- Open Access
Polymerase chain reaction–based assays for the diagnosis of human brucellosis
© Wang et al.; licensee Biomed Central Ltd. 2014
- Received: 24 March 2014
- Accepted: 9 July 2014
- Published: 1 August 2014
Polymerase chain reaction (PCR) is an in vitro technique for the nucleic acid amplification, which is commonly used to diagnose infectious diseases. The use of PCR for pathogens detection, genotyping and quantification has some advantages, such as high sensitivity, high specificity, reproducibility and technical ease. Brucellosis is a common zoonosis caused by Brucella spp., which still remains as a major health problem in many developing countries around the world. The direct culture and immunohistochemistry can be used for detecting infection with Brucella spp. However, PCR has the potential to address limitations of these methods. PCR are now one of the most useful assays for the diagnosis in human brucellosis. The aim of this review was to summarize the main PCR techniques and their applications for diagnosis and follow-up of patients with brucellosis. Moreover, advantages or limitation of the different PCR methods as well as the evaluation of PCR results for treatment and follow-up of human brucellosis were also discussed.
- Polymerase chain reaction techniques
- Human brucellosis
- Brucella spp
- Molecular diagnosis
Brucellosis is a widespread zoonotic disease caused by members of genus Brucella. Its prevalence is more than 10 per 100 000 population in some endemic countries . Transmission of brucellosis from animals to humans occurs mainly through direct contact with infected animals, ingestion of raw dairy products of animal origin, or consumption of infected meat from domestic livestock . Human brucellosis may lead to a variety of clinical presentations, such as fever, sweating, chills, headache, malaise, myalgia and even arthralgia of the large joints . The presentations and phases of the disease may be acute, sub-acute, chronic, relapsed, active or inactive. Antibiotic treatment of human brucellosis often results in high treatment failure and relapse rates. Because the clinical presentation is non-specific, laboratory testing is required for confirmation.
Brucella species are gram-negative, facultative intracellular bacteria, which lack capsules, flagellae, endospores or native plasmids . Currently, the genus Brucella consists of ten species: B. abortus, B. suis, B. ovis, B. melitensis, B. canis, B. neotomae, B. pinnipedialis, B. ceti, B. microti and B. inopinata. Four species of the genus Brucella are pathogenic for humans, namely B. melitensis, B. abortus, B. suis, and B. canis. B. melitensis is considered as the most pathogenic species, followed by B. suis, whereas B. abortus is the mildest type of brucellosis. Additionally, Brucella isolates from marine mammals can cause human infections. DNA-DNA hybridization studies demonstrated that there are high degrees of genetic similarity of Brucella spp. .
At present, there are various assays for diagnosis of human brucellosis such as standard microbiological tests for the isolation of Brucella spp. from blood, tissue specimens, body fluids and bone marrow, serological tests for the detection of anti-Brucella spp. antibodies and molecular methods for the detection of Brucella spp. DNA . The most commonly used methods for detection and segregation of Brucella spp. were culture techniques and serological tests (standard agglutination tube test, anti-human globulin test, Rose-Bengal test, mercaptan-based tests, enzyme-linked immunosorbent assay and brucellacapt) ,. However, isolation of Brucella spp. is associated with a risk of laboratory-acquired infections and time consuming, and culture sampling sensitivity is often low, depending on the culture medium, Brucella species, disease stage and quantity of circulating bacteria. Serological tests seem to be more effective but can be unspecific due to cross reaction or subsensitive reactions in samples from areas with a low or subclinical prevalence of brucellosis . The principles and the main applications of these methods have been well reviewed elsewhere .
To ensure effective brucellosis disease prevention and control, a fast and accurate identification method is necessary. Polymerase chain reaction (PCR) technique offers a sensitive and specific way of detecting Brucella spp. from peripheral blood and other tissues . The first brucellosis PCR-based test was introduced by Fekete et al. in 1990 . They successfully amplified a 635 bp fragment of a 43 kDa outer membrane protein gene from B. abortus strain 19. Several studies have reported that PCR is a very useful tool for the rapid diagnosis of acute brucellosis and a good marker for the posttreatment follow-up and the early detection of relapses ,. Moreover, many studies have developed PCR-based assays to differentiate 10 Brucella species –. Up to data, PCR assays have been used in diagnosis of both animal brucellosis and human brucellosis . To our current knowledge, at least 200 reports have been published dealing with various methods based on PCR for laboratory diagnosis of human brucellosis. This review article highlights various PCR-based methods for the clinical diagnosis of human brucellosis. The principles, advantages or limitation of the different methods are also being discussed together with examples of applications taken from the literatures.
For the diagnosis of human brucellosis, a PCR assay with one pair of primers was developed, which amplifies the target genomic sequence of Brucella species. Primer pairs include the primers for sequences encoding 16S rRNA ,, outer membrane protein (omp2a, omp2b) –, 31-kDa immunogenic Brucella abortus protein (BCSP 31) ,, 16S-23S ribosomal DNA interspace region  and insertion sequence (IS711) ,. Studies showed that standard PCR appeared to be a more sensitive technique than microbiological methods, not only for the diagnosis of a first episode of infection, but also for the early detection of relapses –. Some research groups also assessed the performances of standard PCR as diagnostic tools for human brucellosis with respect to conventional methods. Their results showed that standard PCR is a promising diagnostic tool for patients with clinical signs and symptoms, and negative serological results, allowing an accurate and early diagnosis of human brucellosis ,.
