Brucella is gram-negative, facultative, intracellular bacteria implicated in infectious zoonosis diseases, particularly among domestic animals, that are also transmittable to humans. The genus Brucella is classified based on the primary host preferences, pathogenicity, host preference, and phenotypic characteristics of its species^[1]. The clinical features of brucellosis overlap with those of an extensive range of infectious and non-infectious diseases; therefore, laboratory testing is deemed the most reliable approach to diagnose this infection^[2]. Microbiological culturing and serological examinations are the most common methods for the detection of Brucella. Although the isolation of these bacteria is the ‘gold standard’ approach, the microbial culture often gives false negative results and dependent on the culture medium, the quantity of the circulating bacteria, and the species of Brucella^[3]. Therefore, serological tests such as the standard serum agglutination test (SAT) seem to be more effective for diagnosis, although an occasional case of cross-reaction or false-positive reaction in the samples from areas with subclinical prevalence of brucellosis has been promoted^[4]. The present study is a comprehensive analyses of the genomic nucleotide sequences of Brucella spp. that also developed a nested-polymerase chain reaction (PCR) assay for the identification and genotyping of Brucella, especially for those contained in blood specimens.

A total of 36 reference strains (B. abortus biovars^[1-7], B. melitensis biovars^[1-3], B. suis biovars^[1-5], B. canis; B. ovis; B. neotomae, B. pinnipedialis, and B. microti) were used. All strains of B. abortus, B. melitensis, B. suis, B. canis; B. ovis; and B. neotomae were preserved in Brucellosis Laboratory, CDC, China. The information on the DNA of B. ceti, B. microti, and B. pinnipedialis strains were sourced from the US National Center for Biotechnology Information (NCBI) website. The Rev1, M5, and M28 belonged to the B. melitensis vaccine strains; 104M and S19 to the B. abortus vaccine strains; VBI22 and VacciS2 to the B. suis vaccine strains; and 45/20 and B1119 to the B. abortus rough strains.

A total of 89 clinical strains isolated from the patient's blood samples were typed through the conventional biological methods. A total of 7 blood and sera samples were collected from the patients and sheep. The B114, B115, and lanzhou-26 were clinical human anticoagulant whole blood samples. The serum antibody titer was > 1:100. However, no Brucella species was isolated. The B243, B251, and B252 samples were collected from the sera of sheep from a farm. The B. melitensis biovars 3 strains was isolated from B252 sample. B. canis strain was isolated from BJ10 samples collected from anticoagulant whole blood samples from dogs at the farm. The samples were positive for Brucella antibodies (Table 1)^[5].

A total of 7 non-Brucella DNA were used to verify the specificity of the nested-PCR assay, which included Bacillus cereus, Bacillus anthracis, Escherichia coli o:157, Salmonella, Pseudomonas aeruginosa, Yersinia enterocolitica o:9, and Vibrio cholerae. E. coli o:157 strain was treated as a negative quality control in this study.

The Bacterial Genomic DNA Extraction Kit (Spin column; Tiangen Biotechnology [Beijing] Co., Ltd, China) was used to extract the nucleic acid DNA from the reference strains and the clinical isolates. Blood and serum samples were extracted by using this kit.

Comparative analysis of the whole genome sequences of Brucella spp. from the NCBI databases revealed that the nucleotide sequences in transposase IS711 orfA and orfB of Brucella spp. possess nucleotide polymorphisms and contain Brucella species and biovars specificity. The oligonucleotide primers were designed based on orfA and orfB (Figure 1). A nested-PCR assay was established to identify and genotype the strains of Brucella spp. The primers used included F1: 5′-AACGTAACCATACATAGCGCATG-3′ and R1: 5′-ACAGATGAGCAATGGAACCGGAT-3′ and F2: 5′-GAATGGGTGCAATTTCTCGC-3′ and R2: 5′-ATATCTTCCGGGGCGAGTTGGTA-3′. The first PCR was conducted with the primer pairs F1 and R1 using the extracted DNA as the template. The second PCR was conducted with the primer pairs F2 and R2 using the first PCR amplicons as the template.

Figure 1.  The nucleotide polymorphisms in transposase IS711 orfA and orfB of Brucella spp.* and vertical bar with a blue background: the base mutation site.

