Open Access

High throughput MLVA-16 typing for Brucella based on the microfluidics technology

  • Riccardo De Santis1,
  • Andrea Ciammaruconi1,
  • Giovanni Faggioni1,
  • Silvia Fillo1,
  • Bernardina Gentile1,
  • Elisabetta Di Giannatale2,
  • Massimo Ancora2 and
  • Florigio Lista1Email author
BMC Microbiology201111:60

DOI: 10.1186/1471-2180-11-60

Received: 8 October 2010

Accepted: 24 March 2011

Published: 24 March 2011

Abstract

Background

Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology.

Results

The Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three Brucella samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were de novo genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created.

Conclusion

In this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to one sixth respect to other microfluidics systems as e.g. the Agilent 2100 bioanalyzer.

Finally, this platform can be considered a valid alternative to standard genotyping techniques, particularly useful dealing with a large number of samples in short time. These data confirmed that this technology represents a significative advancement in high-throughput accurate Brucella genotyping.

Background

The members of the genus Brucella are Gram-negative, facultative intracellular bacteria responsible of a considerable human morbidity and in animals of enormous economic losses [1] due to abortion and infertility in livestock (cattle, goats, and sheep). As brucellosis is a zoonotic disease, practically all human Brucella infections develop from direct or indirect contact to animals. In particular, brucellosis in humans occurs as a sub-acute or chronic illness, that is generally not lethal in previously healthy patients, and can result in a wide variety of manifestations and significant morbidity if the diagnosis is unobserved and treatment is not rapidly initiated [2]. There are nine recognized species of Brucella[3] that differ in their host preference [4]. In particular, the nine recognized host-specific Brucella spp. are: B. abortus which preferentially infects cattle; B. melitensis infects sheep and goats; B. suis infects pigs; B. canis the dog; B. ovis, sheep and goats; B. neotomae the desert wood rat; B. microti the common vole [5]; B.ceti, cetaceans [6]; B. pinnipedialis, seals [6, 7]. Recently, an additional novel species, B. inopinata sp., isolated from a human breast implant infection, was described [8]. Currently, the division in species and between biovars of a given species is performed using differential tests based on phenotypic characterization of lipopolysaccharide (LPS) antigens, phage typing, dye sensitivity, requirement for CO2, H2S production, and metabolic properties [9]. The genotyping of Brucella field strain isolated in outbreaks is an essential tool to better understand the epidemiology of the disease and to give support to the trace-back of infection sources. It is also essential to identify the presence of Brucella strains that can affect livestock populations and new strains that were previously considered to be exotic [10], thus improving the outcomes of the national brucellosis eradication programme. Although brucellosis has been eradicated in Northern Europe, Australia, the USA and Canada, this disease remains endemic in most areas of the world [11]. Therefore, the knowledge of the prevailing genotypes of Brucella spp. present in a country is an important epidemiological tool to assess the necessary steps required for the formulation of policies and strategies for the control of brucellosis in animal populations. In addition, Brucella spp. represent potential biological warfare agents due to the high contagious rates for humans and animals, the non-specific symptoms associated with the infection, and the fact that the organism can be readily aerosolized [1214]. Therefore, the discrimination between natural outbreaks and/or intentional release of micro-organism agents may be of crucial importance in the context of the bioterrorism. Brucella species are characterised by >80% interspecies homology by DNA-DNA hybridization studies [15, 16] and >98% sequence similarity by comparative genomics [17]. In fact, the sequencing of 16 S rRNA showed a 100% of identity between all of the Brucella spp. [18]. The simple identification of genus and, in some cases, species by PCR assays [19, 20], is adequate for purposes as diagnosis of human/animal disease or identification of food contamination but not for the tracing of outbreaks or bioterrorist attack. Therefore, the development of strain typing methods is essential in order to investigate the source of an epidemic event. Molecular DNA technology such as repetitive intergenic palindromic sequence-PCR (REP-PCR) [21], random amplified polymorphic DNA-PCR (RAPD-PCR) [22], arbitrary primed-PCR (AP-PCR) [23], amplified fragment length polymorphism (AFLP) [24], single nucleotide polymorphism (SNP) [25, 26], and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [27] has been employed to sub-type Brucella spp.

