It is of great importance to quickly and accurately detect Brucella abortus and Brucella melitensis from clinical and non-clinical samples because of their high prevalence and high risk in causing brucellosis, a life-threating infectious disease affecting both humans and animals.
The current study describes a new method for the detection of brucellosis in clinical samples using real-time polymerase chain reaction (PCR) and high-resolution melt (HRM) curve analysis. This study was conducted on 70 human and 55 animal isolates with more than 1/80 serum antibody titers. Additionally, the accuracy and specificity of the methods were compared.
The mean range [cycles threshold±standard deviation (CT±SD)] for the amplified samples was 15.39–25.15 by real-time PCR. The melting peak range (°C) ±SD for B. abortus and B. melitensis was 90.10±0.4 and 89.70±0.4, respectively, and 10 was reported on peak height.
The results of HRM analysis can be used for species differentiation and bacterial genotyping according to nucleotide polymorphism. This molecular method could help in diagnosing Brucella quickly and precisely. Quick recognition of Brucella species could decrease its prevalence among humans and animals and mitigate economic loss.
Ahmad-Nejad P. Ghebremedhin B.
Brucella species are pathogenic bacteria, which cause a serious infection among humans and animals called brucellosis . Human infection occurs through direct or indirect contact with infected animals or via contaminated meat or dairy products . Direct contact with infected animal tissue or the inhalation of aerosol particles is among the other modes of transmission . These small bacteria are obligate intracellular parasite, aerobic, Gram-negative coccobacilli with 90% homology in nucleic acid sequence . The most prevalent pathogenic species in humans and animals are Brucella melitensis (preferred hosts are goat, sheep and camel) and Brucella abortus (cattle and buffalo) , .
Presently, brucellosis is a major issue in most developing countries and some of their developed counterparts. The outbreak of this infectious disease in the Middle East countries (Saudi Arabia, Iran, Palestine, Syria, Jordan and Oman), Central Asia and Latin America is more than 500,000 new cases reported annually. A quick and accurate diagnosis is of utmost importance in preventing epidemics and the associated morbidity and mortality , . Brucellosis, however, is difficult to diagnose in that it has no specific symptoms. In fact, the diagnosis of brucellosis cannot be established based on clinical symptoms alone, because in most cases it is mistaken for other diseases such as malaria, typhoid and leptospirosis , . The most reliable diagnostic method is the isolation of bacteria from blood, serum, or contaminated tissues by conventional culture methods and phenotypic evaluation. Due to its slow growth, Brucella culture is time-consuming, dangerous and expensive and the result is not always positive . Other diagnostic methods based on biochemical and antigenic features, metabolism differences, carbon dioxide need, tonality, phage sensitivity, H2S production, oxidative metabolic pattern and reaction with antiserum for phenotypic diagnosis and identification of isolates can result in false positives . However, modern molecular methods, such as whole-genome sequencing and various techniques provide an appropriate way for diagnosing Brucella . Moreover, by using high-resolution melt (HRM) curve analysis with real-time polymerase chain reaction (PCR) scanning a segment of the genome with extremely high resolution, the interpretation of results and differentiation of different types of species with genetic proximity is possible . Accordingly, an accurate and quick diagnosis of two common species isolated from samples with brucellosis, is studied in this article. In order to diagnose and differentiate the species, we employ an optimized method of real-time PCR, which is facilitated by high-resolution melt analysis (HRMA). Unlike similar studies, this project initially focused on the amplification of segments by a pair of primers. The designed primers reproduces the segments with different sizes. Difference in size allows us to carry out real-time PCR only once and perform the HRMA technique of strain isolation with more confidence. The present study is applicable for the diagnosis of Brucella species in clinical samples, epidemiology, as well as veterinary studies.
Materials and methods
Collected, stored and extracted DNA samples
This cross-sectional study was conducted on 70 human and 55 animal isolates. The specimens were taken between April 2012 and May 2015. The human specimens consisted of 65 blood, five cerebrospinal fluid (CSF) and 38 animal samples, including eight liver, 10 spleen and 35 blood specimens collected using a simple random sampling method. Sampling was performed for humans by a physician using syringes prepared under sterile conditions from suspected patients with symptoms of brucellosis such as fever, chills, fatigue, body aches, headache, joint pain, low back pain and back pain. The animal specimens were also collected under sterile conditions by a veterinarian from animals suspected of brucellosis. Blood specimens were incubated at 37 °C for 21 days in a blood culture medium (BacT/Alert, BioMérieux, Lyon, France). One hundred microliters of the blood culture medium were then transferred to a Brucella broth and Brucella agar (Merck, Darmstadt, Germany) under sterile conditions and the plates were incubated at 37 °C for 48–72 h. After incubation, diagnosis was carried out at a species level using colony morphology, Gram staining, oxidase, catalase and growth sensitivity to the dyes basic fuchsin and thionin. Samples that were identified as Brucella species were stored in skim milk containing 10% sterilized glycerol at −80 °C until the next stages were performed. Whole DNA of all isolates was extracted from cultured-blood specimens using proteinase K and the phenol-chloroform method. DNA was stored at −20 °C until PCR was conducted. The project was approved by the Iran University Human Ethics committee. (Ethical code: IR.IUMS.REC1393.8812342002).
