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. 2014 Sep 28;22(2):220–226. doi: 10.1016/j.sjbs.2014.09.014

Characterization and evaluation of an arbitrary primed Polymerase Chain Reaction (PCR) product for the specific detection of Brucella species

Jafar A Qasem a,⁎, Sabah AlMomin b, Salwa A Al-Mouqati b, Vinod Kumar b
PMCID: PMC4336441  PMID: 25737656

Abstract

Laboratory detection of Brucella is based largely on bacterial isolation and phenotypic characterization. These methods are lengthy and labor-intensive and have been associated with a heightened risk of laboratory-acquired infection. Antibody based indirect detection methods also suffer from limitations in proper diagnosis of the organism. To overcome these problems, nucleic acid amplification has been explored for rapid detection and confirmation of the presence of Brucella spp. PCR-based diagnostics is useful for screening large populations of livestock to identify infected individuals and confirms the presence of the pathogen. Random Amplification of Polymorphic DNA (RAPD) was performed and identified a 1.3 kb PCR fragment specifically amplifiable from DNA isolated from Brucella. A BLAST search revealed no significant homology with the reported sequences from species other than the members of Brucella. The isolated fragment seems to be a part of d-alanine–d-alanine ligase gene in Brucella sp. Translational BLAST revealed certain degree of homology of this sequence with orthologs of this gene reported from other microbial species at the deduced amino acid level. The sequence information was used to develop PCR based assays to detect Brucella sp. from various samples. The minimum detection limit of Brucella from blood and milk samples spiked with Brucella DNA was found to be 1 ng/ml and 10 ng/ml, respectively. In conclusion, we demonstrated that the PCR based detection protocol was successfully used for the detection of Brucella from various organs and spiked samples of diseased sheep. Diagnosis of Brucellosis by PCR based method reported in this study is relatively rapid, specific and simple.

Keywords: Brucellosis, abortus, melitensis, PCR, Diagnosis, Kuwait

1. Introduction

Brucellosis, a worldwide zoonotic disease caused by member of the genus Brucella, affects a large range of domesticated livestock, wildlife, marine mammals, and humans (Boschirolis et al., 2001). Brucellosis is emerging as a serious animal and public health issue in many parts of the world (Pappas et al., 2005; Park et al., 2005).

Although it has been eradicated in several developed countries, it remains endemic in most areas of the developing world (De Santis et al., 2011) and continues to pose a human health risk especially in developing countries despite efforts in eradicating the disease from domestic animals. It is considered to be an economically important disease in Kuwait (Al-Khalaf et al., 1992; AlMomin et al., 1999). People working in jobs requiring frequent contact with animals, especially the slaughterhouse workers, farmers, and veterinarians are at high risk. There is a need for the early detection of Brucella through better diagnostic procedures to safeguard public health. This is more important in Kuwait because of the growing meat industry. Several incidences of Brucellosis have been related to the consumption of raw milk, contact with farm animals, dessert camping in Kuwait and through contaminated air transmission (Mousa et al., 1987). Lack of effective prevention and management strategies are attributed to various factors such as lack of sufficient knowledge about the disease among the physicians, its either under-diagnosis or misdiagnosis (Thakur et al., 2002).

Current detection methods for Brucellosis include bacteriological and immunochemical assay methods (Al Dahouk et al., 2003; Fekete et al., 1990). Diagnosis remains to be difficult due to misleading non-specific manifestations and unusual presentations. According to an estimate, fewer than 10% of human cases of Brucellosis may be clinically recognized and treated or reported (Mantur et al., 2007).

Molecular based methods have provided powerful tools to identify infectious organisms with high sensitivity and specificity (Marianelli et al., 2008; Sreevatsan et al., 2000; Leal-Klevezas et al., 1995; Hopper et al., 1989). A Brucella diagnostic test that utilizes biotinylated Brucella abortus S19 whole genomic DNA has been reported (Hopper et al., 1989). However, this test is lengthy and can only be used when the number of bacteria is reasonably high (over 105 cells). It is proposed that PCR-RFLP assays of specific gene loci can serve as tools for diagnostic, epidemiological, taxonomic, and evolutionary studies on Brucella sp. (Al Dahouk et al., 2005). Previously, we have reported arbitrarily primed PCR amplification of a 1.3 kb DNA fragment for the specific detection of Brucella sp. (AlMomin et al., 1998).