Efficiency of blood PCR assays
Positive predictive value
Negative predictive value
In fact, blood samples are often used for the diagnosis of human brucellosis by the standard PCR . Several factors were reported to affect PCR results in a blood specimen such as the high concentrations of leukocytes DNA and heme compounds . Additionally, human genomic DNA affect the sensitivity of peripheral-blood PCR assay for the detection of Brucella DNA . Zerva L et al. reported that serum samples should be used preferentially over whole blood for diagnosis of human brucellosis by PCR , but Mitka S et al. revealed that buffy coat and whole blood were the optimal specimens . Moreover, sample volume used and efficient DNA extraction protocol are also the points of concern for the standard PCR to be used in routine laboratory testing for human brucellosis .
Real time PCR
Compared with the standard PCR, real-time PCR is a valuable technique in determining the quantification of nucleic acids in individual blood samples, as well as in automating the data. With the decreasing prices of real-time PCR thermocyclers and the reagents, many more people now have access to this technology to measure DNA copy number, mRNA expression levels and viral titers . Recently, real-time PCR for the rapid detection and differentiation of Brucella species in clinical samples has recently been developed, targeting 16S-23S internal transcribed spacer region (ITS) and the genes coding omp25 and omp31 , BCSP 31 –, and IS711 ,.
Real-time PCR seems to be highly reproducible, rapid, sensitive and specific. Additionally, this assay is easily standardized and minimises the risk of infection in laboratory workers. It is therefore a useful method for both the initial diagnosis of human brucellosis and the differentiation among inactive, seropositive, and active states. Queipo-Ortuño et al. reported that the sensitivities of a SYBR Green I LightCycler-based real-time PCR assay with serum samples was 93.3%, which is higher than 90% and 65% obtained by PCR-ELISA with whole blood samples and blood cultures, respectively . This group further developed a LightCycler-based real-time PCR assay to detect Brucella DNA in serum samples. This assay was found to be 91.9% sensitive and 95.4% specific when tested with 65 negative control samples and 62 serum samples from patients with active brucellosis .
Furthermore, Surucuoglu S et al. compared the TaqMan real time PCR technique to conventional methods using serum samples from patients with different clinical forms of brucellosis. The sensitivity, specificity, positive and negative predictive values of this PCR method were calculated as 88%, 100%, 100%, and 83%, respectively . Alsayed Y et al. further investigated the potential of a combination of several tests (culture, ELISA and real-time PCR) to support the diagnosis in different clinical manifestations of brucellosis with peripheral blood samples. They found that if the agglutination test is negative, real-time PCR, and/or ELISA, and/or culture are recommended . Moreover, using a panel of seven primer sets, Winchell JM et al. developed a real-time PCR method to differentiate members of the Brucella genus isolates, and has the potential to detect novel species . Other studies also reported that real-time PCR allowed the rapid diagnosis of human brucellosis ,. These results suggest that the high species specificity and selectivity of real-time PCR assay make it a useful tool for diagnosis of human brucellosis.
Comparison of conventional and real-time PCR assays lower limit of detection (fg)
IS711 copy number
B. canis RM6/66
B. abortus 544
B. melitensis 16 M
B. ovis 63/290
To overcome the inherent disadvantage of cost of the test, multiplex PCR has been developed to detect viral, bacterial, and/or other infectious agents. The advantages of using multiplex PCR technique are that it minimizes expense and recognizes many pathogens at once . These advances have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of human brucellosis.
Lübeck PS et al. developed and applied a multiplex PCR assay for Brucella diagnostics based on the perosamine synthetase gene in 2003 . El Kholy AA et al. also established a multiplex PCR technique using 2 sets of primers (B4/B5 and JPF/JPR) for the diagnosis of active human brucellosis in Egypt . They found that this technique showed high sensitivity, specificity and accuracy, and could serve as important alternatives to culture methods for diagnosis of human brucellosis. Additionally, a multiplex PCR assay can be used to simultaneously detect and type Brucella species present in clinical samples. In 2007, Imaoka K et al. developed a multiplex PCR procedure to identify four major species of the genus Brucella in one reaction tube. Four pairs of primers targeting bcsp31, omp2b, omp2a and omp31 genes were used. The specific amplification for each Brucella spp. examined in this study was achieved with these primers . Other groups also reported robust and rapid multiplex PCR assays, which were able to identify and differentiate currently recognised Brucella species in a single test of less than an hour and a half –. The timely and accurate information provided by this assay would be valuable to trace sources of infection and may help in rapid diagnosis of human brucellosis. Furthermore, several multiplex PCRs have been described for identification of Brucella partly at the biovar level using different primer combinations. A 19-primer multiplex PCR specifically identified B. neotomae, B. pinnipedialis, B. ceti, and B. microti simultaneously. Also, this method was able to differentiate B. abortus biovars 1, 2, 4 from biovars 3, 5, 6, 9 . A novel multiplex PCR assay for the rapid detection of Brucella genus at the species and at the biovar level has been described. The assay was shown to be ideal method for detection of B. suis at the biovar level and the differentiation of B. suis, B. canis and B. microti. It is well knows that only a few biovars of Brucella species are pathogenic for humans, hence rapid identification of Brucella genus at the biovar level is necessary. Moreover, several multiplex PCRs have been described for the simultaneous detection of Mycobacterium tuberculosis (M. tuberculosis) complex and Brucella spp., targeting the IS711, bcsp31 and omp2a genes for the identification of Brucella spp. and the IS6110, senX3-regX3 and cfp31 genes for the detection of the M. tuberculosis complex –. The results showed that this technique was a practical approach for the differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis.
