amplification of the target Brucella DNA

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Fig. 2
figure 2

Comparison of effective annealing temperature between the DPO-based PCR assay and conventional primer-based PCR assay. The DPO-based PCR assay demonstrates a wider range of annealing temperatures (50–70℃) for efficient target gene amplification, showing a nearly identical pattern across left lanes 1–11. In contrast, the conventional primer-based PCR assay exhibits amplification primarily at a specific temperature, highlighting its narrow optimal temperature range, as seen in right lanes 1–11. M: DNA Marker 2000; lanes 1–11: 49.8℃, 51.5℃, 53.4℃, 55.7℃, 58.3℃, 61.0℃, 63.7℃, 66.1℃, 68.0℃, 69.4℃ and 70.0℃, respectively

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Detection limit of the DPO-based PCR assay

The detection limit of the DPO-based PCR assay for Brucella spp. was determined and result showed that the detection limit of this assay for Brucella spp. was 5.3 × 101 CFU/mL (Fig. 3), which was consistently found in pure cultures and spiked animal tissue samples. The assay showed consistent results in repeated experiments. The DPO-based PCR assay demonstrated excellent reproducibility, with intra-assay and inter-assay coefficients of variation (CV) of less than 5% and 10%, respectively. However, there was no significant difference in detection limit between DPO-based PCR assay and conventional PCR assay.

Fig. 3
figure 3

Detection limit of the DPO-based PCR assay for Brucella spp. M: DNA Marker 2000; 1: 5.3 × 106 CFU/mL; 2: 5.3 × 105 CFU/mL; 3: 5.3 × 104 CFU/mL; 4: 5.3 × 103 CFU/mL; 5: 5.3 × 102 CFU/mL; 6: 5.3 × 101 CFU/mL; 7: 5.3 × 100 CFU/mL

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Specificity of the DPO-based PCR assay

Applying the assay to test B. abortus, B. melitensis, (A) hydrophila, C. jejuni, E. coli, S. aureus, K. pneumoniae, S. enteritidis, S. flexneri, L. monocytogenes, Y. enterocolitica, S. haemolyticus, C. sakazakii, (B) cereus, V. parahaemolyticus, L. casei, L. plantarum, V. cholerae, L. welshimeri, (C) perfringens, V. anguillarum, V. vulnificus, P. aeruginosa and P. vulgaris, only Brucella spp. were positive in this DPO-based PCR assay without non-specific results (Fig. 4a), while conventional primer-based PCR assay showed a lower specificity (Fig. 4b). The results indicated that the DPO-based PCR assay showed no cross-reactivity with non-target species under the tested conditions, while the conventional PCR assay exhibited non-specific amplification in several lanes (Fig. 4b, lanes 7, 11, 14, 21, and 23). This suggests that the DPO system, due to the bubble-like structure of the poly I linker, may offer improved specificity compared to conventional primers.

Fig. 4
figure 4

Specificity of the DPO-based PCR assay for Brucella spp. The DPO-based PCR assay developed in this work has high diagnostic specificity for Brucella spp. (a), while conventional primer-based PCR assay shows a low specificity (b). M: DNA Marker 2000; Lanes 1–24 were B. abortus, B. melitensis, A hydrophila, B cereus, C jejuni, C. sakazakii, C. perfringens, E. coli, K. pneumoniae, L. monocytogenes, L. welshimeri, L. casei, L. plantarum, P. aeruginosa, P. vulgaris, S. aureus, S. enteritidis, S. flexneri, S. haemolyticus, V. parahaemolyticus, V. cholerae, V. anguillarum, V. vulnificus and Y. enterocolitica, respectively

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Diagnostic capability of the DPO-based PCR assay in practice

Applying the assay to 500 samples collected from sheep at livestock slaughter plants revealed that 72 samples were positive detected by the DPO-based PCR assay (Table 3), which were consistent with the results of conventional culture-based assay. Both the DPO-based PCR assay and the conventional culture-based assay showed a detection rate of 14.4% (72/500), while the ELISA method exhibited a significantly lower detection rate of 10.4% (26/250) in blood samples. This discrepancy may be attributed to the low production rate of antibodies in the early stages of the disease, which limits the sensitivity of serological tests. By contrast, the DPO-based PCR assay was more rapid, time-saving, and sensitive, showing a better diagnostic capability and practicality.

Table 3 Practical application of the DPO-based PCR assay
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Discussion

The microbiological diagnosis of brucellosis is based on three key approaches: culture, serology, and nucleic acid amplification tests [7]. This study used liver and blood samples available from sheep at slaughter-aged livestocks to investigate brucellosis, which incuded bacteriology, serology, and molecular detection. Specifically, the conventional culture-based assay, ELISA to detect Brucella antibodies in serum, and a DPO-based PCR assay were used.

