(HRM) Analysis

Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx

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Diagnostic Microbiology and Infectious Disease

journal homepage: www.elsevier.com/locate/diagmicrobio

Identiﬁcation of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis

Jama’ayah Mohamed Zahidi a,⁎,TayBeeYonga,RohaidahHashima, Azura Mohd Noor a, Siti Hawa Hamzah b, Norazah Ahmad a a Bacteriology Unit, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia b Microbiology Unit, Department of Pathology, Hospital Pulau Pinang, Jalan Residensi, 10990 Georgetown, Pulau Pinang, Malaysia article info abstract

Article history: Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Received 9 September 2014 Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was Received in revised form 10 December 2014 used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates Accepted 28 December 2014 from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Available online xxxx Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to Keywords: Brucellosis species level. Brucella © 2015 The Authors Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license Real-time PCR Brucella spp (http://creativecommons.org/licenses/by-nc-nd/4.0/). PCR in Brucella HRM analysis

1. Introduction The laboratory investigation for brucellosis is usually by isolation of the bacteria or detection of anti-Brucella antibodies. Although isolation Brucellosis is caused by Brucella spp. and is a common zoonotic in- of the bacteria is the “gold standard”, it requires long incubation periods fection. Brucella spp. are Gram-negative coccobacilli, nonmotile, and and is seldom successful (Jama’ayah et al., 2011; Rich et al., 2000; non–spore-forming; grow optimally at 37 °C with visible colonies Robichand et al., 2004). Since the identification and differentiation of after 24 h; and may require supplementary CO2 for their growth espe- Brucella are laborious and time consuming, molecular approaches cially on primary isolation (Corbel and Bannai, 2005). There are current- have been explored to overcome these problems. The conventional ly 6 classical species within the genus Brucella,namely,Brucella abortus polymerase chain reaction (PCR) and multiplex PCR typing are capable (preferred hosts are cattle and buffalo), Brucella melitensis (goat, sheep, of identifying Brucella up to species level. Primers B4/B5 based on the and camel), Brucella ovis (sheep), Brucella canis (dogs), Brucella suis gene encoding a 31-kDa B. abortus antigen (BCSP31) has been used for (swine), and Brucella neotomae (desert wood rat). Currently new de- the detection of Brucella in blood samples (Baddour and Alkhalifa, signed species are Brucella pinnipedialis, Brucella ceti, Brucella microti, 2008; De Santis et al., 2011). A multiplex PCR assay, AMOS PCR and Brucella inopinata (Foster et al., 2007; Scholz et al., 2008, 2009). (AMOS is acronym from “abortus, melitensis, ovis, suis”), was able to The main pathogenic species in both animals and human are identify B. abortus, B. melitensis, B. ovis,andB. suis including biovar B. melitensis, B. abortus, B. suis,andB. canis (Araj, 2010; Cloeckaert and level using a combination of different primers. This PCR assay had suc- Vizcaino, 2004). Brucella spp. associated with marine animals have cessfully identified B. abortus biovars 5, 6, and 9 and some field strains also been described (Foster et al., 2007; McDonald et al., 2006). The of biovar 3 B. abortus (Sasa et al., 2005). However, this method needs World Health Organization (WHO) reported an estimated 500,000 at least 5–6 h to perform and required postamplification in handling new cases of human brucellosis each year worldwide (Pappas et al., of PCR product. 2006). Brucella spp. are mostly transmitted to human by direct contact Winchell et al. (2010) developed a molecular technique utilizing with infectious animal tissue, inhalation of aerosolized droplets, or con- real-time PCR followed by high-resolution melt (HRM) curve analysis sumption of unpasteurized milk product (Corbel, 2006). to rapidly identify and discriminate members of Brucella genus. This assay had been successful in the identification and discrimination of B. suis and B. canis but was not able to differentiate B. suis biovar 4 from B. canis (Corbel, 2006; Winchell et al., 2010). In this study, we ⁎ Corresponding author. Tel.: +60-3-26162655; fax: +60-3-2691-9716. aimed to identify and discriminate Brucella spp. isolated from human E-mail address: [email protected] (J.’ Mohamed Zahidi). brucellosis in Malaysia using real-time PCR and HRM curve analysis. http://dx.doi.org/10.1016/j.diagmicrobio.2014.12.012 0732-8893/© 2015 The Authors Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