In this study, we report molecular characterization of the isolated 1.3 kb DNA fragment and use of the sequence information to design PCR based assays to detect Brucella DNA in spiked blood and milk samples and also in the organ tissues of infected animals.

2. Materials and methods

2.1. Source of materials

Brucella and other bacterial strains were purchased from the American Type Culture Collection (ATCC): B. abortus ATCC 23448 (biovar 1), ATCC 23449 (biovar 2), ATCC 23450 (biovar 3), ATCC 23451 (biovar 4), ATCC 23452 (biovar 5), ATCC 23453 (biovar 6), ATCC 23454 (biovar 7) and ATCC 23455 (biovar 9), Brucella melitensis ATCC 23456 (biovar 1), Actinomyces pyrogenes (ATCC 8104), Klebsiella pneumoniae (ATCC 29939), Listeria monocytogenes (ATCC 7647), Ochrobactrum anthropi (ATCC 49188), Pseudomonas aeruginosa (ATCC 10145), Rhizopus stolonifer (ATCC 14037), Serratia marcescens (ATCC 13880), Yersinia enterocolitica (ATCC 29913), and Escherichia coli HB101. Reagents used in the experiments were either of analytical or of molecular biology grade purchased from Sigma Co. and BRL (Bethesda Research Laboratories, Life Technologies). Oligonucleotide primers were obtained from Operon Technologies, Alameda, California, USA.

2.2. Culture conditions

The Brucella cultures were grown on potato agar, under conditions of 10% CO2 and 37 °C for 48 h. Cultures were suspended in sterile 0.01 M Tris, pH 7.3 and killed by heating at 80 °C for 30 min. The non-Brucella bacterial species were cultured according to ATCC media recommendations.

2.3. Genomic DNA isolation, PCR, cloning and sequencing of Brucella specific fragment

Prior to DNA extraction, the bacterial cultures were washed with PBS and pelleted by centrifugation. Total DNA was isolated from heat-killed Brucella cells as described by AlMomin et al. (1999). The DNA from other bacterial species was isolated according to Bassam et al. (1992). Sequence of the random primers used in the study are, OPB-01 (5′-GTTTCGCTCC-3′), OPB-03 (5′-CATCCCCCTG-3′), OPB-05 (5′-TGCGCCCTTC-3′), OPB-19 (5′-ACCCCCGAAG-3′) and OPA-20 (5′-GTTGCGATCC-3′). A standard PCR amplification reaction mixture (25 μl) contained 5 ng genomic DNA, 1.5 mM MgCl2, 1 μM primer, 1.5 units DNA polymerase, 200 μM deoxynucleoside triphosphate (dNTP), in 10 mM Tris/HCl, pH 8.8, 10 mM KCl, 0.002% Tween-10. A negative control that contained all the components of the PCR reaction except the template DNA was included to monitor any possible contamination. PCR was performed in an ABI/Perkin Elmer 9700 thermo cycler. The program of 30 cycles consisted of 1 min at 94 °C, 2 min at 35 °C, and 3 min at 72 °C, and a final extension step at 72 °C for 7 min. Samples were held at 4 °C until electrophoresis. Following PCR amplification, the reaction mixture was analyzed on a 1.5% (w/v) agarose gel containing 0.5 μg/ml of ethidium bromide in TBE buffer (89 mM Tris/HCl, 89 mM boric acid, 2 mM EDTA, pH 8.0). A 100 bp DNA ladder was used as size standard. Gel images were documented using a UV transilluminator.

