A New Speci c Sequence to Distinguish B.canis From Other Brucella by PCR

YinBo YE Shenyang Agricultural University JiangHua YANG Shenyang Agricultural University DongLiang LI Beijing Animal Disease Prevention & Control Center LiHua HAO Division of the Standards, China Institute of Veterinary Drug Control Zhao ZHANG Shenyang Agricultural University SiYao MEI Shenyang Agricultural University Huan ZHANG Shenyang Agricultural University FangYuan DU Shenyang Agricultural University BaoShan LIU Shenyang Agricultural University ZeLiang CHEN (  zeliangchen@yahoo.com ) Shenyang Agricultural University


Introduction
The genus Brucella is one kind of the most widespread bacteria, inducing Brucellosis. They have been gradually expanded by discovering strains from wildlife animal species, such as amphibians and sh [1]. Four major pathogenic Brucella species causing disease in humans are B.abortus (cattle, buffalo), B.melitensis (goats, sheep, camels), B.suis (pigs), and B.canis (Dogs) [2]. Annually more than 500,000 new brucellosis cases are diagnosed worldwide [3].
As the primary pathogen of brucellosis in dogs, B.canis can be transmitted to humans by infected dogs or their secretions. Unlike the infection by other Brucella species, the infection symptoms with B.canis are absent or mild [4]. However, endocarditis or meningitis may develop in some cases [5]. So, the human infection of Brucella canine remains a concern, especially as pets are being raised in large numbers.
The isolation and culture are the most accurate method for brucellosis detection and species identi cation by amino sugar quinovosamine assay [6]. However, they are time-consuming and need to be performed in a laboratory with biosafety level III. Its effectiveness is affected by the infected animal's bacteremia level [7]. Molecular biological detection technology has the characteristics of safety and reliability, high sensitivity, strong speci city and simple operation, and has begun to be popularized and applied to detect Brucella [8,9]. Now there were a variety of PCR methods for detecting canine Brucella [10,11], such as bruce-ladder [11] and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures [12]. Most of them distinguished B.canis by multiple ampli cation products, which required a lot of time to remember each Brucella species' ampli ed bands. More to the point, it needed high quality and concentration genomic DNA, limiting the choice of clinical specimens [13].
In 2014, a PCR method for only detecting B.canis was established based on the 12bp deletion of BCAN_B0548 region 530056 site in chromosome II of B.canis ATCC 23365 [13]. However, it cannot detect other Brucella species and omit probably the brucella infection by other Brucella species [14].
In this study, we found a speci c sequence of B.canis by comparing the genomes of B.canis and B.melitensis and BLASTing in the GeneBank, which is reverse complementary in other Brucella species. PCR of three primers designed based on this sequence can speci cally detect canine Brucella and other Brucella, which simpli ed the clinical identi cation and diagnosis of canine brucellosis and is very conducive to its prevention and control to reduce human infection.

Strains and DNA extraction
All the strains used in this experiment were listed in Table 1 Other common bacterial strains are preserved in this laboratory. Their genetic DNA was extracted with MiniBEST Bacteria Genomic DNA Extraction Kit (Takara, Dalian, China) According to the manufacturer's instructions and measured with an ultraviolet spectrophotometer. The copies of the genome were counted online on the website http://scienceprimer.com/copy-number-calculator-for-realtime-pcr. The extracted DNA was stored at -20°C until use. 86 Cannie blood samples were collected in the pet hospitals in Shenyang. Their DNA was extracted with the MiniBEST Whole Blood Genomic DNA Extraction Kit (Takara, Dalian, China) and stored at -20°C. Taq PCR MasterMix was purchased from Vazyme Biotech Co., Ltd, Nanjing, China.
Screening of the speci c sequence of B.canis Genome sequences of B.canis and other species were downloaded from the NCBI database. Sequences were analyzed by the multiple genome alignment software Mauve 20150226 (The Darling lab at the University of Technology Sydney). The list of gaps and single nucleotide polymorphisms (SNPs) of alignment results of chromosomes 1 and 2 were exported. BLAST was used to compare the candidate gaps of the two strains for the speci c differential sequences of the B.canis. The potential sequences in both genomes of B.canis and B.melitensis were aligned by the software DNAMAN 7.
primer design Primer-blast on the NCBI website was used to design the primers and make the appropriate adjustments to obtain speci c primers. The designed primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China).