2. Methodology Genomic DNA was extracted from approximately 2 × 109 cells of the isolated using Qiagen DNAeasy Blood and Tissue kit. The DNA was eluted in 2.1. Bacterial strains and DNA preparation 50 μL in AE buffer (Tris-EDTA solution) and quantified using NanoDrop™ 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to ensure A total of 41 Brucella spp. isolates from blood culture of 41 patients purity and determine DNA concentration. Purity of all DNA isolates was mea- were obtained from various parts of Malaysia in this study (Table 1). sured at absorbance 260–280 nm, giving a ratio of ~1.8. The DNA was then The isolates were collected between 2009 and 2011 from the Northern stored at −80 °C until further use. Just before the assay, the DNA was diluted West of Peninsular Malaysia. Identification of all isolates was carried out to yield a concentration of 1.0 ng to keep the amount of template used consis- using standard microbiology procedures, namely, Gram stain, oxidase tent between samples. Well-characterized local strains of B. melitensis, and urease test, H2S production, and growth in the presence of basic thi- B. abortus, B. canis,andB. suis were used as control strains. These strains onine and fuchsin. The Agglutination with monospecific antisera for A were kindly supplied by the Veterinary Research Institute, Malaysia. and M antigens (Animal Health Veterinary Laboratory Agency, UK) were also included in identification of genus and species levels of the or- 2.2. Real time PCR and HRM analysis ganism Brucella (Table 1). In addition, 8 isolates of Gram-negative bacteria identified by conventional and commercial biochemical methods The 4 primer sets used for real-time PCR was as described by Winchell were used as negative controls in the HRM analysis. These included 1 et al. (2010). These primer sets were Bmel, (B. melitensis); Bcan, (B. canis); strain of each Escherichia coli 0157, Acinetobacter spp., Proteus mirabilis, Bsui, (B. suis); and Boa, (B. ovis and B. abortus)(Table 2). The real-time PCR Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella pneumoniae, mixture was prepared using Type-it® HRM kit (Qiagen, Hilden, Vibrio cholerae,andSalmonella spp. Germany) containing 12.5 μL of 2× HRM master mix, 0.7 μmol/L final con- All isolates were stored at −70 °C in 15% of glycerol with tryptic soy centration of the forward and reverse primer, 1.0 ng of DNA template, and broth until further testing. Prior to DNA extraction, 10 μL of glycerol nuclease free water (Qiagen) to a total volume of 25 μLperreaction.The stock culture was plated onto Brucella agar and incubated aerobically Type-it® HRM contains HotStarTaq Plus DNA Polymerase, Type-it HRM at 37 °C for 24–72 h. buffer, EvaGreen, Q-Solution, and dNTP mix. The real-time PCR was

Table 1 Brucella spp. isolates used in HRM assays (n = 41) and comparison of the results of phenotypic characteristic and HRM analysis.