In the last years the variable number of tandem repeats (VNTR), allelic hypervariability related to variation in the number of tandemly repeated sequences, were used for the discrimination of bacterial species that display very little genomic diversity. Polymorphic tandem repeat loci have been identified by analysing published genome sequences of B. melitensis 16 M, B. suis 1330, and B. abortus 9-941 [16, 28]. Schemes based on multiple locus VNTR analysis (MLVA) were tested. In Brucella, MLVA schemes with 21 loci (MLVA-21), 15 and 16 loci (MLVA-15 and MLVA-16) were published [12, 16, 29]. The authors used a subset of loci that preserved the clusters corresponding to classical species, comprising markers with repeat unit sizes of 9 bp or greater and good species identification capability ('minisatellites') and markers with repeat unit sizes of up to 8 bp and higher discriminatory power ('microsatellites') [30]. The MLVA band profiles may be resolved by different techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing systems. The most frequently used method is the agarose gel. Recently, a more rapid and inexpensive method based on the Lab on a chip technology has been proposed [31]. This miniaturized platform for electrophoresis applications is able to size and quantify PCR fragments, and was previously used for studying the genetic variability of Brucella spp. [32]. Recently a new high throughput micro-fluidics system, the LabChip 90 equipment (Caliper Life Sciences), was developed. This platform can be considered particularly useful when dealing with a large number of samples in short time. Therefore we evaluated the LabChip 90 system for MLVA typing of Brucella strains applying the selected subset of 16 loci proposed by Al-Dahouk et al. [12] to fifty-three field isolates and ten DNA samples provided in 2006 for Brucella suis ring-trial. Furthermore, twelve DNA samples, provided in 2007 for a MLVA VNTR ring trial and seventeen human Brucella isolates whose MLVA fingerprinting profiles were previously resolved [32, 33], were de novo genotyped.

Results

By means of MLVA-16 on LabChip 90 (Caliper Life Sciences) sixty-three DNA samples, fifty-three field isolates of Brucella (Table 1) and ten DNA provided for Brucella suis ring-trial, were analysed for investigating a broader number of loci. In order to set up the system, DNA samples, previously genotyped by sequencing system and Agilent technology [32, 33], were reanalyzed. DNA from all ninety-two isolates was amplified at 16 loci (MLVA-16 typing assay) to generate multiple band profiles. The LabChip 90 equipment acquires the sample in less than a minute and the analysis of 96 samples in less than an hour. After PCR amplification 5 μl of each reaction was loaded into a 96-well plate and the amplification product size estimates were obtained by the LabChip Gx Software. The data produced by the Caliper system showed band sizing discrepancies compared with data obtained from other electrophoresis platforms. Therefore a conversion table that would allow the allocation of the correct alleles to the range of fragment sizes was created. The table contained for each locus the expected size, the range of observed sizes, including arithmetical average ± standard deviation, and the corresponding allele (Table 2). The variability range for each allele was established experimentally by the analysis of different strain amplification products. Furthermore, in order to look at intra- and interchip variability, each allele was analyzed by repeating five times the analysis on the same chip and different chips. The comparison of the average and standard deviations obtained by the analysis of the intra- and interchip variability by t-test (confidence of interval 95%) shown a P value > 0.005 (data not shown).
Table 1

The fifty-three strains provided by Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise - G. Caporale-(Istituto G. Caporale).