For this study, a pair of 23 bp primers was designed that was able to simultaneously recognize and differentiate B. melitensis at the 398 bp segment and B. abortus at the 520 bp segment. The forward primer sequence was 5′-ATTGACACCTTGCCTGGACGG-3′ and the reverse primer sequence was 5′-GTTGAAAACCAGGGGCTGGC-3′.
Specificity, sensitivity and reliability of the primers and HRM
To evaluate the specificity of the primers, DNA was extracted from those species that were genetically close to Brucella and also from non-infected human blood specimens, cattle and sheep spleen DNA. Serial dilutions (10-1, 10-2, 10-3 and 10-4) of DNA from B. abortus S19 and B. melitensis 16 M were used to evaluate the sensitivity of the primers in PCR based on DNA concentration. Sterilized distilled water was used to dilute the DNA.
The Solis BioDyne 5 FIREpol® Master Mix, containing EvaGreen® saturated color fluorescence was used in real-time PCR. The PCR reactions were performed according to the values provided in Table 1. Amplifications were done in a Rotor-Gene (Corbett Rotor-Gene 6000 Qiagen, Valencia, CA, USA) as follows: an initial holding step at 95 °C for 10 min, 35 cycles of annealing at 95 °C for 10 s, elongation step at 60 °C for 40 s, and then a final elongation at 72 °C for 30 s.
|Master mix (5× HOT FIREPol®EvaGreen®HRM Mix)||12.5|
|Forward and reverse primers||2|
|Total reaction volume||25|
HRM analysis of Brucella abortus and Brucella melitensis
Real-time PCR was performed by conducting HRMA at specific time intervals at 86–93 °C with a rate of 0.1 °C. Data were normalized according to the available information. In addition, B. abortus S19 and B. melitensis M16 strains were used as the standard and control samples.
Primer specificity and sensitivity
Specificity of the primers designed for PCR were measured using DNA extracted from two standard Brucella strains, all studied specimens, 15 pathogenic species of bacteria, human blood and spleen tissue. The results indicated that the lowest limit of detection (LLOD) for B. abortus and B. melitensis were 20–25 ng/μL and 20–30 ng/μL, respectively. In these DNA concentrations, the primers were able to diagnose and differentiate the two Brucella species. Nonspecific bands were not observed on gel electrophoresis and no bands were detected after amplification of other DNA samples (Table 2).
|Strain||PCR identification||Strain (from human)||PCR identification|
|Brucella spp.||*88/88||Pseudomonas aeruginosa||0/1|
|Brucella abortus||16/16||Campylobacter spp.||0/1|
|Brucella melitensis||72/72||Klebsiella pneumoniae||0/1|
|Salmonella enterica ATCC:9270||0/2||Listeria monocytogenes||0/1|
|Agrobacterium tumefaciens PTCC 1654||0/1||Proteus mirabilis||0/1|
|Staphylococcus aureus ATCC:6538||0/1||Salmonella enteritidis||0/1|
|Shigella sonnei ATCC:9290||0/1||Staphylococcus aureus||0/1|
|Shigella flexneri ATCC:12022||0/2||Streptococcus pneumoniae||0/1|
|DNA extraction from human blood||0/2||Escherichia coli O157:H7||0/2|
|DNA extraction from human spleen||0/1||Vibrio cholerae PTCC 1611||0/1|
*The primers are specified for two species of Brucella (Brucella abortus and Brucella melitensis).