Isolates Phenotypic characteristic result HRM result

Species Growth on dye (μg/mL) Serologic agglutination

H2S Urease Basic fuchsin 20 Thionine 20 Thionine 40 A M R BRC 3/09 B. canis − ++ + + −−+ B. canis BRC 4/10 B. melitensis − ++ + + ++− B. melitensis BRC 5/10 B. melitensis − ++ + + ++− B. melitensis BRC1/11 B. melitensis − ++ + + − + − B. melitensis BRC2/11 B. melitensis − ++ + + ++− B. melitensis BRC311 B. melitensis − ++ + + ++− B. melitensis BRC4/11 B. melitensis − ++ + + ++− B. melitensis BRC5/11 B. melitensis − ++ + + ++− B. melitensis BRC6/11 B. melitensis − ++ + + ++− B. melitensis BRC7/11 B. melitensis − ++ + + ++− B. melitensis BRC8/11 B. melitensis − ++ + + ++− B. melitensis BRC9/11 B. melitensis − ++ + + − + − B. melitensis BRC10/11 B. melitensis − ++ + + ++− B. melitensis BRC 11/11 B. melitensis − ++ + + ++− B. melitensis BRC 12/11 B. melitensis − ++ + + ++− B. melitensis BRC 13/11 B. melitensis − ++ + + ++− B. melitensis BRC 14/11 B. melitensis − ++ + + ++− B. melitensis BRC 15/11 B. melitensis − ++ + + ++− B. melitensis BRC 16/11 B. melitensis − ++ + + ++− B. melitensis BRC 17/11 B. melitensis − ++ + + ++− B. melitensis BRC 18/11 B. melitensis − ++ + + ++− B. melitensis BRC 19/11 B. melitensis − ++ + + ++− B. melitensis BRC 20/11 B. melitensis − ++ + + ++− B. melitensis BRC 21/11 B. melitensis − ++ + + ++− B. melitensis BRC 22/11 B. melitensis − ++ + + ++− B. melitensis BRC 23/11 B. melitensis − ++ + + +−−B. melitensis BRC 24/11 B. melitensis − ++ + + ++− B. melitensis BRC 25/11 B. melitensis − ++ + + +−−B. melitensis BRC 26/11 B. melitensis − ++ + + ++− B. melitensis BRC 27/11 B. melitensis − ++ + + ++− B. melitensis BRC 28/11 B. melitensis − ++ + + ++− B. melitensis BRC 29/11 B. melitensis − ++ + + +−−B. melitensis BRC 30/11 B. melitensis − ++ + + ++− B. melitensis BRC 31/11 B. melitensis − ++ + + +−−B. melitensis BRC 32/11 B. melitensis − ++ + + +−−B. melitensis BRC 33/11 B. melitensis − ++ + + ++− B. melitensis BRC 34/11 B. melitensis − ++ + + − + − B. melitensis BRC 36/11 B. melitensis − ++ + + +−−B. melitensis BRC 38/11 B. melitensis − ++ + + +−−B. melitensis BRC 39/11 B. melitensis − ++ + + ++− B. melitensis BRC 40/11 B. melitensis − ++ + + ++− B. melitensis

+, Positive. −, Negative.

Table 2 Real-time PCR primer sequence used for the detection and species identiﬁcation of Brucella spp.

Primer Sequence (5′-3′) Gene target Amplicon size

Bmel (B. melitensis) F-GAGCGATCTTTACACCCTTGT Int-hyp 125 R-GGACGGTGTAATAAACCCATTGG Bcan (B. canis) F-GCAACTACTCTGTTGACCCGA Int-hyp 88 R-TGCCGATCAGGCTGTGTTG Bsui (B. suis) F-TGCGCTATGATCTGGTTACGTT Transposase 69 R-AGCGCGGTTTTCTGAAGGT Boa (B. abortus and B. ovis) F-GACCTCTTCGCCACCTATCTGG glk 163 R-CCTTGTGCGGGGCCTTGTCCT

carried out using Corbett Rotor-Gene Q (Qiagen) with an initial denatur- 2.3. Specificity and sensitivity testing ation step of 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 55 °C for 30 s, with acquisition of data at 72 °C for 10 s in the green chan- Eight Gram-negative bacteria, namely, E. coli 0157, Acinetobacter nel (excitation at 470 nm and detection at 510 nm). After amplification, spp, P. mirabilis, P. aeruginosa, Y. enterocolitica, K. pneumoniae, HRMwasperformedbetween70°Cand95°Cattherateof0.1°C. V. cholerae,andSalmonella spp., were tested for the specificity of the The HRM curves of the primer sets were then normalized using 2 assay. Ten-fold serial dilutions were made from 1.0 ng DNA template normalized regions of the premelt phase (melting phase of the per Brucella isolate, and the dilutions were then subjected to real-time double-stranded DNA) and the postmelt phase (completion of separa- PCR using the 4 primer sets to determine the lower limit of detection tion of the 2-stranded DNA into single-stranded DNA). (LLOD). The assay was carried out in triplicates.