Samples

Species-biovar according MLVA Database Genotypinga

Year

Host

Geographic origin

BruIT200

B.melitensis biovar 3

2002

human

Sardinia, Italy

BruIT201

B.abortus biovar 1

2002

bovine

Piemonte, Italy

BruIT202

B.melitensis biovar 3

2002

bovine

Lazio, Italy

BruIT203

B.abortus biovar 1

2002

bovine

Lazio, Italy

BruIT204

B.abortus biovar 3

2002

bovine

Piemonte, Italy

BruIT205

B.melitensis biovar 3

2002

water buffalo

Campania, Italy

BruIT206

B.melitensis biovar 3

2002

water buffalo

Campania, Italy

BruIT207

B.abortus biovar 1

2003

water buffalo

Campania, Italy

BruIT208

B.melitensis biovar 3

2003

milk

Emilia-Romagna, Italy

BruIT209

B.melitensis biovar 3

2003

bovine

Abruzzo, Italy

BruIT210

B.abortus biovar 3

2001

bovine

Piemonte, Italy

BruIT211

B.abortus biovar 3

2001

bovine

Piemonte, Italy

BruIT212

B.abortus biovar 3

2002

bovine

Piemonte, Italy

BruIT213

B.abortus biovar 3

2007

bovine

Italy

BruIT214

B.abortus biovar 3

2002

bovine

Piemonte, Italy

BruIT215

B.melitensis biovar 3

2001

ovine

Lazio, Italy

BruIT216

B.melitensis biovar 3

2001

ovine

Lazio, Italy

BruIT217

B.melitensis biovar 3

2001

water buffalo

Lazio, Italy

BruIT218

B.melitensis biovar 3

2002

bovine

Campania, Italy

BruIT219

B.melitensis biovar 3

2001

wild boar

Campania, Italy

BruIT220

B.melitensis biovar 3

2001

bovine

Piemonte, Italy

BruIT221

B.melitensis biovar 3

2001

ovine

Piemonte, Italy

BruIT222

B.melitensis biovar 3

2001

ovine

Lazio, Italy

BruIT223

B.melitensis biovar 3

2001

ovine

Lazio, Italy

BruIT224

B.abortus biovar 3

2001

bovine

Lazio, Italy

BruIT225

B.abortus biovar 3

2001

bovine

Piemonte, Italy

BruIT226

B.melitensis biovar 3

2001

human

Lazio, Italy

BruIT227

B.suis biovar 2

2003

hare

Emilia-Romagna, Italy

BruIT228

B.suis biovar 2

2003

hare

Emilia-Romagna, Italy

BruIT239

B.abortus biovar 3

2008

bovine

Molise, Italy

BruIT240

B.abortus biovar 3

2008

bovine

Molise, Italy

BruIT241

B.abortus biovar 3

2008

bovine

Molise, Italy

BruIT242

B.abortus biovar 3

2008

bovine

Molise, Italy

BruIT243

B.abortus biovar 3

2008

bovine

Molise, Italy

BruIT244

B.abortus biovar 3

2008

bovine

Molise, Italy

BruIT245

B.abortus biovar 3

2007

water buffalo

Campania, Italy

BruIT246

B.melitensis biovar 3

2007

water buffalo

Campania, Italy

BruIT247

B.abortus biovar 3

2007

bovine

Calabria, Italy

BruIT248

B.abortus biovar 3

2007

water buffalo

Puglia, Italy

BruIT249

B.abortus biovar 3

2009

bovine

Campania, Italy

BruIT250

B.abortus biovar 3

2009

bovine

Calabria, Italy

BruIT251

B.abortus biovar 3

2009

bovine

Calabria, Italy

BruIT252

B.abortus biovar 6

2009

bovine

Calabria, Italy

BruIT253

B.abortus biovar 6

2009

ovine

Puglia, Italy

BruIT254

B.melitensis biovar 3

2001

bovine

Piemonte, Italy

BruIT255

B.abortus biovar 3

2002

bovine

Piemonte, Italy

BruIT256

B.suis biovar 2

2002

bovine

Piemonte, Italy

BruIT257

B.suis biovar 2

2001

ovine

Lazio, Italy

BruIT258

B.suis biovar 2

2005

water buffalo

Campania, Italy

BruIT259

B.suis biovar 2

2002

wild boar

Piemonte, Italy

BruIT260

B.abortus biovar 1

2007

bovine

Campania, Italy

BruIT261

B.abortus biovar 3

2007

bovine

Italy

BruIT262

B.abortus biovar 1

2007

bovine

Calabria, Italy

aMLVA bank for bacterial genotyping http://mlva.u-psud.fr/[35].

