Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry

YABIO 11212

No. of Pages 6, Model 5G

2 February 2013 Analytical Biochemistry xxx (2013) xxx–xxx 1

Contents lists available at SciVerse ScienceDirect

Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio

2 3 4 5 6 7

8 1 2 0 2 11 12 13 14 15 16 17 18 19 20 21

Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry Olga Leclerc a,⇑, Pierre-Olivier Fraisse a, Guillaume Labarraque a, Caroline Oster a, Jean-Philippe Pichaut a, Maud Baume b, Sophie Jarraud b, Paola Fisicaro a, Sophie Vaslin-Reimann a a b

Department of Medical and Inorganic Chemistry, Laboratoire National de Métrologie et d’Essais, 75015 Paris, France National Reference Centre for Legionella, Hospices Civils de Lyon, Université Lyon 1, Lyon, France

a r t i c l e

i n f o

Article history: Received 7 October 2012 Received in revised form 17 December 2012 Accepted 19 December 2012 Available online xxxx Keywords: Legionella pneumophila DNA ICP–MS analysis Uncertainty evaluation Method validation

a b s t r a c t The development of a method for the quantification of Legionella pneumophila genomic deoxyribonucleic acid is considered. The method is based on the quantification by inductively coupled plasma mass spectrometry (ICP–MS) of the mass fraction of phosphorus, stoichiometrically presented in the DNA molecules. Through the DNA sequencing data, it was possible to convert the ICP–MS analysis results into DNA genome units. L. pneumophila DNA samples were analyzed using ICP–sector field MS and ICP–quadrupole MS with a collision/reaction cell. Spectrophotometric measurements of the absorbance at 260 nm and real-time PCR techniques were used to independently confirm the ICP–MS results. The comparison of the methods showed that the ICP–MS method provides better accuracy with respect to currently applied analytical techniques such as UV spectrophotometry, fluorescent dye methods, and real-time PCR. Moreover, with the use of calibration standards whose values are traceable to the International System of Units and the possibility of evaluating the contribution to the overall uncertainty of each step of the measurement procedure, the method enables long-term comparability of the measurement results. These advantages make the ICP–MS method suitable for nucleic acid investigation, from nucleotides to genomic DNA, as well as for the certification of the reference materials containing nucleic acids. Ó 2013 Elsevier Inc. All rights reserved.

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

Legionella pneumophila, first recognized 35 years ago [1], is a significant cause of pneumonia [2]. L. pneumophila bacteria are found worldwide and cause the majority of all reported cases of legionellosis [3–5]. Large, focal outbreaks of Legionnaires’ disease continue to occur and the need for surveillance for legionellosis exists all over the world. Public water supplies, particularly in hospitals, often contain L. pneumophila, which represents a potential source of severe infections. Therefore, detection and quantification of L. pneumophila seem to be essential to prevent an outbreak of the disease [6]. In France, monitoring of Legionella spp. in water is officially performed using the culture method (French Standard NF T90-431, Agence Francaise de Normalisation, 2003), in compliance with international standard ISO 11731 (International Organization for Standardization, 1998) [7]. Although this method is widely used for the detection of Legionella in environmental water, the analysis is time consuming and has a poor sensitivity. Recently, methods based on the polymerase chain reaction (PCR), especially real-time PCR (or qPCR), were developed for DNA quantification [8–12]. They give only a rough estimation of concentration values. Thus, ⇑ Corresponding author. Fax: +33 1 4043 3737. E-mail address: [email protected] (O. Leclerc).

Standard NF T90-471 (2006), describing the method validation for Legionella detection and quantification by qPCR, was established to reduce the performance differences of the qPCR methods available. Therefore the development of appropriate DNA reference materials is a key point toward standardization. A first batch has been produced and certified by qPCR (SRM_LEGDNA_01) and has been used since 2009. Nowadays, spectroscopy methods are ordinarily used for quantification of nucleic acids [13]. Spectrophotometric analysis of DNA molecules is based on Beer–Lambert’s law (A260 = ecl), where e is the molar absorption coefficient, c is the DNA concentration, and l is the optical path length of the cuvette. At a wavelength of 260 nm the average extinction coefficient for double-stranded DNA is 0.020 (lg/ml)1 cm1 and the absorbance corresponds to a concentration of 50 lg ml1. The presence of RNA, nucleotides, and impurities such as proteins and phenols may affect the estimated concentration. Overestimation of the DNA concentration may be caused by the application of the molar absorption coefficient of double-stranded DNA for the samples containing a significant amount of single-stranded DNA [14]. Another spectroscopy method used for quantification of nucleic acid samples is fluorescence in the presence of a DNA dye such as PicoGreen, which is more sensitive than the A260 measurements. However,