Based on RAPD data, a PCR fragment of 1.3 kb that is specifically amplifiable in DNA template from Brucella was identified. The band was excised and purified using gel extraction kit (Qiagen). The purified PCR fragment was inserted into a pCR2.1-TOPO vector (Invitrogen) using the protocol described in the instruction manual. The ligation mixture was used to transform E. coli TOP10F competent cells. A number of colonies were screened for the presence of 1.3 kb insert. One of the positive clones was sequenced from both strands. DNA sequence data were analyzed by comparison with the published sequences in the NCBI GenBank database using BLAST (Altschul et al., 1990).

Several internal primers were designed and screened to identify primer pairs that would amplify a smaller and more specific DNA fragment from Brucella sp. Sequence of selected internal primers are, KW1 (5′-CGGCTTGTTTCGCTCCATCGG-3′), KW2 (5′-GATTTCATTCAGCACGATACG-3′), KW3 (5′-GCTTCGTGAACGGTGCGCTGG-3′) and KW4 (5′-CGCACGGATTTCATTCTCTAC-3′).

2.4. Preparation of spiked samples

The recovery of pathogen-DNA and sensitivity of the detection method was tested using milk and blood samples containing the pathogen DNA. Whole human blood was obtained from Kuwait Blood Bank (Mubarak Al Kabeer Blood Bank, Kuwait) and used for spiking with Brucella DNA. Various concentrations of Brucella DNA (B. abortus strain Biovar-01–ATCC23448) were seeded in whole human blood with citrate phosphate dextrose anticoagulant. DNA isolation was done according to the published study (Queipo-Ortuno et al., 1997; Solomon and Jackson 1992). Similarly, long-life pasteurized milk (Brand Kuwait Danish Dairy; KDD) was used to seed the milk with Brucella DNA. Total DNA isolation from milk was performed according to the published methods (Rijpens et al., 1996). In both type of samples, Brucella DNA was added at the concentration range of 1 ng to 1 μg per ml. Total DNA was isolated from samples and PCR was performed for the detection of Brucella DNA.

2.5. Evaluation of the PCR based detection method for the presence of Brucella in sheep tissues

Organs from healthy and infected sheep were obtained from a local abattoir. The DNA was isolated from liver, kidney and lymph tissues according the protocol described by (Wards et al., 1995; Gallien et al., 1998; Fekete et al., 1992). PCR was performed using primers KW3 and KW4.

2.6. Ethical statement

All animals were used under the auspices of a protocol approved by Public Authority for Agriculture and Fish Resources (PAAF), Kuwait. The diagnosis of infected animals was carried out at PAAF laboratories for animal facilities, the infected animals were diagnosed positive for Brucellosis by culture as gold stranded method, the positive diagnosis was confirmed by Rose Bengal screening test and serum agglutination test.

3. Results

3.1. RAPD analysis

Five RAPD primers have been used to identify a unique DNA sequence that is highly specific to Brucella sp. Among the tested primers, OPB-01 generated a 1.3 kb band exclusively from DNA isolated from Brucella sp. (AlMomin et al., 1998). This DNA fragment was successfully amplified from genomic DNA of eight biovars of B. abortus and one biovar of B. melitensis. However, this fragment was not amplifiable by PCR from DNA samples of genetically related species such as O. anthropi and other non-Brucella organisms commonly associated with farm animals.