Table 2

Comparison between Brucella product sizes estimated by LabChip GX software (Observed size) and actual sizes obtained by direct sequencing of the PCR product or data available in Genbank (Expected size).

PCR

Locus (UL bps)a

Allele

Expected size

Observed size

x ± sb

Singleplex 1

Bruce08 (18)

2

312

  
  

3

330

346-359

352,63 ± 5,37

  

4

348

369-383

376 ± 4,62

  

5

366

385-410

399,09 ± 6,58

  

6

384

411-434

419,29 ± 6,71

Singleplex 2

Bruce43 (12)

1

170

179-188

183,17 ± 2

  

2

182

191-200

196,07 ± 2,32

  

3

194

  

Singleplex 3

Bruce12 (15)

7

302

  
  

8

317

  
  

9

332

  
  

10

347

359-369

362,8 ± 3,7

  

11

362

379-388

384,13 ± 3,64

  

12

377

390-400

395,16 ± 3,05

  

13

'392

409-420

413 ± 2,55

  

14

407

424-433

428,82 ± 3,05

  

15

422

434-440

438,25 ± 2,87

  

17

452

  

Singleplex 4

Bruce18 (8)

3

130

143

 
  

4

138

150-157

153,57 ± 2,64

  

5

146

159-162

160,33 ± 1,37

  

6

154

164-176

171,62 ± 2,95

  

7

162

178-184

181,65 ± 1,53

  

8

170

187-194

191 ± 2,24

  

9

178

  

Singleplex 5

Bruce11 (63)

2

257

266-270

268 ± 2,82

  

3

320

321-344

337,82 ± 4,31

  

4

383

407-422

410,52 ± 3,56

  

6

509

504-536

515,8 ± 12,52

  

8

635

623-649

639,6 ± 8,71

  

9

698

680-724

696,67 ± 15,6

  

12

887

  
  

15

1076

  

Singleplex 6

Bruce21 (8)

5

140

  
  

6

148

162

 
  

7

156

178-179

178,5 ± 0,71

  

8

164

180-186

182,55 ± 1,19

  

9

172

192-199

194,05 ± 1,94

Singleplex 7

Bruce06 (134)

1

140

151

 
  

2

274

282-294

285,9 ± 3,33

  

3

408

429-454

439,89 ± 6,04

  

4

542

518-624

575,4 ± 24,92

Singleplex 8

Bruce42 (125)

1

164

172-198

175,1 ± 3,13

  

2

289

279-298

288,88 ± 2,14

  

3

414

420-442

428,27 ± 6,18

  

4

539

504-569

529,31 ± 14,1

  

5

664

642-647

644 ± 2,64

  

6

789

695-763

726,4 ± 22,02

  

7

914

  

Singleplex 9

Bruce45 (18)

2

133

  
  

3

151

156-169

162.01 ± 1,93

  

4

169

  
  

5

187

196-206

198,95 ± 2,63

Singleplex 10

Bruce55 (40)

1

193

204-209

207,05 ± 1,67

  

2

233

243-259

248,36 ± 4,09

  

3

273

275-308

282,85 ± 2,5

  

4

313

327

 
  

5

353

  
  

6

393

418-422

420,25 ± 1,7

  

7

433

  

Singleplex 11

Bruce30 (8)

2

119

130

 
  

3

127

132-144

139,29 ± 2,11

  