Specificity and sensitivity of the HRM assay
Figure 1A and B illustrates the result of HRM test specificity. As can be seen in this figure, the DNA of 15 different bacteria, the names of which are not mentioned in Table 2, were not amplified. The DNA of standard B. abortus and B. melitensis sample as well as the clinical samples were amplified. The HRMA test was carried out successfully. The range of mean [cycles threshold±standard deviation (CT±SD)] for the amplified samples was from 15.39 to 25.15. The range of melt peak (°C) ±SD for B. abortus and B. melitensis was 90.10±0.4 and 89.70±0.4, respectively, and 10 was reported on peak. A standard curve was obtained in a linear graph, which had the correlation coefficients of (R2) in 0.987 based on the real-time PCR with serial dilutions (10−1, 10−2, 10−3 and 10−4) of B. melitensis (Figure 1C). No difference was noted in the melting profiles of B. melitensis and B. abortus, at 1:10, 1:100, 1:1000 and 1:10,000 fold differences in template concentration using primer sets of the respective Brucella spp. (Figure 1D).
Real-time PCR results
A total of 50 Brucella positive samples were detected and confirmed by the real-time PCR technique. Twenty-one belonged to women and 29 samples were from men. All serum titers were reported more than 1:80. Among the obtained 38 positive samples from animals, 28 and 10 were obtained from sheep and cows, respectively. Figure 2 indicates the diagram of different Brucella strains. At this stage, genus Brucella was detected, but the Brucella species were not detectable. All 58 human and animal samples were identified by real-time PCR method (Figure 2).
High-resolution melt analysis
Figure 3A shows the analysis of 50 HRMA samples obtained from humans and 38 gathered samples from animals by a primers pair. The normalization of data at 86–93 °C clearly revealed the difference in the amplified segments. At 87.5 °C two species were isolated from each other. Moreover, by placing a standard strain, B. abortus and B. melitensis were differentiated appropriately. The results of the process can be clearly seen in Figure 3B. These two types of Brucella are separable at 89–89.5 °C. No difference was observed between the amplified samples of clinical and standard strains. Figure 3C displays the differentiation of two species using the standard strains. According to the results, among the 88 samples, 16 B. abortus and 72 B. melitensis samples were detected. Among 16 B. abortus samples, six samples were from men and 10 from sheep. Brucella abortus was not detected in cows. Among 72 B. melitensis samples, 38 samples were from humans: nine from women and 29 isolates from men. Thirty-four samples from a total of 38 animal specimens were B. melitensis, 28 were from sheep and 10 were isolated from cows. Furthermore, different genotypes of B. melitensis were reported by analyzing the species and omitting the results of B. abortus. All 72 B. melitensis isolates, which were identified by the HRMA method have shown genotype confidence percentage (GCP). Among the clinical samples demonstrated in Figure 3D, three different genotypes were reported at 85.5–89 °C.
Discussion and conclusions
HRMA is a novel, closed tube and quick method, which enables researchers to analyze genetic variations at the genome level . Using thermal denaturation of double-stranded DNA, the technique extracts significant details and is capable of finding a single nucleotide polymorphism (SNP). Genome scanning is the most salient function of this technique, which is able to specify the new varieties and differentiate them precisely from the PCR products. After segment amplification, the method conducted used real-time PCR. The fruitful attempt of the present study was to employ a pair of primers for the identification of bacterial genus and species. This primer can identify the B. abortus strain by amplifying the 520 bp segment and B. melitensis species by amplifying its 398 bp segment. Difference in segment size and the sequence of the amplified segments is the basic differentiating factor of the two species. Unlike other studies that use the HRMA technique for the identification of different strains of bacteria from several primers, this study only utilizes one pair of primers , , , . This advantage lowers test errors and could bear acceptable results by performing the real-time PCR and HRMA only once. Moreover, the designed pair of primers is able to identify all pathogenic Brucella strains. However, as standard strains of all Brucella species were not available, the identification and optimization of the study for brucellosis, Brucella canis and Brucella ovis was not possible.
The designed primer in this study is specific for a hypothetical protein . The primer substantiates the presence of such a protein in the Brucella and indicates that its size is variable in different types of Brucella. This size variation, however, is at species level and its sequence could be used for the identification and differentiation of Brucella species. Hypothetical proteins are those proteins that are still unidentified and their efficiencies are not established in the function of bacteria, but their sequences reveal that they could have a functional structure. Various sizes of the amplified segments in this study illustrate that hypothetical proteins could have various sizes, functions and structures in different species.
Winchell et al.  designed various primers for each Brucella species. They managed to identify a SNP on the int-hyp gene and differentiate the B. melitensis from B. canis. The study called for certain primers for each Brucella species, a problem which is presently ratified by the evaluation of a new segment of hypothetical protein sequence. Our reports from this study suggest a test, which could identify the genus Brucella by the real-time PCR technique. With a quick stage design, this test, after PCR, is also able to diagnose the prevalent Brucella species in Iran, namely B. abortus and B. melitensis.