The first PCR reaction system included 2×Taq mastermix (12.5 μL, primer F1, and primer R1 [10 μmol/L]; Kangwei Century Biotechnology Co., Ltd, China) 0.6 μL each and DNA template 2 μL, to which 9.3–25.0 μL of distilled water was added. The first PCR reaction conditions were as follows: 94 ℃ for 4 min; 94 ℃ for 45 s, 55 ℃ for 45 s, and 72 ℃ for 60 s, 30 cycles. The final extension was performed at 72 ℃ for 5 min. The second PCR reaction system and reaction conditions were the same as for the first PCR system, except for the template DNA from the first PCR amplicons (nested-PCR). A bright electrophoresis band of approximately 666 bp was amplified for the strains of Brucella spp. by using the nested-PCR assay. A total of 7 non-Brucella DNA could not be amplified by nested-PCR assay. The nested-PCR amplicons were purified and sequenced. Multiple sequences alignments were performed using the Clustal W, and a schematic representation of each locus was generated using the MEGA 5.1 by using the unweighted group average method (UPGMA), Neighbor-Joining tree construction, and Tamura-Nei algorithm with 1,000 bootstraps.

The sensitivity of the nested-PCR assay was determined by using 36 reference strains. All strains could amplify the positive bands. The minimum detection limit was tested by using 2-fold decreasing dilutions of B. suis 1330 DNA. The initial DNA (56.2 ng/μL) was diluted to 0.03 fg/μL. After several testing, the sensitivity of the nested-PCR was 3.35 fg, which was equivalent to 1 copy number of Brucella DNA in a 25-μL reaction system, considering that 24-fg nucleic acid DNA equals to approximately 7 copies of Brucella DNA^[6].

A total of 36 Brucella reference strains, 89 clinical isolates, and 7 blood samples were examined by nested-PCR assay. The nested-PCR products were sequenced, and the sequences were clustered (Figure 2). The cluster analysis was performed as follows:

Figure 2.  The cluster analysis of 36 Brucella reference strains, 89 clinical isolates, and 7 blood samples by nested-PCR. The black background of the code numbers indicates blood samples (B115, B243, B252, BJ10, and Lanzhou-26) in Figure 1.

1) Three strains isolated from marine animals were assigned to 1 group, which included B. ceti 10759, B. ceti 28753, and B. pinnipedialis B2–94.

2) All B. melitensis biovar 2 and B. melitensis biovar 3 strains were clustered together.

3) All B. abortus biovar 1 strains were clustered together, along with 1 B. neotomae 5K33 strain.

4) The B. ovis 63/290 and B. ovis 25840 strains were clustered together, along with 1 B. abortus B1119 rough strains.

5) All B. suis biovar 3 strains were clustered together, along with 1 B. microti 4915 strain.

6) A total of 6 B. canis strains were clustered into 2 groups, 1 group included 4 B. canis strains, while another included 2 B. canis strains along with 3 B. suis biovar 2 strains.

7) The 7 blood samples could not be amplified with a single primer pairs (F1 & R1 or F2 & R2). However, 5 blood samples showed bright electrophoresis bands through the nested-PCR assay with the primer pairs (F1 & R1 → F2 & R2). The cluster analysis revealed that B115 was B. abortus biovar 1, B252 was B. melitensis biovar 3, B243 was B. suis biovar 3, BJ10 was B. canis, and Lanzhou-26 was B. abortus biovar 1 (Figure 3). All 7 blood samples could not be amplified with a single primer B4 and B5^[7] and could not be detected by fluorescent quantitative-PCR^[8].

Figure 3.  The nested-PCR electrophoresis of 7 extracted DNA from blood samples. The B114, B115, B243, B251, B252, BJ10, and Lanzhou-26 were the tested blood and serum samples. 1330 was Brucella suis biovars 1 strain. F1 & R1: PCR products with primers F1 and R1. F2 & R2: PCR products with primers F2 and R2. F1 & R→F2 & R2: nested-PCR products with primers F1 and R1 first and F2 and R2 second.

Recent studies have shown that PCR, either individually or multiplex reactions, can be used routinely to detect DNA from pure bacteria as they are rapid, highly specific, and sensitive. The PCR assay showed the highest sensitivity with the primer pairs B4 and B5^[6], as it detected ≥ 500 CFU^[7]. Due to the effects of inherent inhibitory factors in the blood and other tissues, real-time PCR assay was not deemed suitable for the detection of Brucella DNA^[7]. Therefore, there is an urgent need to develop a simple and sensitive method for the identification of Brucella, especially for pathogenic bacteria in the blood and other tissue samples.

In this study, nested-PCR assay was unaffected by the inhibitory factors, and its detection sensitivity reached one copy number of Brucella DNA. Meanwhile, the species and biovars of infectious Brucella could be identified and typed. Until date, there exists no method that can completely match the biological typing for Brucella. owing to the high consistency of Brucella DNA, such as the recently proposed and widely used multiple locus variable number of tandem repeats (MLVA) analysis^[8]. Fortunately, nested-PCR assay can classify human pathogenic Brucella, including B. abortus, B. melitensis, B. suis, and B. canis. The results of the present study revealed that nested-PCR can be useful for the detection and typing of Brucella DNA, including that of clinical strains and blood specimens.