4

135

146-152

148,87 ± 1,7

  

5

143

155-160

157,77 ± 1,78

  

6

151

165-169

167 ± 2

  

7

159

174

 
  

8

167

  
  

9

175

  
  

10

183

205-206

202,25 ± 0,5

  

11

191

  
  

12

199

  

Singleplex 12

Bruce04 (8)

2

152

161-164

162.5 ± 2.1

  

3

160

169-175

171.6 ± 2

  

4

168

177-182

179.1 ± 1.3

  

5

176

185-191

187.3 ± 1.8

  

6

184

194-198

195.7 ± 1.3

  

7

192

201-207

203.4 ± 2.2

  

8

200

213-214

213.7 ± 0.6

  

9

208

219-222

220.5 ± 2.1

  

10

216

241

 
  

11

224

248-254

250.2 ± 2.4

  

12

232

  
  

13

240

  
  

14

248

  
  

15

256

  
  

17

272

  
  

18

280

  
  

19

288

  
  

20

296

  
  

22

312

  

Singleplex 13

Bruce07 (8)

2

134

  
  

3

142

  
  

4

150

150-154

151.9 ± 1.5

  

5

158

157-162

159.8 ± 1.4

  

6

166

166-171

168.1 ± 1.4

  

7

174

175-178

176.8 ± 1

  

8

182

183-186

184.4 ± 1.1

  

9

190

192-195

195 ± 1.5

  

10

198

200

 
  

11

206

  
  

12

214

  
  

13

222

  
  

14

230

  

Singleplex 14

Bruce 09 (8)

3

124

131-140

135,52 ± 2,6

  

4

132

147

 
  

5

140

155-158

156,33 ± 1,52

  

6

148

162-167

165,4 ± 1,89

  

7

156

172-177

174,42 ± 1,19

  

8

164

182-187

184,42 ± 1,61

  

9

172

191-198

193,75 ± 2,5

  

10

180

201-203

202,12 ± 0,83

  

11

188

209-212

210,75 ± 1,25

  

12

196

220

 
  

13

204

228-230

228,66 ± 1,15

  

14

212

  
  

15

220

  
  

16

228

249-255

252,66 ± 3,21

  

17

236

  
  

18

244

266-271

268,85 ± 1,86

  

19

252

  
  

20

260

  
  

22

276

  
  

23

284

  
  

24

292

  

Singleplex 15

Bruce 16 (8)

2

144

153-157

154,9 ± 1,59

  

3

152

158-166

163,04 ± 2,38

  

4

160

167-172

168,53 ± 1,66

  

5

168

177-185

181,52 ± 2

  

6

176

186-194

189,83 ± 2,55

  

7

184

199-203

200,8 ± 1,4

  

8

192

207-209

207,66 ± 1,15

  

9

200

216-219

217,37 ± 1,18

  

10

208

224-227

224,75 ± 1,5

  

11

216

231

 
  

12

224

242-248

244,75 ± 2,5

  

14

240

  
  

15

248

  

Singleplex 16

Bruce 19 (6)

4

79

  
  

5

85

  
  

6

91

  
  

15

145

  
  

16

151

  
  

18

163

173-177

175 ± 1,4

  

19

169

180-183

182,5 ± 0,5

  

20

175

184-188

186 ± 1,8

  

21

181

189-193

190,6 ± 1,2

  

22

187

194-201

197,9 ± 1,1

  

23

193

202

 
  

25

205

  

a Unit Length size

b Arithmetic average (x) ± standard deviation (σ) of the observed sizes

The required precision is directly related to the repeat unit size of the loci. Only data with a standard deviation lower than the 50% of the repeat unit size were considered valid. The LabChip 90 equipment MLVA-16 products were separated and DNA fragment sizes were correlated to the alleles by the conversion table. Generally, close alleles were not observed to overlap allowing to assign the correct allele to each observed value. However, the markers Bruce 08, Bruce 21, Bruce 16 and Bruce 19 showed continuity between some neighboring range which may lead to incorrect assignment of allele to the observed value (Table 2). The identified species were compared with the results of the previous analysis [32, 33], obtaining a full concordance for 15 markers while the marker Bruce 19 did not show agreement with the results obtained by the different analysis systems. For the loci including alleles spanning into ambiguous ranges, we performed sequencing of the amplicons showing on Caliper maximum or minimum allele values. Furthermore we performed some random sequencing of the amplicons obtaining a confirmation of the correct assignment (data not shown).