The primer pairs have substantial effect on the multiplex PCR efficiency. The presence of more than one primer pair in the multiplex PCR increases risk for primer-dimers. Thus, nonspecific products may be obtained . Ideally, all the primer pairs in a multiplex PCR should not only eliminate non-specific PCR products, but also enable similar amplification efficiencies for their respective target. Therefore, the multiplex PCR requires laborious optimization .
Nested and semi-nested PCR
The nested PCR means that two different pairs of PCR primers are used for a single locus. The first pair is an amplified sequence. The second pair of primers (nested primers) is complementary to the sequence amplified by the first pair primers and produces a second PCR product that will be shorter than the first one . Same as nested, semi-nested PCR has two different pairs of PCR primers, but the second pair of primers has one primer identical to the first pair . The nested PCR and semi-nested PCR amplify only the specific sequences sought and are more specific than the standard PCR. Recently, nested PCR and semi-nested PCR assays were developed for identifying Brucella in samples of human blood and then to explore their clinical practice for the diagnosis of human brucellosis.
Two nested PCR assays have been applied for the diagnosis of human brucellosis in Kuwait. Two pairs of primers derived from IS711 were used. The results showed that the use of nested primers gave increased sensitivity and higher specificity providing a better molecular diagnostic approach for human brucellosis ,. Lin GZ et al. also reported a nested PCR for the laboratory diagnosis of human brucellosis . Moreover, a semi-nested PCR for diagnosis of human brucellosis were developed and evaluated with whole blood. The primers were from IS6501 and bcsp31 genes . We are now performing nested PCR combined with real-time PCR approach for the diagnosis of human brucellosis with the primers from bcsp31 and VirB11 genes. B. abortus and B. melitensis can be rapidly identified. The results have not been reported. According to the results, this assay was sensitive and could be used for the diagnosis of human brucellosis in the clinical laboratory.
However, the nested PCR and semi-nested PCR have some disadvantages. For example, the assays increased risk of primer dimerization and cross-react of PCR products. In addition, the nested PCR or semi-nested PCR will only identify a set of Brucella bacteria, not a single specific species.
Other PCR-based assays
In addition to standard PCR and its derivatives (nested and semi-nested, multiplex and real-time PCR), there are other significant PCR-based assays have been developed in the last years. Fekete et al. developed the arbitrarily primed polymerase chain reaction (AP-PCR) to distinguish 25 different Brucella strains according to the banding patterns of their amplified DNA on agarose gels. The degrees of relatedness among these strains of the genus Brucella were revealed through the calculated similarity coefficients . In 1994, AMOS PCR assays were used to identify vaccine strains from strains that cause infections based on the number and sizes of products amplified by PCR ,. In 1996, Tcherneva et al. reported the REP-PCR as a promising fingerprinting method for the evaluation of Brucella outbreak . Also, various PCR-RFLPs display sufficient polymorphism to distinguish Brucella species and biovars, and can serve as tools for diagnostic, epidemiological, taxonomic, and evolutionary studies . In addition, Multiple-locus variable-number tandem-repeat assays (MLVA) were used to study the molecular epidemiological characterization of Brucella isolates from humans . PCR methods have been used successfully to identify all Brucella species and most of the biovars, offering an improvement over conventional molecular genotyping methods ,. Furthermore, Bruce-ladder multiplex PCR assay was evaluated using 625 Brucella strains. This method can differentiate in a single step all of the classical Brucella species, including strains from marine mammals and the S19, RB51, and Rev.1 vaccine strains . Recently, the microfluidic Lab-on-Chip was also proposed as a rapid and specific detection method for the characterization of Brucella isolates ,.
PCR-based assays were shown to be valuable tools for detecting Brucella strains. PCR approaches have several advantages for the diagnosis of human brucellosis, such as speed, safety, high sensitivity and specificity . This technique might be considered complementary to the traditional methods and followed up by serology and/or culture . However, its disadvantages such as the higher cost, issues of quality control and quality assurance must be further evaluated on clinical samples before PCR can be used in routine laboratory testing for human brucellosis ,.