3.2. Isolated fragment sequence and characterization

The isolated fragment was sequenced and its homology to other reported sequences was examined. Sequence analysis at the nucleotide level (BLASTN) (Altschul et al., 1990) revealed high degree of homology (over 97%) to d-alanine–d-alanine ligase sequence from various biovars of B. abortus, Brucella microti CCM 4915, Brucella suis 1330, Brucella canis ATCC 23365, Brucella ovis ATCC 25840 and Brucella pinnipedialis B2/94. The 1.3 kb fragment isolated from Brucella did not show similarity to published sequences of any organisms other than Brucella sp. at the nucleotide sequence level. This result indicates that the isolated fragment may be unique to Brucella sp. at the nucleotide level. Homology at the deduced amino acid level was determined by BLASTX analysis (Altschul et al., 1997). A translation blast through BLASTX revealed 46–66% homology to d-alanine–d-alanine ligase sequence from various other bacterial species (Fig. 1). Fig. 2 depicts the degree of homology of d-alanine–d-alanine ligase of selected bacterial species. The bacterial species that demonstrate certain degree of sequence similarity to d-alanine–d-alanine ligase of Brucella sp. at the deduced amino acid level are Rhizobium leguminosarum (66%), Agrobacterium radiobacter (57%), Klebsiella variicola (53%), Mesorhizobium opportunistum (54%), Bradyrhizobium sp. (50%), E. coli (46%) and Salmonella enterica (46%). Therefore, in addition to primer specificity and PCR conditions, the low degree of homology of the isolated fragment from Brucella with sequences reported from other microbes at the nucleotide level could be one of the reason for high specificity of PCR based detection.

Figure 1.

Figure 1

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Alignment of amino acid sequence deduced from the nucleotide sequence of 1.3 kb fragment of Brucella with d-alanine–d-alanine ligase sequences reported from various bacterial species.

Figure 2.

Figure 2

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Dentogram showing sequence homology of d-alanine–d-alanine ligase of Brucella with unrelated naturally occurring bacterial species associated with sheep. The homology tree depicts the percentage homology among different species. Species used are Brucella abortus, Rhizobium leguminosarum, Agrobacterium radiobacter, Escherichia coli and Salmonella enterica.

Primer pair KW3 and KW4 produced a consistent PCR amplification of the DNA template from Brucella sp. Same primer pairs were also used to detect the pathogen from spiked samples of milk and whole blood. Figs. 3 and 4 show the detection limit of PCR based screening method to amplify Brucella specific amplicon from total DNA isolated from spiked samples of raw milk or whole blood respectively. Presence of Brucella DNA can be detected at concentrations as low as 1 ng/ml and 10 ng/ml of blood and milk respectively.

Figure 3.

Figure 3

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Amplification of Brucella specific DNA fragment (B1-1000) using primer pair KW1/KW2 and total DNA isolated from milk sample spiked with Brucella DNA. Lane 1: 100 bp DNA molecular marker; Lane 2: 10 μg DNA; Lane 3: 1 μg DNA; Lane 4: 100 ng DNA; Lane 5: 10 ng DNA.

3.3. Brucella detection

To test this detection method for the screening of sheep for the presence of Brucella infection, several organ tissues from healthy and infected sheep were collected from the abattoir. PCR was performed using DNA isolated from organ tissues. Primer pair KW3 and KW4 was used in this experiment. The results of the screening are presented in Fig. 5 and Table 1. The PCR based detection method was successfully used with 3 types of test tissues (Fig. 5). A 700 bp amplicon was observed in the PCR reaction performed with DNA isolated from animal tissues that were suspected to be positive for Brucella infection. Lymph tissue exhibited the highest level of detection 78%, however the lowest detection percent was in the liver tissue 11%.

Figure 5.

Figure 5

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Amplification of Brucella specific DNA fragment (B1-700) using primer pair KW3/KW4 and total DNA isolated from various organs of animals. Lane 1: 100 bp DNA molecular marker; Lane 2–7: DNA isolated from kidney; Lane 8–15: DNA isolated from lymph; Lane 16–21: DNA isolated from liver; Lane 22: DNA isolated from Brucella (positive control).

Table 1.

Detection of Brucella by PCR analysis of DNA isolated from organ tissues of infected sheep using primers KW3 and KW4.

Infected animal number Total
PCR results
93 456 335 195 437 412 417 402 476
Infected tissues
Liver − − − − + na na − na 1/9 (11%)
Kidney − + − + + na na − na 3/9 (33%)
Lymph + + na + + + + + − 7/9 (78%)
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na = not available.