Discussion

Many methods have been developed to differentiate Brucella strains but MLVA currently represents one of the most promising technologies regarding the epidemiology of bacteria with a high genetic homogeneity, such as Brucella ssp. In 2003 Bricker et al [28] published a MLVA based on eight locus scheme. In 2006 Whatmore et al [16] described a new scheme that included the eight of the original loci of Bricker as well as an additional 13 newly VNTR loci to give a 21 locus scheme, VNTR-21, that allowed to provide some resolution at the species level. In the same year a scheme labelled MLVA-15, based on a subset of 15 loci that comprises 8 markers with good species identification capability and 7 with higher discriminatory power, was published [29], and followed by MLVA-16, a slight modification of MLVA-15 [12]. The different alleles, amplified by standard PCR techniques, can be analysed by several electrophoretic techniques as agarose gel, or capillary electrophoresis sequencing. In this paper the attention was addressed on the LabChip 90 equipment (Caliper), a platform based on microfluidics technology specifically developed for measuring the length of DNA fragments and that do not require fluorescent primers. This electrophoresis machine represents a compromise between the more expensive capillary electrophoresis apparatus and the traditional agarose gel electrophoresis. In spite of a lower precision respect to the automated capillary electrophoresis, the ability to acquire 96 amplification product sizes in less than a hour represent an increased time-reduction over the traditional ethidium bromide slab gel electrophoresis, with 40-50 amplification product sizes for the same analysed markers acquired in a higher time [34]. The LabChip 90 represents also a significant improvement respect to other microfluidics systems as e.g. the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Ca). In effect the LabChip 90 allows performing the strain genotyping in a time equal to one sixth respect to Agilent. Furthermore this system requires less handling as a single plate can be read directly after the PCR reaction, while the Agilent equipment needs a manual charge of the single PCR products for each single chip well. Finally, the LabChip GX software improves efficiency of data acquiring by automating the data flows. In fact, the software allows to export the summary of analysis results to a spreadsheet application, with the consequent elimination of the paper-based flows. As described previously [31, 32] the sizing proposed by the Lab on chip technology does not correspond to the real size, resulting in a shift of a variable value (offset) respect to the real size estimated by sequencing. Therefore, a correspondence table which allows for each range of observed values to assign the expected size and corresponding allele (Table 2) was created. We did not observe in general the overlap among close alleles, allowing to unambiguously assign the correct allele to each observed value. However, for some contiguous alleles we observed a continuity between ranges which may lead to incorrect assignment of allele to the observed value (Table 2). Furthermore, the instrument was not in agreement with the results obtained by the different analysis systems for the marker Bruce 19. The reduced discriminatory ability could be due to the different resolution achieved by such platform related to the fragment sizes (routinely ± 10% in a 150-500 -bp range, ± 15% in a 100-150 -bp range and in a 500-1500 -bp range and ± 20% in a 1500-5000 -bp range). However, the comparison of the results obtained by the MLVA-16 method on the Caliper LabChip 90 platform and those previously resolved by capillary electrophoresis sequencing system and the Lab on a chip technology (Agilent Technologies) showed a good size correlation. Therefore, this platform can be considered a valid alternative to standard genotyping technique, particularly useful dealing with a large number of samples in short time.