Evaluation of PCR results for treatment and follow-up of patients
Most patients with brucellosis suffer a relapse after receive the duration and combination of antibiotic therapy. Therefore, it is necessary to evaluate the progress towards therapeutic failure or relapse . The conventional methods are difficult for the diagnosis of these relapses. Several previous studies reported the applications of PCR for the diagnosis of post treatment follow-up and relapses. Queipo-Ortuño MI et al. examined the usefulness of PCR assay in post treatment follow-up and relapse of patients with brucellosis. They showed positive PCR tests for the relapse as well as negative once the relapse treatment was completed ,. Nimri LF obtained the positive PCR results in the relapse cases, indicating that the assay could be a useful tool to confirm a relapse in cases of a treated brucellosis . Navarro E et al. also developed a real-time PCR assay to monitor the evolution of Brucella melitensis DNA load in blood during therapy and post-therapy follow-up in patients with brucellosis. This assay showed 100% analytical sensitivity for both initial infections and relapses . Moreover, Mitka S et al. showed that PCR assays were negative in all follow-up samples from patients who had completed a successful treatment and were positive in all follow-up samples from patients who had relapses in the first year after therapy, including the times of the relapses . However, dead phagocytosed bacteria may present in the circulating mononuclear cells in certain patients who have concluded successful treatment. Because PCR cannot differentiate between DNA from live and dead organisms, therefore, the ability to amplify the DNA of Brucella DNA from dead or phagocytized cells should be considered when interpreting the results.
At present, PCR-based assays could allow rapid and more-sensitive identification of Brucella genus at the species and at the biovar level, compared with traditional techniques. The implementation of PCR-based assays into the clinical setting will likely improve therapeutic outcomes. However, PCR protocols lack standardization. As new methods for Brucella spp. identification and typing, PCR tests are still being developed and still await validation for use with clinical samples. For instance, the sensitivity and specificity of most PCR-based methods is associated with inhibitors in DNA samples such as EDTA, RNase or DNase, heme, heparin, phenol, and probably a host of other reagents. There is still a great deal of work required for standardization before any of these methods may be used in routine laboratory testing for brucellosis. Future studies should focus on the integration of these techniques into clinical decision making.
This work was support in part by grants from the National Natural Science Foundation of China (No.81360242), Natural Science Foundation of Inner Mongolia Autonomous Region of China (No. 2014JQ04, No. 2014MS0808) and Scientific Research Fund in Universities of Inner Mongolia Autonomous Region (No. NJZY14528).
- Buzgan T, Karahocagil MK, Irmak H, Baran AI, Karsen H, Evirgen O, Akdeniz H: Clinical manifestations and complications in 1028 cases of brucellosis: a retrospective evaluation and review of the literature. Int J Infect Dis. 2010, 14 (6): e469-e478.PubMedView ArticleGoogle Scholar
- Minas M, Minas A, Gourgulianis K, Stournara A: Epidemiological and clinical aspects of human brucellosis in Central Greece. Jpn J Infect Dis. 2007, 60 (6): 362-366.PubMedGoogle Scholar
- Fanni F, Shahbaznejad L, Pourakbari B, Mahmoudi S, Mamishi S: Clinical manifestations, laboratory findings, and therapeutic regimen in hospitalized children with brucellosis in an Iranian Referral Children Medical Centre. J Health Popul Nutr. 2013, 31 (2): 218-222.PubMedPubMed CentralView ArticleGoogle Scholar
- Moreno E, Cloeckaert A, Moriyón I:Brucella evolution and taxonomy. Vet Microbiol. 2002, 90 (1–4): 209-227.PubMedView ArticleGoogle Scholar
- Galińska EM, Zagórski J: Brucellosis in humans–etiology, diagnostics, clinical forms. Ann Agric Environ Med. 2013, 20 (2): 233-238.PubMedGoogle Scholar
- Tiller RV, Gee JE, Frace MA, Taylor TK, Setubal JC, Hoffmaster AR, De BK: Characterization of novel Brucella strains originating from wild native rodent species in North Queensland, Australia. Appl Environ Microbiol. 2010, 76 (17): 5837-5845.PubMedPubMed CentralView ArticleGoogle Scholar
- Sakran W, Chazan B, Koren A: Brucellosis: clinical presentation, diagnosis, complications and therapeutic options. Harefuah. 2006, 145 (11): 836-840. 860PubMedGoogle Scholar