Fig. 1. (a) HRM melting profile for determination of Bsui primers specificity using Gram-negative isolates and (b) no amplification for Gram-negative isolates, but only B. suis control strain being amplified using Bsui primer. (c) A standard curve of B. melitensis performed in a linear graph with R2 0.995. (d) Normalized melt curve analysis in serial dilution of B. melitensis DNA (amplicon “1X” is equivalent to 100 ng).

Table 3 Sensitivity of 4 primers with mean ± SD of crossing point cycle and melting points for each strain.

Primer Strain LLOD (pg) Range of mean CT ±SD Range of melt peak (°C) ± SD Bmel B. melitensis 0.1 11.0 ± 0.25 to 34.2 ± 0.50 86.33 ± 0.04 Bcan B. canis 1.0 12.5 ± 0.20 to 33.0 ± 0.20 81.79 ± 0.06 Bsui B. suis 1.0 13.3 ± 0.30 to 30.4 ± 0.60 78.80 ± 0.01 Boa B. abortus and B. ovis 1.0 14.3 ± 0.09 to 29.7 ± 0.90 88.27 ± 0.19

3. Results testingofprimerBsuiwasshowninFig. 1a and b, which showed no amplification during real-time PCR assay with any of the bacteria. The LLOD of each the primer set for the de- 3.1. Specificity and sensitivity of the HRM assay tection of B. abortus, B. melitensis, B. canis, and B. suis was shown in Table 3. Four well- characterized strains (B. abortus, B. melitensis, B. canis, and B. suis) generated 1 melting For specificity, none of the 8 Gram-negative isolates were positive with the HRM anal- peak at 88.27 °C, 86.33 °C, 81.79 °C, and 78.80 °C, respectively. The LLOD for each primer ysis using the 4 primer sets of Brucella spp. An example of the HRM analysis for specificity set was 1.0 pg; however, the LLOD for B. melitensis was lower at 0.1 pg. This result was

Fig. 2. Specificidentification of Brucella spp. isolates in real-time PCR followed by HRM analysis. (a) Normalize graph of HRM assay and (b) melting profile for separation B. melitensis and other Brucella spp. using Bmel primer. (c) HRM curve and (d) melting profile of B. canis and BRC 3/09 isolate, which showed reduced the fluorescence in the presence of double stranded DNA. (e) Melting curve displaying B. suis only using Bsui primer. (f) HRM curve of B. abortus (green curve), which indicate an early melting curve compared to other Brucella spp. using Boa primer.

Table 4 Melting proﬁles for each Brucella spp. isolates in HRM analysis.

Isolates Melting peak Melting peak Melting peak Melting peak Isolates Melting peak Melting peak Melting peak Melting peak (°C) Bmela (°C) Bcanb (°C) Bsuic (°C) Boad (°C) Bmela (°C) Bcanb (°C) Bsuic (°C) Boad