0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.12.023

Please cite this article in press as: O. Leclerc et al., Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry, Anal. Biochem. (2013), http://dx.doi.org/10.1016/j.ab.2012.12.023

60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

YABIO 11212

No. of Pages 6, Model 5G

2 February 2013 2

Method development for Legionella DNA quantification / O. Leclerc et al. / Anal. Biochem. xxx (2013) xxx–xxx

The DNA was extracted from the cell material and purified according to the following extraction protocol to eliminate all extra sources of phosphorus. Initially, the lysis of the cells was carried out, aiming at disrupting and disassociating the cellular components for subsequent purification. Five milligrams of cell material was dissolved in 2700 ll of ATL sterile buffer solution. Three hundred microliters of lysis solution (10 mg ml1 lysozyme, 10 mmol Tris–HCl, pH 8.0, 1 mmol ethylenediaminetetraacetic acid (EDTA), 1% Triton) was added, then the sample was incubated at 37 °C and centrifuged. The protein digestion and nuclease deactivation were performed with a proteinase K solution (300 ll). The solution was incubated at 56 °C for 1 night in a water bath. Further addition of 60 ll of RNase A at room temperature enabled the lysis of the RNA under slight stirring to make homogenization of the sample mixture possible. The DNA was finally extracted and purified with a QIAmp DNA Maxi Kit (Qiagen, France). The number of columns used depended on the initial mass of cells and was based on the optimal DNA extraction yield obtained for 5 mg of cells per column. The salts were removed with Amicon columns (Millipore, Molsheim, France). DNA samples were diluted for further analysis by ICP–MS. Gravimetrically prepared aliquots (about 0.8 ml) in AE buffer (10 mmol L1 Tris–Cl, 0.5 mmol L1 EDTA, pH 9) were stored at 20 °C until analysis.

145

Measurement methods

170

Spectrophotometric DNA analysis Absorbance of the DNA samples was measured on a Lambda 25 UV/Vis (Perkin–Elmer) spectrophotometer over a scan range of 220–320 nm with a scan speed of 60 nm/min at 1-nm intervals. For each measurement, 5 ll of the extracted DNA sample was pipetted into a cuvette (Hellma quartz TrayCell, 8200; Mulheim, Germany). The estimated DNA concentration was based on the measurement of the extinction coefficient at A260. The absorbance of DNA was measured and corrected by blank solution absorbance. Blank solutions were run every six samples. The DNA amount is expressed in mass concentration (ng ll1). Spectrophotometric measurements were also used to evaluate the purity of the nucleic acids, using the following equations: A260/A280 = (A260 reading  A320 reading)/(A280 reading  A320 reading) and A260/A230 = (A260 reading  A320 reading)/(A230 reading  A320 reading). Data were processed using UV–Visible WinLab and Microsoft Excel.

171

128

the performances of these DNA quantification methods indicate an evident lack of measurement accuracy and precision at low DNA concentration values [15,16]. Other methods such as mass spectrometry with different ionization modes were also applied for high-weight biomolecular measurements. Thus, matrix-assisted laser desorption/ionization and high-performance liquid chromatography with mass spectrometry [17,18] have been used to quantify oligodeoxynucleotides. However, these techniques are still hampered by the complex nature of the ionization of polyionic DNA material that causes substantial uncertainty in quantitative analysis. Inductively coupled plasma–mass spectrometry/optical emission spectroscopy (ICP–MS/OES), as the most sensitive and versatile analytical techniques in elemental and isotopic measurements, have become attractive methods for DNA analysis. Most elements in the periodic table can be ionized in the ICP source, including biologically important elements such as phosphorus and sulfur. As a result, biomolecules containing nucleic acids and amino acids can be detected respectively by their phosphorus and sulfur contents. The determination of biomolecules such as DNA, phosphorylated proteins, and RNA, using ICP–MS on the basis of phosphorus content measurement, reported earlier [19–28], is the more sensitive one. The ICP–OES method is also applied despite the lower sensitivity and the significant amount of sample required (mg range) [14,29–32]. The aim of this study was the development and validation of an analytical method based on ICP–MS for the accurate quantification of genomic DNA of L. pneumophila. The method is based on the quantification of the mass fraction of phosphorus, stoichiometrically present in the DNA strains. Through the DNA sequencing data, it is possible to convert the ICP–MS analysis results into DNA genomic units, making the method useful for the certification of DNA reference materials used in molecular biology applications such as qPCR. The main advantages of this method are that it provides highly accurate (small combined uncertainty) results and ensures metrological traceability. According to the International Vocabulary of Basic and General Terms in Metrology (VIM) [33] definition, metrological traceability is the ‘‘property of a measurement result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty.’’ When this reference is the International System of Units (SI) via SI quantities and units [34], this can be formulated as SI traceable or traceable to SI. If properly obtained, such results are invariant with respect to time and place and are independent of the particular measurement procedure used.

