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TIAN Guo Zhong. A Nested-Polymerase Chain Reaction Assay to Identify and Genotype Brucella[J]. Biomedical and Environmental Sciences, 2021, 34(3): 227-231. doi: 10.3967/bes2021.028
Citation: TIAN Guo Zhong. A Nested-Polymerase Chain Reaction Assay to Identify and Genotype Brucella[J]. Biomedical and Environmental Sciences, 2021, 34(3): 227-231. doi: 10.3967/bes2021.028

A Nested-Polymerase Chain Reaction Assay to Identify and Genotype Brucella

doi: 10.3967/bes2021.028
Funds:  This work was supported by the National Key R&D Program of China [Grant 2017YFC1200403], National Science and Technology Major Project [2017ZX10303401003]
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  • Corresponding author: TIAN Guo Zhong, male, born in 1965, ME, majoring in the pathogen of brucellosis, Tel: 86-10-58900767, E-mail: tianguozhong@icdc.cn
  • Received Date: 2020-05-25
  • Accepted Date: 2020-10-10
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  • [1] Franco MP, Mulder M, Gilman RH, et al. Human brucellosis. Lancet Infect Dis, 2007; 7, 775−86. doi:  10.1016/S1473-3099(07)70286-4
    [2] Araj GF. Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents, 2010; 36, 12−7. doi:  10.1016/j.ijantimicag.2010.06.014
    [3] Bauer S, Aubert AC, Richli M, et al. Blood cultures in the evaluation of uncomplicated cellulitis. Eur J Intern Med, 2016; 36, 50−6. doi:  10.1016/j.ejim.2016.07.029
    [4] Pabuccuoglu O, Ecemis T, El S, et al. Evaluation of serological tests for diagnosis of brucellosis. Jpn J Infect Dis, 2011; 64, 272−6.
    [5] Tian GZ, Zheng M, Cui BY, et al. Analysis of pathogens and genetic characteristics of Brucella canis. Chin Endemiol, 2016; 35, 707−12.
    [6] Sidor IF, Dunn JL, Tsongalis GJ, et al. A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals. J Vet Diagn Invest, 2013; 25, 72−81. doi:  10.1177/1040638712470945
    [7] Baily GG, Krahn JB, Drasar BS, et al. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg, 1992; 95, 271−5.
    [8] Tian GZ, Cui BY, Piao DR, et al. Multi-locus variable-number tandem repeat analysis of Chinese Brucella strains isolated from 1953 to 2013. Infect Dis Poverty, 2017; 6, 89. doi:  10.1186/s40249-017-0296-0
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A Nested-Polymerase Chain Reaction Assay to Identify and Genotype Brucella

doi: 10.3967/bes2021.028
Funds:  This work was supported by the National Key R&D Program of China [Grant 2017YFC1200403], National Science and Technology Major Project [2017ZX10303401003]
    Corresponding author: TIAN Guo Zhong, male, born in 1965, ME, majoring in the pathogen of brucellosis, Tel: 86-10-58900767, E-mail: tianguozhong@icdc.cn
TIAN Guo Zhong. A Nested-Polymerase Chain Reaction Assay to Identify and Genotype Brucella[J]. Biomedical and Environmental Sciences, 2021, 34(3): 227-231. doi: 10.3967/bes2021.028
Citation: TIAN Guo Zhong. A Nested-Polymerase Chain Reaction Assay to Identify and Genotype Brucella[J]. Biomedical and Environmental Sciences, 2021, 34(3): 227-231. doi: 10.3967/bes2021.028
  • 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].

    NO.Strain IDBiovarsNote NO.Strain DBiovarsNoteNO.Strain DBiovarsNote NO. Strain DBiovarsNote
    1A13334A1REF3463/290OvisREF6713106M3Isolate10078037M2Isolate
    22308A1REF3525840OvisREF6813107M3Isolate10179029M3Isolate
    39-941A1REF36B2-94PinniREF6913108M3Isolate10279054M3Isolate
    486/8/59A2REF37208S1Isolate7013138S1Isolate10379077M3Isolate
    5TulyaA3REF38226S3Isolate7113139S2Isolate10479104M3Isolate
    6292A4REF391048A3Isolate7213147S1Isolate10579130M3Isolate
    7B3196A5REF401057A1Isolate7313160A1Isolate10680345M3Isolate
    8870A6REF411148A3Isolate7413195M3Isolate10780347M3Isolate
    963/75A7REF423001M3Isolate7513248M2Isolate10880355M2Isolate
    10C86A9REF435017M3Isolate7613331S2Isolate10980356M2Isolate
    11104MAVacci447033M3Isolate7713332S1Isolate11080359A3Isolate
    12S19AVacci458257A1Isolate7813341M3Isolate11180392A9Isolate
    1345/20AREF468416A9Isolate7923385CanisIsolate11281006M3Isolate
    14B1119AREF479057S3Isolate8052141CanisIsolate11381011A7Isolate
    1516MM1REF4810009S3Isolate8157002A3Isolate11481033M3Isolate
    1663/9M2REF4910036CanisIsolate8260033A1Isolate11584001M3Isolate
    17EherM3REF5010062S3Isolate8361005M3Isolate11686028M1Isolate
    18NIMREF5111038CanisIsolate8463006M2Isolate11786028S1Isolate
    19M5MVacci5211055A3Isolate8563031A1Isolate11888040S1Isolate
    20M28MVacci5311059M3Isolate8664008S3Isolate11990001A3Isolate
    21Rev1MVacci5412088M3Isolate8764027M2Isolate12092008M3Isolate
    221330S1REF5512099M3Isolate8864029A1Isolate121B257A1Isolate
    23ThomsenS2REF5612110M3Isolate8965050M2Isolate122HN-13M1Isolate
    24686S3REF5713031M3Isolate9065066M2Isolate123HNLGM3Isolate
    2540S4REF5813034M3Isolate9165079A1Isolate124HNLRM3Isolate
    26513S5REF5913051M3Isolate9266148M2Isolate125HNMBM3Isolate
    27VacciS2SVacci6013067M3Isolate9372003M2Isolate126B114/Blood
    28VBI22SVacci6113080M3Isolate9473011M3Isolate127B115A1Blood
    29RM6/66CanisREF6213081M3Isolate9573096S3Isolate128BJ10CanisBlood
    3010759CetiREF6313083M3Isolate9673099M3Isolate129Lanzhou-26A1Blood
    3128753CetiREF6413096S3Isolate9773101M3Isolate130B243S3Serum
    324915MicroREF6513102M3Isolate9873162M3Isolate131B251/Serum
    335K33NeotoREF6613105M3Isolate9978014M3Isolate132B252M3Serum
      Note. A: B. abortus biovar, M: B. melitensis biovar, S: B. suis biovar, Canis: B. canis, Ceti: B. ceti, Pinni: B. pinnipedialis, Neoto: B. neotomae, Ovis: B. ovis, Micro: B. microti, REF: Reference strain, Isolate: Clinical isolate. /: Brucella was not detected in the blood samples by culturing or nested-PCR assay. REF, reference.

    Table 1.  The 36 reference strains, 89 clinical isolates, and 7 blood and sera samples used in this study

    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.

  • The author are sincerely grateful to all participants in this study.

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