The efficiency of this test in diagnosing human and animal specimens is also substantiated. Among a total of 88 diagnosed human and animal specimens using this method, 81.8% were B. melitensis and 18.2% B. abortus. Other epidemiological studies have also reported a higher prevalence rate of B. melitensis than B. abortus. Winchell et al.  in a similar study discussed the quick diagnosis of Brucella genus and species. That study was on standard specimens, while the present article is on the diagnosis and differentiation of this technique in standard, clinical and animal samples. Moreover, the present study has decreased the total test time by HRMA optimization and lowered the rating time.
Use of high quality and proper fluorescent dyes is also essential to the HRMA test. This study used the EvaGreen dye, which has extremely lower toxicity than other saturated fluorescent dyes . In addition, these dyes are not expensive and give more powerful signals than other unsaturated dyes and probe-based methods. The previous studies revealed the better quality of this type of dye than other saturated dyes, such as SYBR Green. By using this type of dye we could carry out the PCR and HRMA stages continuously. By attaching it to the amplifying DNA segments, EvaGreen could maintain its stability and fluorescence emission , , .
In this paper, detection of other Brucella spp. was not possible due to lack of related reports in Iran. However, using the test designed by Mohamed Zahidi et al.  in Malaysia, has diagnosed 41 clinical samples of B. melitensis. Moreover, according to the primers designed by Winchell et al. , diagnosis, differentiation and optimization was made for B. suis, as well. Another study, Gopaul et al.  managed to find SNP points by scanning the Brucella species genome using the minor-groove binding-based method. The present article, however, by presenting a novel approach for the distinction and differentiation of species based on the size of the produced segments, but not on the polymorphism points and SNPs, could differentiate two species of B. melitensis and B. abortus in clinical and animal samples, appropriately. As the study is on positive samples, we recommend that CT be between 15 – 30 for achieving acceptable responses from the HRMA sigmoid curve, although in the case of extracted clinical samples from human blood, the responses will be acceptable. For better results, we also recommend that the PCR cycles be performed at their maximum limit.
The analysis of HRMA results can be used for species differentiation and bacterial genotyping. This molecular method could help diagnose Brucella quickly and precisely. A quick recognition of the brucellosis agent could prevent its prevalence among humans and animals and mitigate substantial economic loss. Moreover, controlling infection and managing the causative agents calls for the utilization of this accurate method. The present study designed a quick and diagnostic molecular method, by which we are able to interpret the results and control infection caused by the prevalent strains of the brucellosis agent.
The authors are greatly thankful to the director and principal of Iran University of Medical Sciences especially to all the faculty members of the Microbiology department for their constant encouragement and support of research for this study. We also wish to thank Serve Pirouzi for help with the English language version of this paper.
Author contributions: FMJ contributed to the conception and design of the work; the acquisition, analysis and interpretation of data for the work. RM contributed to data collection and interpretation of data for the work. GRI contributed to the design of the work, data collection and final approval of the version to be published. VP and MK contributed to data analysis, drafting the work and revising it critically for important intellectual content. NAM and TZS contributed in the revising the draft and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. MS contributed in revising the article and final approval of the version to be published. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
1. Pishva E, Salehi R, Hoseini A, Kargar A, Taba FE, Hajiyan M, et al. Molecular typing of Brucella species isolates from human and livestock bloods in Isfahan province. Adv Biomed Res 2015;4:104.Search in Google Scholar PubMed