129

Materials and methods

Table 1 Microwave digestiona and ICP–MS operating conditions

130

Extraction and purification of DNA samples

131

The preparation of genomic nucleic acid samples was performed from cell culture material of L. pneumophila serogroup 1 Philadelphia strain (ATCC 33152) kindly provided by the National Reference Centre of Legionella (France). The cell material contains DNA, RNA, and proteins as major constituents. DNA preparations were performed in an ISO 7 clean room (ISO 14644-1). Tests for possible risk of nucleic acid cross-contamination due to handling were performed by culture method and showed negligible levels (total microbial flora 26 UFC m3 per day of DNA preparation at 22 °C). The moisture content in the cell powder was determined by drying the samples at 70 °C until a constant weight was reached and estimated at 10.5%. The mass of cell material was determined using a calibrated balance.

83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

132 133 134 135 136 137 138 139 140 141 142 143 144

ICP–MS system

Plasma conditions RF power (W) Resolving power Plasma gas, argon (L min1) Nebulizer flow rate (L min1) Collision cell gas flow (ml min1) Cell entrance (V) Integration time (s) Detection mode

Thermo Axiom SF MS

Agilent 7700 QMS

1350 3000 14 0.7–0.8 (concentric) –

1550 (DM = 1 amu) 15 0.98 (Micromist) Helium, 10

–

130

0.100 (dwell time) Counting

3 Counting

a The microwave digestion power control program consisted of 250 W (2 min) ? 0 W (2 min) ? 250 W (5 min) ? 0 W (2 min) ? 400 W (5 min) ? 0 W (2 min) ? 600 W (10 min).

Please cite this article in press as: O. Leclerc et al., Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry, Anal. Biochem. (2013), http://dx.doi.org/10.1016/j.ab.2012.12.023

146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169

172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188

YABIO 11212

No. of Pages 6, Model 5G

2 February 2013 Method development for Legionella DNA quantification / O. Leclerc et al. / Anal. Biochem. xxx (2013) xxx–xxx 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252