- Nielsen K, Yu WL: Serological diagnosis of brucellosis. Prilozi. 2010, 31 (1): 65-89.PubMedGoogle Scholar
- Al Dahouk S, Nöckler K: Implications of laboratory diagnosis on brucellosis therapy. Expert Rev Anti Infect Ther. 2011, 9 (7): 833-845.PubMedView ArticleGoogle Scholar
- Pabuccuoglu O, Ecemis T, El S, Coskun A, Akcali S, Sanlidag T: Evaluation of serological tests for diagnosis of brucellosis. Jpn J Infect Dis. 2011, 64 (4): 272-276.PubMedGoogle Scholar
- Araj GF: Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents. 2010, 36 (Suppl 1): S12-S17.PubMedView ArticleGoogle Scholar
- Bricker BJ: PCR as a diagnostic tool for brucellosis. Vet Microbiol. 2002, 90 (1–4): 435-446.PubMedView ArticleGoogle Scholar
- Fekete A, Bantle JA, Halling SM, Sanborn MR: Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. J Appl Bacteriol. 1990, 69 (2): 216-227.PubMedView ArticleGoogle Scholar
- Mitka S, Anetakis C, Souliou E, Diza E, Kansouzidou A: Evaluation of different PCR assays for early detection of acute and relapsing brucellosis in humans in comparison with conventional methods. J Clin Microbiol. 2007, 45 (4): 1211-1218.PubMedPubMed CentralView ArticleGoogle Scholar
- Al Ajlan HH, Ibrahim AS, Al Salamah AA: Comparison of different PCR methods for detection of Brucella spp. in human blood samples. Pol J Microbiol. 2011, 60 (1): 27-33.PubMedGoogle Scholar
- 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-PubMedPubMed CentralView ArticleGoogle Scholar
- López Goñi I, García Yoldi D, Marín CM, De Miguel MJ, Muñoz PM, Blasco JM, Jacques I, Grayon M, Cloeckaert A, Ferreira AC, Cardoso R, de Sá MI C, Walravens K, Albert D, Garin-Bastuji B: Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J Clin Microbiol. 2008, 46 (10): 3484-3487.PubMedPubMed CentralView ArticleGoogle Scholar
- Hinić V, Brodard I, Thomann A, Cvetnić Z, Makaya PV, Frey J, Abril C: Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems. J Microbiol Methods. 2008, 75 (2): 375-378.PubMedView ArticleGoogle Scholar
- Yu WL, Nielsen K: Review of detection of Brucella spp. by polymerase chain reaction. Croat Med J. 2010, 51 (4): 306-313.PubMedPubMed CentralView ArticleGoogle Scholar
- Romero C, Gamazo C, Pardo M, López-Goñi I: Specific detection of Brucella DNA by PCR. J Clin Microbiol. 1995, 33 (3): 615-617.PubMedPubMed CentralGoogle Scholar
- Nimri LF: Diagnosis of recent and relapsed cases of human brucellosis by PCR assay. BMC Infect Dis. 2003, 3: 5-PubMedPubMed CentralView ArticleGoogle Scholar
- Leal Klevezas DS, Martínez Vázquez IO, López Merino A, Martínez Soriano JP: Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol. 1995, 33 (12): 3087-3090.PubMedPubMed CentralGoogle Scholar
- Sifuentes-Rincón AM, Revol A, Barrera-Saldaña HA: Detection and differentiation of the six Brucella species by polymerase chain reaction. Mol Med. 1997, 3 (11): 734-739.PubMedGoogle Scholar
- Bardenstein S, Mandelboim M, Ficht TA, Baum M, Banai M: Identification of the Brucella melitensis vaccine strain Rev.1 in animals and humans in Israel by PCR analysis of the PstI site polymorphism of its omp2 gene. J Clin Microbiol. 2002, 40 (4): 1475-1480.PubMedPubMed CentralView ArticleGoogle Scholar
- Matar GM, Khneisser IA, Abdelnoor AM: Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA. J Clin Microbiol. 1996, 34 (2): 477-478.PubMedPubMed CentralGoogle Scholar
- Queipo-Ortuño MI, Morata P, Ocón P, Manchado P, Colmenero JD: Rapid diagnosis of human brucellosis by peripheral-blood PCR assay. J Clin Microbiol. 1997, 35 (11): 2927-2930.PubMedPubMed CentralGoogle Scholar
- Fox KF, Fox A, Nagpal M, Steinberg P, Heroux K: Identification of Brucella by ribosomal-spacer-region PCR and differentiation of Brucella canis from other Brucella spp. pathogenic for humans by carbohydrate profiles. J Clin Microbiol. 1998, 36 (11): 3217-3222.PubMedPubMed CentralGoogle Scholar
- Cloeckaert A, Grayon M, Grepinet O: An IS711 element downstream of the bp26 gene is a specific marker of Brucella spp. isolated from marine mammals. Clin Diagn Lab Immunol. 2000, 7 (5): 835-839.PubMedPubMed CentralGoogle Scholar
- Elfaki MG, Uz-Zaman T, Al-Hokail AA, Nakeeb SM: Detection of Brucella DNA in sera from patients with brucellosis by polymerase chain reaction. Diagn Microbiol Infect Dis. 2005, 53 (1): 1-7.PubMedView ArticleGoogle Scholar
- Morata P, Queipo-Ortuño MI, Reguera JM, García-Ordoñez MA, Pichardo C, Colmenero JD: Posttreatment follow-Up of brucellosis by PCR assay. J Clin Microbiol. 1999, 37 (12): 4163-4166.PubMedPubMed CentralGoogle Scholar