BRC 3/09 86.67 81.55 0.00 88.17 BRC 21/11 86.45 82.10 0.00 88.20 BRC 4/10 86.45 82.12 0.00 88.10 BRC 22/11 86.48 82.02 0.00 88.18 BRC 5/10 86.42 82.13 0.00 88.12 BRC 23/11 86.40 82.05 0.00 88.15 BRC 1/11 86.42 82.10 0.00 88.15 BRC 24/11 86.35 82.05 0.00 88.18 BRC 2/11 86.40 82.10 0.00 88.15 BRC 25/11 86.45 82.07 0.00 88.17 BRC 3/11 86.40 82.13 0.00 88.17 BRC 26/11 86.40 82.05 0.00 88.20 BRC4/11 86.38 82.05 0.00 88.12 BRC 27/11 86.37 82.03 0.00 88.10 BRC 5/11 86.37 82.10 0.00 88.18 BRC 28/11 86.35 82.10 0.00 88.12 BRC 6/11 86.40 82.10 0.00 88.13 BRC 29/11 86.37 82.10 0.00 88.10 BRC 7/11 86.37 82.08 0.00 88.23 BRC 30/11 86.43 82.05 0.00 88.20 BRC 8/11 86.42 82.13 0.00 88.13 BRC 31/11 86.35 82.05 0.00 88.10 BRC 9/11 86.37 82.10 0.00 88.20 BRC 32/11 86.35 82.07 0.00 88.15 BRC 10/11 86.45 82.07 0.00 88.08 BRC 33/11 86.37 82.02 0.00 88.13 BRC 11/11 86.45 82.13 0.00 88.13 BRC 34/11 86.40 82.05 0.00 88.17 BRC 12/11 86.45 82.08 0.00 88.10 BRC 36/11 86.43 82.05 0.00 88.22 BRC 13/11 86.45 82.10 0.00 88.15 BRC 38/11 86.37 82.10 0.00 88.13 BRC 14/11 86.47 82.05 0.00 88.12 BRC 39/11 86.35 82.05 0.00 88.18 BRC 15/11 86.47 82.07 0.00 88.17 BRC 40/11 86.35 82.05 0.00 88.20 BRC 16/11 86.45 82.13 0.00 88.17 BRC 17/11 86.43 82.13 0.00 88.20 B. melitensis 86.38 82.10 0.00 88.15 BRC 18/11 86.43 82.15 0.00 88.18 B. canis 86.70 81.47 0.00 88.20 BRC 19/11 86.37 82.08 0.00 88.12 B. suis 86.77 82.10 78.73 88.20 BRC 20/11 86.40 82.13 0.00 88.18 B. abortus 86.82 82.07 0.00 87.93

a Primer sets Bmel, B. melitensis. b Primer sets Bcan, B. canis. c Primer sets Bsui, B. suis. d Primer sets Boa, B. ovis and B. abortus.

shown in the crossing point cycles of DNA template ranging (mean ± SD) from 11.0 ± temperature melting profile, which was also observed in all isolates except isolate BRC3/ 0.25 to 34.2 ± 0.50, when it was performed in the amount ranging from 100 ng to 0.1 pg. 09 (Table 4). The BRC3/09 strain showed a higher temperature melting profile with the A standard curve was obtained in a linear graph, which gave correlation coefficients Bmel primers (Fig. 2b) indicating that this strain was not B. melitensis. When using Bcan (R2) in 0.995 based on real-time PCR with serial dilutions of B. melitensis (Fig. 1c). No dif- primer on BRC3/09, a lower temperature melting profile was observed for this strain ference was noted in the melting profiles of B. melitensis B. abortus, B. canis,andB. suis from and B. canis control strain. This was not exhibited by the other 40 strains (Table 1, 1:10, 1:100, 1:1000, 1:10,000, and 1:100,000 fold differences in template concentration Fig. 2c and d). All the 40 isolates identified as B. melitensis showed genotype confidence using primer sets of the respective Brucella spp. (Fig. 1d). percentage (GCP) equal or greater than 95 using Bmel primers (Table 5). The GCP for BRC3/09 was 96.44 using Bcan primers, but only 17.09 using Bmel primers. 3.2. HRM analysis Only B. suis control strain displayed positive amplification curve with Bsui primers, while none of 41 Brucella spp. isolates was amplified. The melting peak for B. suis control The HRM analysis on all the 41 Brucella isolates using the 4 primer sets was shown in strain was detected at approximately 78.73 °C (Fig. 2e), while other isolates failed to gen- Fig. 2a–f. The HRM assay using Bmel primer exhibited a melting pattern that began its sep- erate any melting peak with the Bsui primer. All 41 isolates showed GCP 0.00 for with the aration approximately at 84.80 °C and ends at approximately 88.75 °C (Fig. 2a), resulting Bsui primer (Table 5). When Boa primer set was used, all Brucella spp. isolates displayed in 2 separate groups in this HRM curve. The B. melitensis control strain showed a lower temperature melting curves that shifted to the right, which indicates that B. abortus