Phosphorus ICP–MS analysis Sector field (SF) ICP–MS AXIOM (Thermo) and quadrupole ICP–MS (ICP–QMS) (Agilent 7700) equipped with a collision/reaction cell were used to quantify L. pneumophila DNA samples. The detailed ICP–MS conditions are presented in Table 1. The fundamental limitation in the detection of phosphorus by ICP–MS is the existence of polyatomic isobaric interferences formed in the inductively coupled plasma ion source. These interferences were overcome with the use of the SF ICP–MS with resolving power of 3000 or collision/reaction cell with the ICP–QMS. Aliquots of about 1 g of solution of extracted DNA were prepared for digestion with nitric acid (1 ml/aliquot) (Suprapur, Merck) using the Ethos 900 microwave (Milestone, Italy). The microwave digestion conditions are listed in Table 1. All reagents used were at least analytical grade purity and the solutions were prepared in a 10,000 class clean laboratory. Ultrapure water (18.2 MX cm1) was obtained by using a Milli-Q system (Millipore) equipped with BioPack ultrafiltration cartridge. The presence of phosphorus in the solvent containing the organic buffer additives can affect the detection limit of the method. To consider its influence, blank solutions were prepared at the same time as DNA samples, using the same Qiagen protocol of extraction and purification used for the cell material. The analyte signal was then corrected by the blank solution signal for each sample. For all ICP–MS measurements, the phosphorus was quantified using the standard addition calibration method, using Phosphorus ICP Standard 1002 mg kg1 from Merck CertiPUR, traceable to SI though NIST SRM 3130a reference material. The solutions for the ICP–MS analysis were prepared gravimetrically. Each sample was diluted to 30–50 ml with Milli-Q ultrapure water and then divided into three aliquots and two of them were gravimetrically spiked with different standard additions. The phosphorus amount was expressed as mass fraction per gram of solution. Real-time PCR analysis The PCR measurements were run on an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Specific primers and probe were designed from the L. pneumophila partial mip (macrophage infectivity potentiator) gene sequence, strain ATCC 33152 (GenBank Accession No. 59668631) using eprimer3 freeware. Some genes have multiple copies in the Legionella genome (ARNr 16S and ARNr 5S); in contrast the mip gene is present as a monocopy [10]. The primer sequences of LegF and LegR are 50 AATTGAGCGCCACTCATAGC-30 and 50 -ACCGATGCCACATCATTAGC30 , respectively. The size of the qPCR products predicted with this primer pair is 148 bp. The qPCR mixture (25 ll) included 12.5 ll of Power SYBR Green MasterMix (Life Technologies), 300 nmol L1 of each forward and reverse primer (MWG), 2 ll of sample or standard DNA, and PCR-grade sterile water (Sigma). After the activation steps at 50 °C for 2 min and 95 °C for 10 min, 40 amplification cycles (15 s at 95 °C and 45 s at 58 °C) were performed. The DNA sample, DNA standard (2.12  105 to 2.12 copies), and no-template control were included in the qPCR plate. The DNA standard solution was prepared using the reference material SRM-LEGDNA-01 (CNRL, France). A comparison was conducted on lyophilized and nonlyophilized samples, in parallel with ICP–MS analysis. The DNA amount obtained by qPCR was expressed as genome units (GU) per milligram of solution. Measurement uncertainties All the uncertainties were calculated according to the principles of the ISO Guide to the Expression of the Uncertainty in Measurement (GUM) [35].

3

Expanded uncertainties, U, were determined by using the equations

uc ¼

p

ðu21 þ u22 þ u23 þ . . .Þ;

U ¼ kuc ;

253 254

255

ð1Þ ð2Þ

257

where uc is the combined uncertainty, ui represents the individual components of uncertainty, and k is the coverage factor, chosen equal to 2, defining a level of confidence of approximately 95%.

258

Results and discussion

261

Development of the ICP–MS method

262

Preliminary ICP–MS measurements were performed on extracted DNA samples to optimize the analytical method. As an example, some results obtained with SF ICP–MS are reported here. The complete 31P separation from the main mass interferences 15 16 + N O and 14N16O 1H+ obtained by SF ICP–MS is shown in Fig. 1. The phosphorus mass fraction quantification was performed on three samples and each sample was analyzed three times. Table 2 shows the DNA mass fractions (ranging from 306 to 343 ng of DNA per gram of solution), calculated according to Eq. (3). Measurement standard deviations less than 1% were found, indicating a good measurement repeatability. The relative expanded uncertainty, calculated according to Eq. (5), was about 3%. High sensitivity and signal-to-noise ratio of SF ICP–MS enables the detection limit for phosphorus in DNA samples in the range of some 0.1 ng g1. Consistent results were obtained over a wide range of mass fractions correlated to the initial amount of the DNA sample.

263

Characterization of the purity of L. pneumophila DNA using spectrophotometric and ICP–MS measurements

280

The DNA samples were prepared by extraction from a cell material, as described under Materials and methods. For this reason it was essential to ensure the sample was sufficiently free of all the potential sources of contamination, particularly those of phosphorus. The extraction protocol was tested on three independent samples and the purity was evaluated using UV absorbance measurements, through the A260/A280 and A260/A230 ratios, where each reading was corrected by the value at A320. The results reported in Fig. 2 are the averages of six replicates measured over 4 days. Pure nucleic acid samples are expected to have an A260/A280 ratio in the range of 1.7–2.0 [36]. The ratio oversteps the limits of the range if the proteins contaminate a nucleic acid sample since the aromatic amino acids presented in protein have maximum absorbance at 280 nm. For our samples, a mean value of the ratio 1.81 ± 0.02 was found, proving the absence of protein contamination. On the other hand, an A260/A230 ratio value between 1.5 and 2.0 usually indicates the absence of solvent and of contamination

282

Fig.1. Phosphorus SF ICP–MS mass spectrum of L. pneumophila DNA.