- Elfaki MG, Al-Hokail AA, Nakeeb SM, Al-Rabiah FA: Evaluation of culture, tube agglutination, and PCR methods for the diagnosis of brucellosis in humans. Med Sci Monit. 2005, 11 (11): MT69-MT74.PubMedGoogle Scholar
- Briones Lara E, Palacios Saucedo Gdel C, Martínez Vázquez IO, Morales Loredo A, Bilbao Chávez Ldel P: Response to the treatment of brucellosis among children. Evaluation with Huddleson reaction and PCR. Rev Med Inst Mex Seguro Soc. 2007, 45 (6): 615-622.PubMedGoogle Scholar
- Asaad AM, Alqahtani JM: Serological and molecular diagnosis of human brucellosis in Najran, Southwestern Saudi Arabia. J Infect Public Health. 2012, 5 (2): 189-194.PubMedView ArticleGoogle Scholar
- Gemechu MY, Gill JP, Arora AK, Ghatak S, Singh DK: Polymerase chain reaction (PCR) assay for rapid diagnosis and its role in prevention of human brucellosis in Punjab, India. Int J Prev Med. 2011, 2 (3): 170-177.PubMedPubMed CentralGoogle Scholar
- Baddour MM, Alkhalifa DH: Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood. Can J Microbiol. 2008, 54 (5): 352-357.PubMedView ArticleGoogle Scholar
- Navarro E, Escribano J, Fernández J, Solera J: Comparison of three different PCR methods for detection of Brucella spp in human blood samples. FEMS Immunol Med Microbiol. 2002, 34 (2): 147-151.PubMedView ArticleGoogle Scholar
- Navarro E, Fernandez JA, Escribano J, Solera J: PCR assay for diagnosis of human brucellosis. J Clin Microbiol. 1999, 37 (5): 1654-1655.PubMedPubMed CentralGoogle Scholar
- Morata P, Queipo-Ortuño MI, de Dios CJ: Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J Clin Microbiol. 1998, 36 (9): 2443-2446.PubMedPubMed CentralGoogle Scholar
- Zerva L, Bourantas K, Mitka S, Kansouzidou A, Legakis NJ: Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. J Clin Microbiol. 2001, 39 (4): 1661-1664.PubMedPubMed CentralView ArticleGoogle Scholar
- Ginzinger DG: Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol. 2002, 30 (6): 503-512.PubMedView ArticleGoogle Scholar
- Kattar MM, Zalloua PA, Araj GF, Samaha-Kfoury J, Shbaklo H, Kanj SS, Khalife S, Deeb M: Development and evaluation of real-time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human brucellosis. Diagn Microbiol Infect Dis. 2007, 59 (1): 23-32.PubMedView ArticleGoogle Scholar
- Colmenero JD, Queipo-Ortuño MI, Reguera JM, Baeza G, Salazar JA, Morata P: Real time polymerase chain reaction: a new powerful tool for the diagnosis of neurobrucellosis. J Neurol Neurosurg Psychiatry. 2005, 76 (7): 1025-1027.PubMedPubMed CentralView ArticleGoogle Scholar
- Debeaumont C, Falconnet PA, Maurin M: Real-time PCR for detection of Brucella spp. DNA in human serum samples. Eur J Clin Microbiol Infect Dis. 2005, 24 (12): 842-845.PubMedView ArticleGoogle Scholar
- Queipo-Ortuño MI, Colmenero JD, Bravo MJ, García-Ordoñez MA, Morata P: Usefulness of a quantitative real-time PCR assay using serum samples to discriminate between inactive, serologically positive and active human brucellosis. Clin Microbiol Infect. 2008, 14 (12): 1128-1134.PubMedView ArticleGoogle Scholar
- Cerekci A, Kılıç S, Bayraktar M, Uyanık MH, Yaşar E, Esen B: Comparison of conventional methods and real-time multiplex polymerase chain reaction for identification and typing of Brucella isolates of human origin. Mikrobiyol Bul. 2011, 45 (3): 392-400.PubMedGoogle Scholar
- Zhang B, Wear DJ, Stojadinovic A, Izadjoo M: Sequential real-time PCR assays applied to identification of genomic signatures in formalin-fixed paraffin-embedded tissues: a case report about brucella-induced osteomyelitis. Mil Med. 2013, 178 (1): 88-94.PubMedView ArticleGoogle Scholar
- Queipo-Ortuño MI, Colmenero JD, Baeza G, Morata P: Comparison between LightCycler Real-Time Polymerase Chain Reaction (PCR) assay with serum and PCR-enzyme-linked immunosorbent assay with whole blood samples for the diagnosis of human brucellosis. Clin Infect Dis. 2005, 40 (2): 260-264.PubMedView ArticleGoogle Scholar
- Queipo-Ortuño MI, Colmenero JD, Reguera JM, García-Ordoñez MA, Pachón ME, Gonzalez M, Morata P: Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. Clin Microbiol Infect. 2005, 11 (9): 713-718.PubMedView ArticleGoogle Scholar
- Surucuoglu S, El S, Ural S, Gazi H, Kurutepe S, Taskiran P, Yurtsever SG: Evaluation of real-time PCR method for rapid diagnosis of brucellosis with different clinical manifestations. Pol J Microbiol. 2009, 58 (1): 15-19.PubMedGoogle Scholar