Conclusion

In this paper we evaluated high throughput system as the LabChip 90 for MLVA-16 typing of Brucella strains. The MLVA typing data obtained on this equipment showed accurate correlation for those obtained by capillary electrophoresis sequencing and the Agilent 2100 Bioanalyzer, with the exception of Bruce 19. This new platform represents a significant improvement of the genotyping techniques in terms of turnaround times and computational efficiency.

Methods

Brucella strains and DNA extraction

In this study fifty-three field isolates submitted for typing by the Istituti Zooprofilattici Sperimentali to the National Reference Laboratory for brucellosis at the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise-G. Caporale (Istituto G. Caporale) during the 2001-2008 period (Table 1), ten DNA samples, collected in UK, provided at the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise-G. Caporale (Istituto G. Caporale) for Brucella suis ring-trial 2006 (COST 845-Brucellosis in man and animals), seventeen Brucella strains isolated from Sicilian hospitalized patients with acute brucellosis [33], and twelve DNA samples, provided by Dr. Falk Melzer for the Ring trial Brucella 2007 [32], were analysed. The provided DNA samples were extracted by Maxwell 16 Cell DNA purification kit (Promega), according to the manufacturer's instructions.

VNTR amplification

VNTR amplifications were performed according to the method described by Le Flèche et al. [29] and then adapted by Al Dahouk et al [12]. Sixteen sets of primers previously proposed were used in sixteen singleplex: Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, Bruce55 (panel 1), Bruce18, Bruce 19, Bruce21, Bruce04, Bruce07, Bruce09, Bruce16, and Bruce30 (panel 2). Amplification reaction mixtures were prepared in 15 μl volumes using 1U FastStart polymerase Taq (Roche) and containing 1 ng of DNA, 1 × PCR Roche reaction buffer (10 mM Tris-HCl, 2,5 mM MgCl2, 50 mM KCl pH 8.3), 0.2 mM dNTPs (Roche) and 0.3 μM of each flanking primer. The amplification was run in a Peltier Thermal Cycler DNA Engine DYAD (MJ Research) thermocycler as follows: an initial heating at 95°C for 5 min, 35 cycles denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 70°C for 60 sec. A final extension was performed at 70°C for 5 min [32].

MLVA-16 analysis

The amplification was performed in 96-well or 384-well PCR plates. The chip was prepared according to manufacturer recommendations (Caliper HT DNA 5 K Kit). Each chip contains 5 active wells: 1 for the DNA marker and 4 for gel-dye solution. For each run it was prepared also a strip well with the ladder (containing eight MW size standards of 100 300 500 700 1100 1900 2900 4900 bp) that was inserted into the appropriate groove of the instrument. The number of samples per chip preparation is 400, equivalent or four 96-well plates or one 384-well plate. After gel preparation, the sample plate was loaded into the plate carrier attached to the robot of the Caliper LabChip 90. During the separation of the fragments, the samples were analyzed sequentially and electropherograms, virtual gel images and table data were shown. Amplification product size estimates were obtained by using the LabChip GX (Caliper Life Sciences). The software allows importing the data to a spreadsheet software and subsequently to the conversion table that, by a special macro set up by our laboratory, allows to assign each size to the corresponding allele. The maximum and minimum value of the observed sizes for each allele was thus established experimentally while the arithmetic average and the corresponding standard deviation (Table 2) were calculated by a statistical function.

Sequencing analysis

The PCR amplicons were purified and sequenced by CEQ 8000 automatic DNA Analysis System (Beckman-Coulter, Fullerton, CA, USA) using a commercial Kit (GenomeLab™ DTCS-Quick Start Kit, Beckman-Coulter) according to the manufacturer instructions.

Declarations

Acknowledgements

This work was part of the European Defence Agency (EDA) project B0060 involving biodefence institutions from Sweden, Norway, the Nederlands, Germany, France and Italy.

Authors’ Affiliations

(1)
Histology and Molecular Biology Section,, rmy Medical and Veterinary Research Center
(2)
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", (Istituto G. Caporale)

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