Please cite this article in press as: O. Leclerc et al., Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry, Anal. Biochem. (2013), http://dx.doi.org/10.1016/j.ab.2012.12.023

259 260

264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279

281

283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299

YABIO 11212

No. of Pages 6, Model 5G

2 February 2013 4

Method development for Legionella DNA quantification / O. Leclerc et al. / Anal. Biochem. xxx (2013) xxx–xxx

Table 2 Quantification of the mass fractions of phosphorus and genomic Legionella pneumophila DNA with SF ICP–MS P mass fraction (ng g1)a

DNA mass fraction (ng g1)b

30.8 ± 0.1 36.0 ± 0.3 34.5 ± 0.2

306.4 ± 7.6 358.2 ± 10.5 342.8 ± 9.2

a

All values based on SF ICP–MS measurements with their standard deviations. b The DNA mass fraction calculated values with the associated expanded uncertainties (k = 2).

300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332

due to a high concentration of salts [34], even though the use of AE buffer as matrix solution could decrease this value, since EDTA has a high absorption at 230 nm. A mean value of 1.73 ± 0.08 was found in our experiments. Spectrophotometric data indicate sufficient DNA purity of the samples. The uncertainties were estimated through the intermediate precision based on a relative standard deviation of measured value for 1 day and then for 4 days using a mean value of DNA concentration. The uncertainties found in our study are in good agreement with those from other studies [15,30]. Important issues for the reliable quantification of DNA samples by ICP–MS are the complete removal of phosphorus-containing impurities such as RNAs and proteins and the recovery of phosphorus through a microwave-aided acid digestion. To ensure that the purification method was quantitatively efficient in the removal of these impurities, the following tests were performed. Two batches of cell samples (nine samples per batch) were treated to evaluate the efficiency of the extraction and purification protocol. One batch was doped with RNA (ribonucleic acid from Saccharomyces cerevisiae, Sigma) and the other with phosphorylated proteins. Both batches followed the previously described purification procedure and were compared with not-doped cell samples extracted and purified in the same manner. The samples were analyzed by both ICP–MS and UV spectrophotometry. The ratios wDNA/mcell shown in Fig. 3 were calculated from the DNA mass fraction (wDNA) calculated from the phosphorus measurements by ICP–MS and the mass of the initial dried cell sample (mcell). Furthermore, the results were statistically treated with the Student t test. The ratios of DNA extracted to initial mass of cells wDNA/mcell varied between 1.27 and 1.34% for not-doped independent samples, while they were included between 1.16 and 1.55% for RNA-doped cells. This shows the efficiency of the extraction and

Fig.2. Measurement of a stock DNA solution over 4 days at 230, 260, and 280 nm to evaluate the purity of the samples. Blue, A260/A280 ratio; gray, A260/A230 ratio. The error bars represent expanded uncertainties for each measurement (n = 6).

purification protocol, which was able to remove the RNA that was added as wRNA/mcell = 11.8%. This ratio is superior to that naturally present in a bacterial cell sample (in the range of 6% of the cell weight) [37]. The spectrophotometric data for the same series of DNA purified samples presented in Fig. 3 (j), exhibited higher values for both doped and not-doped samples with respect to ICP–MS values, but the two spectrophotometric values are compatible within the method uncertainty. To validate the extraction protocol also with respect to the purification from the phosphorus-containing proteins, pure HSP27 phosphorylated protein (three phosphorus atoms per molecule chain—27,000 amu) was used. The results obtained on ICP–MS signal intensities of phosphorus enabled us to consider the DNA purification protocol (removing 95% of proteins, according to producer indications) sufficient for phosphorylated protein elimination.

333

Quantification of L. pneumophila DNA

349

Once the ICP–MS analysis was optimized and the extraction and purification procedure validated, a new sample batch was prepared and characterized, to validate the overall quantification procedure. Here the data obtained by ICP–QMS with the collision/reaction cell are reported. In fact, a quadrupole mass spectrometer has lower sensitivity compared to SF ICP–MS; however, it is characterized by a shorter measurement cycle. Data from ICP–MS analysis expressed as mass fractions of phosphorus per gram of solution were converted into DNA mass fraction using the equation