Table 5 Genotype conﬁdence percentage using the speciﬁc primers for each Brucella spp. isolate in HRM analysis.

Isolates GCP Bmela GCP Bcanb GCP Bsuic GCP Boad Isolates GCP Bmela GCP Bcanb GCP Bsuic GCP Boad

BRC 3/09 17.09 96.44 0.00 13.70 BRC 18/11 98.05 0.12 0.00 11.28 BRC 4/10 98.50 0.03 0.00 12.83 BRC 19/11 98.93 0.95 0.00 17.36 BRC 5/10 98.69 0.09 0.00 23.18 BRC 20/11 99.21 0.08 0.00 9.52 BRC 1/11 97.70 0.26 0.00 11.49 BRC 21/11 97.61 0.02 0.00 2.02 BRC 2/11 98.48 0.12 0.00 13.70 BRC 22/11 98.62 0.06 0.00 27.70 BRC 3/11 98.22 0.09 0.00 17.44 BRC 23/11 96.84 0.04 0.00 18.80 BRC4/11 98.67 0.11 0.00 18.76 BRC 24/11 99.69 0.05 0.00 14.37 BRC 5/11 99.58 0.26 0.00 9.65 BRC 25/11 98.92 0.18 0.00 18.03 BRC 6/11 98.00 0.35 0.00 16.81 BRC 26/11 96.71 0.27 0.00 9.70 BRC 7/11 95.47 0.18 0.00 4.33 BRC 27/11 99.18 0.29 0.00 7.28 BRC 8/11 95.13 0.23 0.00 16.46 BRC 28/11 96.16 0.82 0.00 9.88 BRC 9/11 99.09 0.39 0.00 8.17 BRC 29/11 98.52 0.19 0.00 15.44 BRC 10/11 97.93 0.28 0.00 14.05 BRC 30/11 97.24 1.53 0.00 5.18 BRC 11/11 97.90 0.13 0.00 22.46 BRC 31/11 99.63 0.14 0.00 12.48 BRC 12/11 98.59 0.43 0.00 22.31 BRC 32/11 97.95 0.18 0.00 9.97 BRC 13/11 99.85 0.11 0.00 12.94 BRC 33/11 99.17 0.11 0.00 11.19 BRC 14/11 96.01 0.31 0.00 23.73 BRC 34/11 97.36 0.67 0.00 7.68 BRC 15/11 99.2 0.24 0.00 11.43 BRC 36/11 97.57 0.42 0.00 3.97 BRC 16/11 96.79 0.34 0.00 11.69 BRC 38/11 99.82 0.2 0.00 7.33 BRC 17/11 98.65 0.12 0.00 5.85 BRC 39/11 99.16 0.45 0.00 8.42 BRC 40/11 95.73 0.19 0.00 4.93

a Primer sets: Bmel, B. melitensis. b Primer sets Bcan, B. canis. c Primer sets Bsui, B. suis. d Primer sets Boa, B. ovis and B. abortus.