According to the literature, the detection limits of Brucella in blood by isolation and culture methods were within the range of 103-104 CFU/mL, while the detection limits of the same specimens by real-time PCR were 104-105 CFU/mL. The main factors affecting the detection limits of real-time PCR were fat and protein [22]. To address these limitations, another study developed a propidium monoazide-quantitative PCR (PMA-qPCR) method to detect viable Brucella by targeting its BCSP31 gene. This novel, rapid method has a higher sensitivity of 103 CFU/mL, utilizing PMA, a DNA-binding dye that can form covalent cross-links with DNA upon photolysis, to selectively amplify DNA from viable cells and inhibit amplification of DNA from dead cells [23]. In our own study, the detection limit of the DPO-based PCR assay for Brucella spp. was even lower, at 5.3 × 101 CFU/mL. Moreover, droplet digital PCR (ddPCR), as a novel PCR technique that partitions the sample into thousands of nanoliter-sized droplets, allowing for absolute quantification of target nucleic acids without the need for standard curves or reliance on amplification efficiency. The ddPCR assay developed in another recent study demonstrated a limit of detection of 1.87 copies per reaction with high repeatability, and exhibited promising diagnostic performance, detecting Brucella DNA in 88.5% of SAT positive patients and 57.6% of suspected sero-negative samples, suggesting its potential as a valuable diagnostic and prognostic tool for brucellosis [24].

Considering Brucella may infect any body organ or tissue, blood cultures is the most common clinical specimen. The bacteriology and serology detection were used as the consistency examination. According to the results, the positive rate was 14.4% by the traditional bacterial culture method on liver and blood samples. The ELISA method was used to detect antibodies in serum, and the positive detection rate was 10.4% in blood samples. We hypothesize that the low serological positive detection rate is due to the ability of blood culture to detect Brucella organisms during the early stage of the disease, when serological test results may still be negative or show low antibody titers [25]. While the DPO-based PCR assay demonstrated superior performance compared to ELISA, a more meaningful comparison would involve other molecular methods, such as conventional PCR or real-time PCR. Future studies should include these methods to provide a comprehensive evaluation of the DPO assay’s diagnostic capabilities.

Although the DPO-based PCR assay demonstrates high sensitivity and specificity, there remains a limitation in its inability to differentiate between Brucella spp. Further optimization of primer design is necessary to accurately distinguish between subtypes of Brucella, such as B. abortus, B. melitensis, B. ovis, and B. suis, in cattle, sheep, goats, and pigs.

While the DPO-based PCR assay demonstrated high specificity against a panel of bacterial strains, it should be noted that potential cross-reactivity with other clinically relevant pathogens, such as Mycobacterium spp. or Coxiella burnetii, was not evaluated. These pathogens share environmental niches with Brucella spp. and may pose a risk of cross-reactivity [26, 27]. Future studies should include these pathogens to further validate the specificity of the assay.

In conclusion, the development of a dual priming oligonucleotide (DPO)-based PCR detection system for Brucella spp. in slaughter-aged livestock, specifically targeting B.abortus and B.melitensis, has shown promising results. To the best of our knowledge, this is the first report of using a DPO-based PCR detection system for detecting Brucella spp.The optimized DPO-based PCR assay exhibited a wide range of annealing temperatures for specific detection, with a low analytical detection limit and high specificity. The high specificity and sensitivity demonstrated by the DPO-based PCR assay suggest its utility in enhancing the diagnosis and surveillance of brucellosis, offering a valuable contribution to the field of veterinary and public health. Although the DPO-based PCR assay showed improved performance over ELISA, further studies are needed to compare its diagnostic accuracy with other molecular methods, such as conventional PCR and real-time PCR.

Data availability

All data generated or analyzed during this study are publicly available and included in this published article.

References

  1. Byndloss MX, Tsolis RM. Brucella spp. Virufactorsactorimmunitymunity. Annu Rev Anim Biosci. 2016;4:111–27.

    Article  PubMed  CAS  Google Scholar 

  2. Wang Y, Vallee E, Heuer C, Wang Y, Guo A, Zhang Z, Compton C. A scoping review on the epidemiology and public significance of Brucella abortus in Chinese dairy cattle and humans. One Health. 2024;18:100683.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV. The new global map of human brucellosis. Lancet Infect Dis. 2006;6(2):91–9.