- Alsayed Y, Monem F: Brucellosis laboratory tests in Syria: what are their diagnostic efficacies in different clinical manifestations?. J Infect Dev Ctries. 2012, 6 (6): 495-500.PubMedView ArticleGoogle Scholar
- 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 (3): 697-702.PubMedPubMed CentralView ArticleGoogle Scholar
- Navarro-Martínez A, Navarro E, Castaño MJ, Solera J: Rapid diagnosis of human brucellosis by quantitative real-time PCR: a case report of brucellar spondylitis. J Clin Microbiol. 2008, 46 (1): 385-387.PubMedPubMed CentralView ArticleGoogle Scholar
- Colmenero JD, Clavijo E, Morata P, Bravo MJ, Queipo-Ortuño MI: Quantitative real-time polymerase chain reaction improves conventional microbiological diagnosis in an outbreak of brucellosis due to ingestion of unpasteurized goat cheese. Diagn Microbiol Infect Dis. 2011, 71 (3): 294-296.PubMedView ArticleGoogle Scholar
- Bounaadja L, Albert D, Chenais B, Henault S, Zygmunt MS, Poliak S, Garin Bastuji B: Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet Microbiol. 2009, 137 (1–2): 156-164.PubMedView ArticleGoogle Scholar
- Queipo-Ortuño MI, De Dios CJ, Macias M, Bravo MJ, Morata P: Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clin Vaccine Immunol. 2008, 15 (2): 293-296.PubMedPubMed CentralView ArticleGoogle Scholar
- Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE: Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev. 2000, 13 (4): 559-570.PubMedPubMed CentralView ArticleGoogle Scholar
- Lübeck PS, Skurnik M, Ahrens P, Hoorfar J: A multiplex PCR-detection assay for Yersinia enterocolitica serotype O:9 and Brucella spp. based on the perosamine synthetase gene. Application to Brucella diagnostics. Adv Exp Med Biol. 2003, 529: 451-453.PubMedView ArticleGoogle Scholar
- El Kholy AA, Gomaa HE, El Anany MG, Abd El Rasheed E: Diagnosis of human brucellosis in Egypt by polymerase chain reaction. East Mediterr Health J. 2009, 15 (5): 1068-1074.PubMedGoogle Scholar
- Imaoka K, Kimura M, Suzuki M, Kamiyama T, Yamada A: Simultaneous detection of the genus Brucella by combinatorial PCR. Jpn J Infect Dis. 2007, 60 (2–3): 137-139.PubMedGoogle Scholar
- Rees RK, Graves M, Caton N, Ely JM, Probert WS: Single tube identification and strain typing of Brucella melitensis by multiplex PCR. J Microbiol Methods. 2009, 78 (1): 66-70.PubMedView ArticleGoogle Scholar
- Mayer-Scholl A, Draeger A, Göllner C, Scholz HC, Nöckler K: Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. J Microbiol Methods. 2010, 80 (1): 112-114.PubMedView ArticleGoogle Scholar
- Kumar S, Tuteja U, Sarika K, Singh D, Kumar A, Kumar O: Rapid multiplex PCR assay for the simultaneous detection of the Brucella Genus, B. abortus, B. melitensis and B. suis. J Microbiol Biotechnol. 2011, 21 (1): 89-92.PubMedView ArticleGoogle Scholar
- Garofolo G, Ancora M, Di Giannatale E: MLVA-16 loci panel on Brucella spp. using multiplex PCR and multicolor capillary electrophoresis. J Microbiol Methods. 2013, 92 (2): 103-107.PubMedView ArticleGoogle Scholar
- 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 (6): 453-456.PubMedView ArticleGoogle Scholar
- Kamal IH, Al Gashgari B, Moselhy SS, Kumosani TA, Abulnaja KO: Two-stage PCR assay for detection of human brucellosis in endemic areas. BMC Infect Dis. 2013, 13: 145-PubMedPubMed CentralView ArticleGoogle Scholar
- Huber B, Scholz HC, Lucero N, Busse HJ: Development of a PCR assay for typing and subtyping of Brucella species. Int J Med Microbiol. 2009, 299 (8): 563-573.PubMedView ArticleGoogle Scholar
- López-Goñi I, García-Yoldi D, Marín CM, de Miguel MJ, Barquero-Calvo E, Guzmán-Verri C, Albert D, Garin-Bastuji B: New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis. Vet Microbiol. 2011, 154 (1–2): 152-155.PubMedView ArticleGoogle Scholar
- Queipo-Ortuño MI, Colmenero JD, Bermudez P, Bravo MJ, Morata P: Rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis using a multiplex real-time PCR assay. PLoS One. 2009, 4 (2): e4526-PubMedPubMed CentralView ArticleGoogle Scholar
- Colmenero JD, Morata P, Ruiz Mesa JD, Bautista D, Bermúdez P, Bravo MJ, Queipo Ortuño MI: Multiplex real-time polymerase chain reaction: a practical approach for rapid diagnosis of tuberculous and brucellar vertebral osteomyelitis. Spine (Phila Pa 1976). 2010, 35 (24): E1392-E1396.View ArticleGoogle Scholar
- Sanjuan Jimenez R, Colmenero JD, Bermúdez P, Alonso A, Morata P: Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis. PLoS One. 2013, 8 (3): e58353-PubMedPubMed CentralView ArticleGoogle Scholar
- Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S: The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 1997, 25: 3235-3241.PubMedPubMed CentralView ArticleGoogle Scholar