Please cite this article as: Mohamed Zahidi J’, et al, Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2014.12.012 6 J’. Mohamed Zahidi et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx produced earlier melting curve at 87.90 °C when compared to other Brucella spp. isolates binding to double-stranded DNA (Gudnason et al., 2007; Sharma et al., at approximately 88.15 °C (Fig. 2f). This implied that none of the 41 Brucella isolates was 2013). As the temperature increased, the presence of double-stranded B. abortus. DNA decreased, thus reducing the fluorescent. This observation was plotted by the level of fluorescence versus the temperature to generate 4. Discussion a melting curve. EvaGreen displayed higher and sharp peak in the melt curve, as shown in Fig. 2b. Another advantage of EvaGreen was a shorter Our study indicated that HRM analysis can give rapid and accurate amplification program where only 1-step initial real-time PCR denatur- identification of B. melitensis, B. abortus, B. canis,andB. suis. The develop- ation condition was required when compared to 2 necessary different ment of molecular technique, which utilizes HRM assay, has the advan- temperatures of an initial denaturation step when SYBR Green was uti- tage of being sensitive and can rapidly differentiate members of the lized (Winchell et al., 2010). Brucella genus. It is a simple method for genotyping, mutation scanning, HRM analysis has been used for the identification of different micro- and sequence matching without requiring labeled probes (Reed et al., organisms such as bacteria, virus, and fungi. It has been shown to pro- 2007). Primers B4 and B5, which amplified 223-bp fragment of the vide the simplest method for genotyping and identification down to gene encoding BCSP 31, have been used in conventional PCR assay for species level. Brucella spp. is a biosafety level 3 bacteria and needs highly the diagnosis of human Brucella in Malaysia. This assay showed high skilled personnel to handle the live bacteria. Conventional culture sensitivity and was able to detect all species and biovar of Brucella method, which is the gold standard for identification of Brucella spp., (Baddour and Alkhalifa, 2008; Baily et al., 1992). However, this conven- is highly risky and time consuming for laboratory personnel because tional PCR method requires the use of gel electrophoresis to document of frequent subculturing and several days may lapse before the bacteria the amplified PCR products. In contrast, the use of real-time PCR has is finally identified. The use of HRM for the identification and differenti- the major advantage of obtaining results in a shorter time frame and ation of Brucella spp. isolated from clinical samples in Malaysia can, does not require electrophoresis analysis (Queipo-Ortuno et al., 2005; therefore, help to reduce exposure to frequent handling of this bacteri- Redkar et al., 2001). Real-time PCR using TaqMan probes for the identi- um and the results can be obtained within 2 h. Saturation dye used in fication and differentiation of Brucella up to the species level is expen- this assay is also low cost as compared to Taqman probes. sive (Bounaadja et al., 2009; Foster et al., 2008). However, real-time This HRM assay managed to differentiate the Brucella spp. using a PCR, which utilizes HRM curve analysis, does not require Taqman minimal amount of template DNA. Identification can be carried out probes because the analysis is based on monitoring the saturating dye using as low as 1 pg of DNA. However, some general characteristics fluorescence in the presence of double-stranded DNA to identify and need to be considered when running the HRM assay. This includes differentiate the species of Brucella. using consistent amount of DNA in all samples to ensure optimal cycle