    Article  PubMed  Google Scholar 

  4. Dadar M, Tiwari R, Sharun K, Dhama K. Importance of brucellosis control programs of livestock on the improvement of one health. Vet Q. 2021;41(1):137–51.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Djokic V, Freddi L, de Massis F, Lahti E, van den Esker MH, Whatmore A, Haughey A, Ferreira AC, Garofolo G, Melzer F, et al. The emergence of Brucella canis as a public health threat in Europe: what we know and what we need to learn. Emerg Microbes Infect. 2023;12(2):2249126.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Franco MP, Mulder M, Gilman RH, Smits HL. Human brucellosis. Lancet Infect Dis. 2007;7(12):775–86.

    Article  PubMed  CAS  Google Scholar 

  7. Yagupsky P, Morata P, Colmenero JD. Laboratory diagnosis of human brucellosis. Clin Microbiol Rev. 2019;33(1):e00073–00019.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Di Bonaventura G, Angeletti S, Ianni A, Petitti T, Gherardi G. Microbiological Laboratory diagnosis of human brucellosis: an overview. Pathogens. 2021;10(12):1623.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Dal T, Kara SS, Cikman A, Balkan CE, Acikgoz ZC, Zeybek H, Uslu H, Durmaz R. Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis. J Infect Public Health. 2019;12(3):337–42.

    Article  PubMed  Google Scholar 

  10. Alamian S, Amiry K, Bahreinipour A, Etemadi A, Yousefi AR, Dadar M. Evaluation of serological diagnostic tests for bovine brucellosis in dairy cattle herds in an endemic area: a multicenter study. Trop Anim Health Prod. 2023;55(2):104.

    Article  PubMed  CAS  Google Scholar 

  11. Baddour MM, Alkhalifa DH. Evaluation of three polymerase chain reaction techniques for detection of. DNA Peripheral Hum Blood Can J Microbiol. 2008;54(5):352–7.

    CAS  Google Scholar 

  12. Finamore-Araujo P, Faier-Pereira A, Ramon do Nascimento Brito C, Gomes Peres E, Kazumy de Lima Yamaguchi K, Trotta Barroso Ferreira R, Moreira OC. Validation of a novel multiplex real-time PCR assay for. Detect Quantification acai pulp PLoS One. 2021;16(2):e0246435.

    PubMed  CAS  Google Scholar 

  13. Schmoock G, Ehricht R, Melzer F, Elschner M, Tomaso H, Neubauer H, Al Dahouk S. Development of a diagnostic multiplex polymerase chain reaction microarray assay to detect and differentiate Brucella spp. Diagn Microbiol Infect Dis. 2011;71(4):341–53.

    Article  PubMed  CAS  Google Scholar 

  14. Mol JPS, Guedes ACB, Eckstein C, Quintal APN, Souza TD, Mathias LA, Haddad JPA, Paixao TA, Santos RL. Diagnosis of canine brucellosis: comparison of various serologic tests and PCR. J Vet Diagn Invest. 2020;32(1):77–86.

    Article  PubMed  Google Scholar 

  15. Mazwi KD, Kolo FB, Jaja IF, Byaruhanga C, Hassim A, van Heerden H. Polyphasic characterization of Brucella spp. in Livestock slaughtered from abattoirs in Eastern Cape, South Africa. Microorganisms. 2024;12(1):223.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Bricker BJ, Halling SM. Differentiation of. Bv 1 2 4 Brucella melitensis Brucella ovis Brucella suis bv 1 PCR J Clin Microbiol. 1994;32(11):2660–6.

    CAS  Google Scholar 

  17. Hindiyeh M, Levy V, Azar R, Varsano N, Regev L, Shalev Y, Grossman Z, Mendelson E. Evaluation of a multiplex real-time reverse transcriptase PCR assay for detection and differentiation of influenza viruses a and B during the 2001–2002 influenza season in Israel. J Clin Microbiol. 2005;43(2):589–95.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Xu YG, Sun B, Zhao HY, Liu ZM, Jiang YP, Wang L, Qiao XY, Li YJ, Tang LJ. Development and evaluation of a dual priming oligonucleotide system-based multiplex PCR assay for simultaneous detection of six foodborne pathogens. Eur Food Res Technol. 2017;243(4):555–63.

    Article  CAS  Google Scholar 

  19. Wang M, Yan Y, Wang R, Wang L, Zhou H, Li Y, Tang L, Xu Y, Jiang Y, Cui W et al: Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System. Front Microbiol 2019, 10:2884.

  20. Kim SR, Ki CS, Lee NY. Rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex PCR assay. J Virol Methods. 2009;156(1–2):111–6.