350

DNAmass fraction ¼

Pmass fraction  genomemass  NA ; Pmol mass  Nbnucleotides

ð3Þ

where Pmol mass is the molar mass of phosphorus, genomemass is the mass of the genome calculated from L. pneumophila DNA sequencing data [38], NA is Avogadro’s number, and Nbnucleotide is the number of nucleotides in the L. pneumophila genome, which is equal to the number of phosphorus atoms in the genome due to the stoichiometric presence of one phosphorus atom per nucleotide. Thus,

DNAGU ¼ DNAmass fraction =genomemass ;

ð4Þ

where DNAGU is the amount of DNA expressed in genomic units per gram of solution. The DNA mass fraction, determined as the average of four aliquots of the same sample analyzed five times and each aliquot on 3 days, was 1.09 ± 0.05 lg g1. To confirm the ICP–MS values, a comparison with qPCR and spectrophotometric measurements was also performed, although attention should be paid to the fact that the measured may not necessarily be the same using different analytical techniques and the sensitivities of the three methods are considerably different. Five DNA samples were analyzed in parallel by ICP–MS and qPCR, giving quite consistent results, i.e., 1.56  107 ± 7.11  105 GU mg1 for ICP–MS and 1.37  107 ± 7.73  106 GU mg1 for Q1 qPCR. Finally, the DNA mass fraction determined with A260 spectrophotometric measurements of a solution that was 58 times more concentrated than the one analyzed by ICP–MS was 62.753 lg g1. The relationship of absorbance unit of 50 lg of DNA per ml solution was applied for a mass fraction calculation. Fig. 4 shows the comparison of the results, converted into genomic units, obtained by the three methods. Even if the results showed a good consistency, it is worth noticing that the uncertainty associated with the result obtained by qPCR is about 11 times higher than that obtained by ICP–MS, because of the high uncertainty of the L. pneumophila standard used for the calibration. This confirms that a reference material characterized by a refer-

Please cite this article in press as: O. Leclerc et al., Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry, Anal. Biochem. (2013), http://dx.doi.org/10.1016/j.ab.2012.12.023

334 335 336 337 338 339 340 341 342 343 344 345 346 347 348

351 352 353 354 355 356 357 358 359

360 362 363 364 365 366 367 368

369 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397

YABIO 11212

No. of Pages 6, Model 5G

2 February 2013 Method development for Legionella DNA quantification / O. Leclerc et al. / Anal. Biochem. xxx (2013) xxx–xxx

5

Fig.3. Ratio of L. pneumophila DNA to the cell material determined for pure cells and cells doped with RNA by SF ICP–MS phosphorus determination (i) and UV measurements (j).

400

ence method able to provide values with a smaller uncertainty, such as the ICP–MS method, would be a great advantage for the users applying the qPCR method.

401

ICP–MS measurement uncertainty assessment

402 403

The relative uncertainty of DNA mass fraction was determined using a model including five components:

406

U relðDNA mass fractionÞ ¼ vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u uu2 ðPmass fraction Þ u2 ðPmolar mass Þ u2 ðN A Þ u2 ðgenomemass Þ2 u2 ðNbnucleotides Þ2 t þ þ þ : 2 genome2mass P 2mass fraction P 2molar mass N 2A Nbnucleotides

398 399

404

407 408

ð5Þ

The relative standard uncertainty of the phosphorus mass fraction (uP mass fraction) was calculated from the standard deviation of three

Fig.4. Comparison of L. pneumophila DNA sample quantification obtained with ICP– MS, qPCR, and UV spectrophotometry (A260). The error bars represent the combined expanded uncertainties (k = 2) of the methods. As the spectrophotometric value is an indicative estimation, it is therefore shown without uncertainty.

replicates of phosphorus measurements. It corresponds to the sum of the phosphorus mass fraction precision and a precision of the standard addition preparation. The combined standard uncertainty for DNA mass fraction determination by the SF ICP–MS method was estimated at 1.34, and 2.28% for the ICP–QMS. The relative standard uncertainty of the molar mass of phosphorus (uP molar mass) is 3.8  106% (IUPAC Table of the Isotopic Composition of the Elements); the relative standard uncertainty of Avogadro’s number (uNA) is 4.4  108% (CODATA database); that of the mass of the genome (ugenome mass) is 3  103%. The uncertainty value of a genome number of nucleotides for L. pneumophila is considered negligible because of the lack of information on the uncertainty associated with sequencing data. The analysis of the DNA mass fraction expanded uncertainty showed that the main component in the complete uncertainty budget was the phosphorus mass fraction determined by ICP–MS.