4. Discussion

Brucellosis still remains as one of the major zoonotic diseases in the developing countries. Early detection is important for the effective control of the disease. In Kuwait extensive vaccination is encouraged to reduce losses due to Brucellosis related abortions and mortality. Although, antibody based methods are routinely used for the diagnosis of Brucellosis, the methods are of low specificity due to the presence of identical antigens (lipopolysaccharide) in various other gram negative bacterial pathogens (Asif et al., 2009). Another disadvantage of serological test is that, even after the recovery period the infected individual will show positive reaction in serological testes (Ariza et al., 1992). Growing number of scientific literature suggest that, development of PCR based detection methods would greatly improve the efficiency of detection (Gemechu et al., 2011; Maas et al., 2007; Al-Attas et al., 2000; Keid et al., 2007; Mitka et al., 2007; Bricker 2002; Navarro et al., 1999; Asif et al., 2009).

To develop an efficient PCR based detection method, a number of random primers have been tested to select a primer that can produce consistent amplification of Brucella specific DNA fragment (AlMomin et al., 1998). The DIG-labeled 1.3 kb DNA fragment was able to detect nano-gram scale of genomic DNA from B. abortus biotype 1, B. abortus (strain S-19), B. melitensis biotype 1 and B. melitensis RebI (AlMomin et al., 1999). Interestingly, no cross reactivity signals were observed for DNA samples of bacterial species naturally occurring in sheeps other than Brucella species such as R. stolonifer, E. coli HB101, K. pneumoniae, L. monocytogenes, O. anthropi, P. aeruginosa, Salmonella choleraesuis, Serratia marcescens and Y. enterocolitica (AlMomin et al., 1999). These results indicate that the sequence of 1.3 kb DNA fragment isolated from Brucella sp. can be used for PCR based detection.

PCR reactions using the primers reported in this study demonstrate its utility in the detection of Brucella DNA isolated from the tissues of infected sheep. The study also demonstrates PCR based detection of Brucella from the samples of milk and blood spiked with known amount of the pathogen DNA.

Sequencing of the Brucella specific DNA fragment revealed some interesting information. Based on the deduced amino acid sequence analysis, the isolated fragment belongs to d-alanine–d-alanine ligase gene. The sequence had no homology to published sequences reported from non-Brucella species at the nucleotide level. This could be one of the reasons for the high specificity of the PCR. Some degree of homology can be noticed only at the deduced amino acid level to d-alanine–d-alanine ligases of other bacterial species. Hence this PCR based method can be utilized effectively in the detection of Brucellosis.

Degree of sensitivity of PCR based method is a key issue for its effective use in the diagnosis of Brucellosis (Mukherjee et al., 2007). Internal primers were designed for use in the routine detection of Brucella infection. The results described in this paper, clearly show that KW3/KW4 produces consistent results in the detection of Brucella. Furthermore, experiments on milk and blood samples spiked with Brucella DNA, clearly show a high sensitivity level of detection method.

5. Conclusion

In conclusion the application of the developed method at the field level, for the screening of the disease in animal tissues, demonstrate its utility in the detection of the disease. Furthermore, in comparison with the culture based methods, PCR based method of detection is more safe to the staff involved in the diagnosis. Efforts are under progress on the development of diagnostic kits based on the sequence and primers described in this report.

Figure 4.

Figure 4

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Amplification of Brucella specific DNA fragment (B1-700) using primer pair KW3/KW4 and total DNA isolated from blood spiked with Brucella DNA. Lane 1: 100 bp DNA molecular marker; Lane 2: 10 μg DNA; Lane 3: 1 μg DNA; Lane 4: 100 ng DNA; Lane 5: 10 ng DNA; Lane 6: 1 ng DNA.

Acknowledgements

The authors are grateful to the Kuwait Foundation for the Advancement of Sciences (KFAS) for the partial funding of the project (FB013C). The support of the Animal department-Public Authority of Agriculture and Fisheries (PAAF) is gratefully acknowledged.

Footnotes

Peer review under responsibility of King Saud University.

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