    Article  PubMed  CAS  Google Scholar 

  21. Lee HJ, Choi J, Hwang TS, Shong YK, Hong SJ, Gong G. Detection of BRAF mutations in thyroid nodules by allele-specific PCR using a dual priming oligonucleotide system. Am J Clin Pathol. 2010;133(5):802–8.

    Article  PubMed  CAS  Google Scholar 

  22. Kaden R, Ferrari S, Jinnerot T, Lindberg M, Wahab T, Lavander M.: determination of survival times and evaluation of methods for detection in several matrices. BMC Infect Dis. 2018;18(1):259.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Zhang SJ, Wang LL, Lu SY, Hu P, Li YS, Zhang Y, Chang HZ, Zhai FF, Liu ZS, Li ZH, et al. A Novel, Rapid, and simple PMA-qPCR Method for detection and counting of viable Brucella organisms. J Vet Res. 2020;64(2):253–61.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Liu J, Song Z, Ta N, Tian G, Yang X, Zhao H, Piao D, Fan Y, Zhang Y, Jiang H. Development and evaluation of a droplet digital PCR assay to detect. Hum Whole Blood PLoS Negl Trop Dis. 2023;17(6):e0011367.

    Article  CAS  Google Scholar 

  25. Shemesh AA, Yagupsky P. Limitations of the standard agglutination test for detecting patients with. Bacteremia Vector Borne Zoonotic Dis. 2011;11(12):1599–601.

    Article  PubMed  Google Scholar 

  26. Leahy E, Shome R, Deka RP, Sahay S, Grace D, Mazeri S, Lindahl JF. Risk factors for. spp Coxiella burnetii Infect among Small Ruminants East India Infect Ecol Epidemiol. 2020;10(1):1783091.

    Google Scholar 

  27. Gonzalez-Espinoza G, Arce-Gorvel V, Memet S, Gorvel JP. Brucella: reservoirs and niches in animals and humans. Pathogens 2021;10(2).

Download references

Funding

This work was supported by the Scientific Research Startup Fund of the Affiliated Hospital of Southwest Medical University (grant number 24080), Natural Science Foundation of Southwest Medical University(grant number 2024ZKY080) and the grants from the National Key Research and Development Program of China (No. 2023YFD1800401).

Author information

Author notes

  1. Dandan Li, Wenxing Xu, Shengnan Huang and Wei An have contributed equally to this work.

Authors and Affiliations

  1. Animal & Plant Inspection and Quarantine technology center, Haikou Customs District, Haikou, China

    Dandan Li

  2. Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou, China

    Wenxing Xu

  3. Haikou Customs District P.R.China, Haikou, China

    Shengnan Huang

  4. Technology Center of Chengdu Customs, Chengdu, China

    Wei An

  5. Institute of Animal Science and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou, China

    Zhe Chao

  6. Technology Center of Shenzhen Customs District P.R.China, Shenzhen, China

    Fang Zhao

  7. Kunming Customs District P.R.China, Kunming, China

    Jun Ai

  8. Animal & Plant Inspection and Quarantine technology center, Shenzhen Customs District, Shenzhen, China

    Junxing Yang

  9. College of Animal Husbandry & Veterinary Medicine, Jinzhou Medical University, Jinzhou, China

    Shenyang Gao

  10. Department of Respiratory Critical Care, The Affiliated Hospital of Southwest Medical University, Luzhou, China

    Yuying Li

  11. Inflammation & Allergic Diseases Research Unit, The Affiliated Hospital of Southwest Medical University, Luzhou, China

    Yuying Li & Guofeng Xu

  12. Technology Center of Nanning Customs, Nanning Customs District P.R.China, Nanning, China

    Lijun Chen

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  1. Dandan Li
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  9. Shenyang Gao
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  10. Yuying Li
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  11. Lijun Chen
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  12. Guofeng Xu
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Contributions

LD, XW, HS and AW developed the DPO-based PCR detection system for the identification of Brucella spp. CZ and ZF optimized the reaction conditions. AJ and YJ collected the clinical samples. GS detected the clinical samples. CL and XG conceived the project. LD, CL, LY and XG was the grant holder and drafted the manuscript. All authors read, revised, and approved the final manuscript.

Corresponding authors

Correspondence to Yuying Li, Lijun Chen or Guofeng Xu.

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Li, D., Xu, W., Huang, S. et al. Specific detection of Brucella spp. from slaughter-aged livestock using a dual priming oligonucleotide system. BMC Microbiol 25, 110 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03807-w

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03807-w

Keywords

BMC Microbiology

ISSN: 1471-2180

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