- Cha RS, Thilly WG: Specificity, efficiency, and fidelity of PCR. PCR Methods Appl. 1993, 3: S18-S29.PubMedView ArticleGoogle Scholar
- Porter-Jordan K, Rosenberg EI, Keiser JF, Gross JD, Ross AM, Nasim S, Garrett CT: Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. J Med Virol. 1990, 30 (2): 85-91.PubMedView ArticleGoogle Scholar
- Seah CL, Chow VT, Chan YC: Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens. Clin Diagn Virol. 1995, 4 (2): 113-120.PubMedView ArticleGoogle Scholar
- Al Nakkas AF, Wright SG, Mustafa AS, Wilson S: Single-tube, nested PCR for the diagnosis of human brucellosis in Kuwait. Ann Trop Med Parasitol. 2002, 96 (4): 397-403.PubMedView ArticleGoogle Scholar
- Al-Nakkas A, Mustafa AS, Wright SG: Large-scale evaluation of a single-tube nested PCR for the laboratory diagnosis of human brucellosis in Kuwait. J Med Microbiol. 2005, 54 (Pt 8): 727-730.PubMedView ArticleGoogle Scholar
- Lin GZ, Zheng FY, Zhou JZ, Gong XW, Wang GH, Cao XA, Qiu CQ: Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp. Mol Cell Probes. 2011, 25 (2–3): 126-129.PubMedView ArticleGoogle Scholar
- Cirak MY, Hizel K: The value of polymerase chain reaction methods targeting two different gene regions for the diagnosis of brucellosis. Mikrobiyol Bul. 2002, 36 (3–4): 271-276.PubMedGoogle Scholar
- Fekete A, Bantle JA, Halling SM, Stich RW: Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers. J Bacteriol. 1992, 174 (23): 7778-7783.PubMedPubMed CentralGoogle Scholar
- Bricker BJ, Halling SM: Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin Microbiol. 1995, 33 (6): 1640-1642.PubMedPubMed CentralGoogle Scholar
- Bricker BJ, Halling SM: Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol. 1994, 32 (11): 2660-2666.PubMedPubMed CentralGoogle Scholar
- Tcherneva E, Rijpens N, Naydensky C, Herman L: Repetitive element sequence based polymerase chain reaction for typing of Brucella strains. Vet Microbiol. 1996, 51 (1–2): 169-178.PubMedView ArticleGoogle Scholar
- Al Dahouk S, Tomaso H, Prenger-Berninghoff E, Splettstoesser WD, Scholz HC, Neubauer H: Identification of brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Crit Rev Microbiol. 2005, 31 (4): 191-196.PubMedView ArticleGoogle Scholar
- Marianelli C, Graziani C, Santangelo C, Xibilia MT, Imbriani A, Amato R, Neri D, Cuccia M, Rinnone S, Di Marco V, Ciuchini F: Molecular epidemiological and antibiotic susceptibility characterization of Brucella isolates from humans in Sicily, Italy. J Clin Microbiol. 2007, 45 (9): 2923-2928.PubMedPubMed CentralView ArticleGoogle Scholar
- Marianelli C, Ciuchini F, Tarantino M, Pasquali P, Adone R: Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping. Microbes Infect. 2006, 8 (3): 860-865.PubMedView ArticleGoogle Scholar
- Scholz HC, Tomaso H, Al Dahouk S, Witte A, Schloter M, Kämpfer P, Falsen E, Neubauer H: Genotyping of Ochrobactrum anthropi by recA-based comparative sequence, PCR-RFLP, and 16S rRNA gene analysis. FEMS Microbiol Lett. 2006, 257 (1): 7-16.PubMedView ArticleGoogle Scholar
- De Santis R, Ciammaruconi A, Faggioni G, Fillo S, Gentile B, Di Giannatale E, Ancora M, Lista F: High throughput MLVA-16 typing for Brucella based on the microfluidics technology. BMC Microbiol. 2011, 11: 60-PubMedPubMed CentralView ArticleGoogle Scholar
- De Santis R, Ciammaruconi A, Faggioni G, D'Amelio R, Marianelli C, Lista F: Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis. BMC Microbiol. 2009, 9: 66-PubMedPubMed CentralView ArticleGoogle Scholar
- Navarro E, Casao MA, Solera J: Diagnosis of human brucellosis using PCR. Expert Rev Mol Diagn. 2004, 4 (1): 115-123.PubMedView ArticleGoogle Scholar
- Al-Attas RA, Al-Khalifa M, Al-Qurashi AR, Badawy M, Al-Gualy N: Evaluation of PCR, culture and serology for the diagnosis of acute human brucellosis. Ann Saudi Med. 2000, 20 (3–4): 224-228.PubMedGoogle Scholar
- Al Dahouk S, Tomaso H, Nöckler K, Neubauer H, Frangoulidis D: Laboratory-based diagnosis of brucellosis–a review of the literature. Part I: Techniques for direct detection and identification of Brucella spp. Clin Lab. 2003, 49 (9–10): 487-505.PubMedGoogle Scholar
- Navarro E, Segura JC, Castaño MJ, Solera J: Use of real-time quantitative polymerase chain reaction to monitor the evolution of Brucella melitensis DNA load during therapy and post-therapy follow-up in patients with brucellosis. Clin Infect Dis. 2006, 42 (9): 1266-1273.PubMedView ArticleGoogle Scholar
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