Optimization of the PCR Ampli cation condition
In the PCR reaction, 2×Taq PCR MasterMix ten μL, each primer (10 μM) 1 μL and ve μL water were mixed in the 200 μL PCR tube. Then one μL Brucella DNA template or one μL distilled water were added as templates or negative control. The PCR ampli cation conditions were as follows: pre-denaturation at 94°C for 5 minutes, then 35 cycles at 95 °C for 15 s, 60 °C for 15 s and 72 °C for 30 s, followed by a nal extension at 72 °C for 10 min. The PCR ampli cation products were identi ed by electrophoresis in 1.5% agarose gel.
For determining better ampli cation conditions, PCR reaction was optimized by annealing in temperature 60°C, 64°C, 68°C, 72°C. The optimum annealing temperature was determined according to the stray band's existence and the ampli ed band's brightness.
Speci city and sensitivity of the assay The DNA template was diluted from 10 4 to 1 copies/μL with sterilized distilled water. One μL diluted DNA was used as the template in PCR ampli cation.
Electrophoresis was performed to determine the sensitivity of the PCR assay.
For verifying the PCR assay's speci city, DNAs of other Brucella strains and bacteria and vaccine in Table 1 were used as templates for PCR ampli cation under the best annealing temperature. The ampli cation products were analyzed in a 1.5% electrophoresis gel and observed under ultraviolet light.

Detection and veri cation
For evaluating the clinical e cacy of the established PCR method, 66 DNAs from B.canis-negative blood samples were spiked double-blindly with the DNA of B.canis and other Brucella were tested. The number of samples spiked with positive brucella DNAs was listed in Table 3. The spiked samples were detected by the developed PCR and the results were analyzed by 1.5% electrophoresis gel. BLAST was used to compare the candidate gaps of the two strains for the speci c differential sequences of the B.canis. It was found that a 132bp gap at 943403rd site on B.canis chromosome 1 corresponds to the same gap at 303367th site on B.melitensis chromosome 1 ( Fig.2A). By aligning, the gap sequence in both genomes of B.canis and B.melitensis has a very low similarity (Fig.2B). Further analysis found that they are reverse complementary sequences (Fig.2C). The gap and adjacent sequence's alignment showed good speci city in BLAST, only having a high similarity and high score with B.canis (Fig.2D). It was suggested that this sequence is a characteristic of B.canis and can be potential for identifying B.canis.

Primer design
Three primers (Table 2) in the region 943226-943581 of chromosome I of B.canis strain RM6/66 were designed by the online primers design programs Primer-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The position of each primer was shown in gure 2C. PCR with these three primers should amplify 310bp and 413bp fragments using B.canis and other Brucella species as templates, respectively.

Optimization of the PCR Ampli cation condition
For determining better ampli cation, annealing temperature in PCR was optimized. The results showed that evident PCR bands could be seen at annealing temperature 60 ℃ to 68 ℃ (Fig.3A). However, the ampli cation band at 64 ℃ was the brightest and the ampli cation effect was the highest. So, 64 ℃ was used as the reaction temperature in the subsequent speci city and sensitivity tests.
The sensitivity of the assay For determining the sensitivity of the PCR method, it was executed using the gradient dilution DNA of B.canis strain RM6/66 as a template. The results showed that the reaction solution with 10 4 -10 2 copies of DNA showed PCR ampli cation bands in the electrophoresis (Fig. 3B), indicating that the established PCR assay could be detected a minimum of 100 copies of Brucella DNA.
The speci city of the assay Respectively using DNA of the B.canis strain RM6/66, strain A19, B.suis strain S2, B.melitensis strain 16M, B.abortus strain 2308, B.suis strain 1330, Vanguard® Plus 5-CVL vaccine, Fel-O-Vax® PCT vaccine (Zoetis, USA) and other bacterial as a template, PCR assay was conducted with best annealing temperature 64℃. The results show that the reaction product with the B.canis DNA and other brucella DNA existed a speci c 310bp and 413bp band in the electrophoresis, respectively (Fig. 3C). At the same time, the reaction products with other DNA are no speci c bands. It showed that the established PCR detection method had excellent speci city. Meantime, it can not only detect Brucella but also distinguish B.canis from other Brucella species.