In the differentiation of Brucella spp. isolates, Winchell et al. (2010) threshold (CT) values between 15 and 35. The samples must be spun found a single nucleotide polymorphism (SNP) within the int-hyp down to remove air bubbles, which may give variability in signal CT gene at the extreme 5′ of a hypothetical protein for identification between samples. B. melitensis and B. canis. The SNP transition from G to T occurred in The determination of Brucella spp. would allow the investigation and B. melitensis isolates, while the transition from G to A occurred in prevention of spread of the disease in human and animal. This assay can B. canis isolates using the Bmel primers and Bcan primers, respectively. be used for rapid detection of infection, which will greatly help in the in- In this study, identification of 40 of the B. melitensis and 1 isolate as vestigation and management of sporadic cases and outbreaks. The spe- B. canis was successfully conducted indicating the sensitivity and speci- cies identification of Brucella is also essential for epidemiological studies ficity of the method. Most of patients identified as B. melitensis mainly of brucellosis. consumed raw milk due to the traditional belief that gives hidden ben- fi e ts. The patients also were noted to be working with farm animals es- Acknowledgments pecially sheep and goats in Malaysia. The HRM assay in this study was also able to identify an isolate We would like to thank the Director-General Ministry of Health, (BRC3/09) as B. canis. Initially, this isolate was reported as Brucella Malaysia, for the permission to publish this article. This work was spp. by conventional PCR using B4 and B5 primers. Real-time analysis supported by Ministry of Health Malaysia grant NMRR 11-699-10195/ fi with Bmel and Boa primers showed ampli cation; however, HRM anal- JPP IMR 11–035. We also thank the Director of Veterinary Research ysis using the same primers showed that this isolate was neither Institute, Ipoh, Malaysia, for the Brucella spp. control strains used in B. melitensis nor B. abortus. When the isolate was further analyzed this study. with HRM using Bcan primers, the HRM curve produced was similar to the B. canis control strain, suggesting that it could be B. canis. B. suis References biovar 3 and biovar 4 have previously been reported to exhibit genotype identical to B. canis by multiple-locus variable-number analysis (MLVA). Araj GF. Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents Some studies also suggested that this may be a unique biovar of B. suis 2010;36S:S12–7. Baddour MM, Alkhalifa DH. Evaluation of three polymerase chain reaction techniques for (Foster et al., 2009; Huynh et al., 2008; Whatmore et al., 2007; Winchell detection of Brucella DNA in peripheral human blood. Can J Microbiol 2008;54:352–7. et al., 2010). However, when the BRC3/09 was subjected to HRM analy- Baily GC, Kraahn JB, Drasar BS, Stoker NG. Detection of Brucella melitensis and Brucella sis using Bsui primer, no amplification was observed. Therefore, this abortus by DNA amplification. J Trop Med Hyg 1992;95:271–5. fi Bounaadja L, Albert D, Chenais B, Henault S, Zygmunt MS, Poliak S, et al. Real-time PCR for nding ruled out the possibility of B. suis biovar 3 and biovar 4 and con- identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target firmed the identity of the isolate as B. canis. genes. Vet Microbiol 2009;137:156–64. In order to perform good HRM analysis, saturating fluorescent dyes Cloeckaert A, Vizcaino N. DNA polymorphism and taxonomy of Brucella species. In: need to be used in HRM assay to monitor the denaturation process of Lopez-Goni I, Moriyon I, editors. Brucella molecular and cellular biologyHorizon bio- science; 2004. p. 1–24. DNA sample. The fluorescent dyes are low cost and allow dissociation Corbel MJ. Brucellosis in human and animals. Geneva: WHO press, World Health Organi- melting curve analysis of the PCR product. The SYBR Green has been zation; 2006. used widely in real-time PCR. This nonsaturation dye gave very strong Corbel MJ, Bannai M. Genus I. Brucella Meyer and Shaw 1920, 173 AL. In: George G, Don JB, James TS, Noel RK, David RB, Paul DV, Michael G, Fred AR, Karl-Heinz S, editors. signal but showed low reproducibility during melting reaction Bergey's manual of systematic bacteriology. 2nd ed. New York: Springer Science+ (Gudnason et al., 2007). This occurs because SYBR Green must be used Business Media; 2005. p. 370–86. at low concentration to prevent inhibition in the PCR reaction De Santis R, Ciammaruconi A, Pomponi A, Fillo A, Lista F. Brucella: molecular diagnostic techniques in response to bioterrorism threat. J Bioterror Biodef 2011;S2:1–8. (Eischeid, 2011). EvaGreen, which is used as the saturation dye in this Eischeid AC. SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR. BMC study, has a unique property of becoming highly fluorescent upon 2011;4:1–5.

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