2. Assenga JA, Matemba LE, Muller SK, Malakalinga JJ, Kazwala RR. Epidemiology of Brucella infection in the human, livestock and wildlife interface in the Katavi-Rukwa ecosystem, Tanzania. BMC Vet Res 2015;11:189.10.1186/s12917-015-0504-8Search in Google Scholar PubMed
3. Soares Cde P, Teles JA, dos Santos AF, Silva SO, Cruz MV, da Silva-Junior FF. Prevalence of Brucella spp in humans. Rev Lat Am Enfermagem 2015;23:919–26.10.1590/0104-1169.0350.2632Search in Google Scholar PubMed
4. Piranfar V, Sharif M, Hashemi M, Vahdati AR, Mirnejad R. Detection and discrimination of two Brucella species by multiplex real-time PCR and high-resolution melt analysis curve from human blood and comparison of results using RFLP. Iran J Basic Med Sci 2015;18:909–14.Search in Google Scholar PubMed
5. Wareth G, Melzer F, Tomaso H, Roesler U, Neubauer H. Detection of Brucella abortus DNA in aborted goats and sheep in Egypt by real-time PCR. BMC Res Notes 2015;8:212.10.1186/s13104-015-1173-1Search in Google Scholar PubMed
6. Mirnejad R, Mohamadi M, Piranfar V, Mortazavi SM, Kachuei R. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples. Asian Pac J Trop Med 2013;6:453–6.10.1016/S1995-7645(13)60073-5Search in Google Scholar PubMed
7. Aloufi AD, Memish ZA, Assiri AM, McNabb SJ. Trends of reported human cases of brucellosis, Kingdom of Saudi Arabia, 2004–2012. J Epidemiol Glob Health 2016;6:11–8.10.1016/j.jegh.2015.09.001Search in Google Scholar PubMed PubMed Central
8. Tumwine G, Matovu E, Kabasa JD, Owiny DO, Majalija S. Human brucellosis: sero-prevalence and associated risk factors in agro-pastoral communities of Kiboga District, Central Uganda. BMC Public Health 2015;15:900.10.1186/s12889-015-2242-zSearch in Google Scholar PubMed PubMed Central
9. Arabestani MR, Rastiany S, Kazemi S, Mousavi SM. Conventional, molecular methods and biomarkers molecules in detection of septicemia. Adv Biomed Res 2015;4:120.10.4103/2277-9175.158027Search in Google Scholar PubMed PubMed Central
10. Kianmehr Z, Kaboudanian Ardestani S, Soleimanjahi H, Fotouhi F, Alamian S, Ahmadian S. Comparison of biological and immunological characterization of lipopolysaccharides from brucella abortus RB51 and S19. Jundishapur J Microbiol 2015;8:e24853.10.5812/jjm.24853Search in Google Scholar PubMed PubMed Central
11. Naze F, Desvars A, Picardeau M, Bourhy P, Michault A. Use of a new high resolution melting method for genotyping pathogenic leptospira spp. PLoS One 2015;10:e0127430.10.1371/journal.pone.0127430Search in Google Scholar PubMed PubMed Central
12. Gopaul KK, Sells J, Lee R, Beckstrom-Sternberg SM, Foster JT, Whatmore AM. Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species. BMC Res Notes 2014;7:903.10.1186/1756-0500-7-903Search in Google Scholar PubMed PubMed Central
13. Winchell JM, Wolff BJ, Tiller R, Bowen MD, Hoffmaster AR. Rapid identification and discrimination of brucella isolates by use of real-time PCR and high-resolution melt analysis. J Clin Microbiol 2010;48:697–702.10.1128/JCM.02021-09Search in Google Scholar PubMed PubMed Central
14. Ramazanzadeh R, McNerney R. Variable number of tandem repeats (VNTR) and its application in bacterial epidemiology. Pak J Biol Sci 2007;10:2612–21.10.3923/pjbs.2007.2612.2621Search in Google Scholar PubMed
15. Ko KY, Kim JW, Her M, Kang SI, Jung SC, Cho DH, et al. Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis. Vet Microbiol 2012;156:374–80.10.1016/j.vetmic.2011.11.011Search in Google Scholar PubMed
16. Chiaraviglio L, Kirby JE. Evaluation of impermeant, DNA-binding dye fluorescence as a real-time readout of eukaryotic cell toxicity in a high throughput screening format. Assay Drug Dev Technol 2014;12:219–28.10.1089/adt.2014.577Search in Google Scholar PubMed PubMed Central
17. Mao F, Leung WY, Xin X. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol 2007;7:76.10.1186/1472-6750-7-76Search in Google Scholar PubMed PubMed Central
18. Li YD, Chu ZZ, Liu XG, Jing HC, Liu YG, Hao DY. A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants. J Integr Plant Biol 2010;52:1036–42.10.1111/j.1744-7909.2010.01001.xSearch in Google Scholar PubMed
19. Mohamed Zahidi J, Bee Yong T, Hashim R, Mohd Noor A, Hamzah SH, Ahmad N. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis. Diagn Microbiol Infect Dis 2015;81:227–33.10.1016/j.diagmicrobio.2014.12.012Search in Google Scholar PubMed
20. Gopaul KK, Koylass MS, Smith CJ, Whatmore AM. Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis. BMC Microbiol 2008;8:86.10.1186/1471-2180-8-86Search in Google Scholar PubMed PubMed Central
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