Detection and veri cation
For verifying the e cacy of the PCR assay, 66 DNAs of clinical blood samples spiked randomly with the DNA of B.canis or other Brucella were tested (Fig. 4). The result showed that the developed assay detected 43 of the 44 spiked samples, with a detection accuracy of 95.5% (21/22) for B.canis and 100% (22/22) for other Brucella, respectively (Table 3). These results indicated that the developed assay had a good feature for the detection of B.canis or other Brucella.

Discussion
The current study aimed to develop a speci c PCR assay for detecting canine Brucellosis, Whether the pathogen was B.canis or any other Brucella. Firstly, a differential sequence of B.canis was found by genome comparison analysis and was analyzed by BLAST. Then a differential PCR method was established using speci c primers in the sequence and tested for clinical application. The developed PCR method had good speci city and sensitivity and can be used to detect canine brucellosis caused by various Brucella, which is conducive to the prevention and control of canine brucellosis and the protection of human safety.
With the development of sequencing, databases, and networking technologies, vast amounts of genomic and sequence data are now available at individual terminals. How to make good use of these data has become an essential aspect of improving research e ciency. Desktop or web bioinformatics software addresses this problem very well. Multiple Genome Alignment Software Mauve can quickly analyze rearrangements, insertions, deletions and changes between genome sequences and determine the differences between genes from a macro and micro perspective [15,16]. BLAST is a general alignment procedure that compares sequences similar to target sequences in Genebank databases to discover target sequences' speci city [17]. The combination of the two software can be used to screen target sequences. Primer-Blast is a procedure for Primer design and comparison, which can effectively analyze the speci city of primers [18]. The combined use of these software can effectively improve the speci city and success rate of PCR, which is widely used [19]. The same process was used in this study and satisfactory results were obtained. [9,20,21]. However, there were a few PCR methods for detecting canine Brucella [10,11]. Bruce-ladder assay is a common method [11], which distinguished B.canis by multiple ampli cation products. However, it required much time to remember each brucella species' ampli ed bands and high quality and concentration genomic DNA limiting clinical specimens' choice [13]. A PCR method for only detecting B.canis was established in 2014, which was based on the 12bp deletion of chromosome II of B.canis ATCC 23365 and had a detection limit of 3 × 10 3 colony-forming units (CFU) [13]. However, the canine brucellosis can be caused by other Brucella species [14], so their infection could be omitted by the PCR method. In this study, a B.canis speci c sequence was found, which showed reverse complementary in other Brucella. The established PCR based on the speci c sequence can detect B.canis and other Brucella species simultaneously without amplifying other common bacteria and viruses. Its detection limit was 100 copies of the B.canis genome, higher than the above PCR method.The PCR assay was evaluated with spiked DNA samples. Of 66 DNA samples, the accuracy was 97.5% (43/44). It might be a low concentration that one sample was identi ed as a false negative. This work could give a more convenient veri cation for PCR method without comparative standard methods due to the spiked samples.

Molecular biological detection of Brucella is abundant
B.canis mostly cause canine brucellosis. However, other commonly exposed brucella strains, such as B.suis, B.abortus and B.melitensis, can also cause it [14]. Unlike B.canis, they have high pathogenicity to humans. People who contact with dogs infected these Brucella are at high risk. Therefore, it is necessary to strengthen the detection of brucellosis. The method established in this study will more comprehensively detect the pathogen of canine brucellosis and provide important methods and means for preventing and controlling the disease.
In conclusion, the study found a specif sequence of B.canis and developed a PCR detection method to detect canine brucellosis caused by B.canis or other Brucella species. It will serve for the prevention and control of canine brucellosis to reduce human brucellosis.

Declarations
Ethics approval and consent to participate The experiment was licensed by the Institutional Animal Care and Use Committee of Shenyang Agricultural University (IACUC Issue No.2020070307). Informed consent was obtained from owners for animal sample collection. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated and/or analysed during the current study are available in the NCBI database(https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP007758.