A Nested-Polymerase Chain Reaction Assay to ldentify and Genotype Brucella＊

TIAN Guo Zhong

Brucellais gram-negative, facultative,intracellular bacteria implicated in infectious zoonosis diseases, particularly among domestic animals, that are also transmittable to humans. The genusBrucellais 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 ofBrucella. 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 ofBrucella[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 ofBrucellaspp . that also developed a nested-polymerase chain reaction (PCR)assay for the identification and genotyping ofBrucella, especially for those contained in blood specimens.

A total of 36 reference strains (B. abortusbiovars[1-7],B. melitensisbiovars[1-3],B. suisbiovars[1-5],B. canis;B. ovis;B. neotomae,B. pinnipedialis, andB.microti) were used. All strains ofB. abortus,B.melitensis,B. suis,B. canis;B. ovis; andB. neotomaewere preserved in Brucellosis Laboratory, CDC,China. The information on the DNA ofB. ceti,B.microti, andB. pinnipedialisstrains were sourced from the US National Center for Biotechnology Information (NCBI) website. TheRev1, M5,andM28belonged to theB. melitensisvaccine strains;104MandS19to theB. abortusvaccine strains;VBI22andVacciS2to theB. suisvaccine strains; and45/20andB1119to theB. abortusrough 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, noBrucellaspecies was isolated. The B243, B251, and B252 samples were collected from the sera of sheep from a farm. TheB. melitensisbiovars 3 strains was isolated from B252 sample.B. canisstrain was isolated from BJ10 samples collected from anticoagulant whole blood samples from dogs at the farm. The samples were positive forBrucellaantibodies (Table 1)[5].

A total of 7 non-BrucellaDNA were used to verify the specificity of the nested-PCR assay, which includedBacillus cereus,Bacillus anthracis,Escherichia colio:157,Salmonella,Pseudomonas aeruginosa,Yersinia enterocoliticao:9, andVibrio cholerae.E. colio: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 ofBrucellaspp. from the NCBI databases revealed that the nucleotide sequences in transposase IS711orfAandorfBofBrucellaspp.possess nucleotide polymorphisms and containBrucellaspecies and biovars specificity. The oligonucleotide primers were designed based onorfAandorfB(Figure 1). A nested-PCR assay was established to identify and genotype the strains ofBrucellaspp. 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.

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

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 °C for 4 min; 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 60 s, 30 cycles. The final extension was performed at 72 °C 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 ofBrucellaspp. by using the nested-PCR assay. A total of 7 non-BrucellaDNA 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 ofB. suis1330 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 ofBrucellaDNA in a 25-μL reaction system, considering that 24-fg nucleic acid DNA equals to approximately 7 copies ofBrucellaDNA[6].

A total of 36Brucellareference 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 1. The nucleotide polymorphisms in transposase IS711 orfA and orfB of Brucella spp.* and vertical bar with a blue background: the base mutation site.

1) Three strains isolated from marine animals were assigned to 1 group, which includedB. ceti10759,B. ceti 28753, andB. pinnipedialisB2-94.

2) AllB. melitensisbiovar 2 andB. melitensisbiovar 3 strains were clustered together.

3) AllB. abortusbiovar 1 strains were clustered together, along with 1B. neotomae5K33 strain.

4) TheB. ovis63/290 andB. ovis25840 strains were clustered together, along with 1B. abortusB1119 rough strains.

5) AllB. suisbiovar 3 strains were clustered together, along with 1B. microti4915 strain.

6) A total of 6B. canisstrains were clustered into 2 groups, 1 group included 4B. canisstrains, while another included 2B. canisstrains along with 3B.suisbiovar 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 wasB. abortusbiovar 1, B252 wasB. melitensisbiovar 3, B243 wasB. suisbiovar 3, BJ10 wasB. canis, and Lanzhou-26 wasB. abortusbiovar 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 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.

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 ofBrucellaDNA[7].Therefore, there is an urgent need to develop a simple and sensitive method for the identification ofBrucella, 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 ofBrucellaDNA.Meanwhile, the species and biovars of infectiousBrucellacould be identified and typed. Until date,there exists no method that can completely match the biological typing forBrucella.owing to the high consistency ofBrucellaDNA, 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 pathogenicBrucella, includingB. abortus,B.melitensis,B. suis, andB. canis. The results of the present study revealed that nested-PCR can be useful for the detection and typing ofBrucellaDNA,including that of clinical strains and blood specimens.

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.

ACKNOWLEDGMENTS

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

#Correspondence should be addressed to TIAN Guo Zhong, male, born in 1965, ME, majoring in the pathogen of brucellosis, Tel: 86-10-58900767, E-mail: tianguozhong@icdc.cn

Received: May 25, 2020;

Accepted: October 10, 2020