409

Conclusions

425

A method for DNA quantification, applied to a sample of L. pneumophila, has been presented. The quantification is based on phosphorus mass fraction determination by SF ICP–MS and ICP–QMS and the conversion of the results in DNA mass fraction and DNA genomic units, using the genomic sequencing data. The method was validated on a DNA sample extracted from cell culture material. The validity of the extraction and purification protocols was proven to ensure the complete removal of any possible contaminations of phosphorus, which would have biased the final results. For the sake of confirmation of the ICP–MS values, the ICP–MS results were compared with two routine methods in the field of microbiology, such as UV spectrophotometry and qPCR. The ICP–MS method shows a satisfactory accuracy (within 3–5%, depending on the ICP–MS instrument used) and the possibility of working with a small amount of sample (about 2 ml of DNA solution at about 30 ng g1 P) per measurement. Finally, with the

426

Please cite this article in press as: O. Leclerc et al., Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry, Anal. Biochem. (2013), http://dx.doi.org/10.1016/j.ab.2012.12.023

410 411 412 413 414 415 416 417 418 419 420 421 422 423 424

427 428 429 430 431 432 433 434 435 436 437 438 439 440 441

YABIO 11212

No. of Pages 6, Model 5G

2 February 2013 6

Method development for Legionella DNA quantification / O. Leclerc et al. / Anal. Biochem. xxx (2013) xxx–xxx

455

use of phosphorus calibration standards whose values are traceable to the SI and the possibility of evaluating the contribution of each step of the measurement procedure to the overall uncertainty, the method will potentially provide measurement results traceable to the SI. These characteristics make the method potentially suitable for the characterization of reference materials of nucleic acids, for species other than L. pneumophila. In fact, for species such as bacteria and viruses, for which rapid and precise analyses are needed and are generally performed by qPCR, reliable calibration standards would be necessary. This paper shows that the ICP–MS method can be applied to the characterization of such calibrants, providing that the sequencing data are available, to be able to convert mass fraction values into genomic units.

456

Acknowledgment

457 458

Catherine Perrot is acknowledged for the tests performed by the culture method during sample preparation.

459

References

460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508

[1] D.V. Fraser, T.R. Tsai, W. Orenstein, W.E. Parkin, H.J. Beecham, R.G. Sharrar, J. Harris, G.F. Mallisson, S.M. Martin, J.E. McDade, C.C. Shepard, P.S. Brachman, Legionnaires’ disease: description of an epidemic of pneumonia, N. Engl. J. Med. 297 (1977) 1189–1197. [2] V.L. Yu, J.F. Plouffe, M.C. Pastoris, J.E. Stout, M. Schousboe, A. Widmer, J. Summersgill, T. File, C.M. Health, D.L. Paterson, A. Chereschsky, Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey, J. Infect. Dis. 186 (2002) 127–128. [3] B.J. Marston, H.B. Lipman, R.F. Breiman, Surveillance for Legionnaires’ disease: risk factors for morbidity and mortality, Arch. Intern. Med. 154 (1994) 2417– 2422. [4] A. Doleans, H. Aurell, M. Reyrolle, G. Lina, J. Freney, F. Vadenesch, J. Etienne, S. Jarraud, Clinical and environmental distributions of Legionella strains in France are different, J. Clin. Microbiol. 42 (2004) 458–460. [5] R.P. Cooke, Hazards in water, J. Hosp. Infect. 57 (2004) 290–293. [6] K.D. Ricketts, C.A. Joseph, The impact of new guidelines in Europe for the control and prevention of travel-associated Legionnaires’ disease, Int. J. Hyg. Environ. Health 209 (2006) 547–552. [7] F. Morio, S. Corvec, N. Caroff, F. Le Gallou, H. Drugeon, A. Reynaud, Real-time PCR assay for the detection and quantification of Legionella pneumophila in the environmental water samples: utility for daily practice, Int. J. Hyg. Environ. Health 211 (2008) 403–411. [8] A.L. Ballard, N.K. Fry, L. Chan, S.B. Surman, J.V. Lee, T.G. Harrison, K.J. Towner, Detection of Legionella pneumophila using a real-time PCR hybridization assay, J. Clin. Microbiol. 38 (2000) 4215–4218. [9] K. Levi, J. Smedly, K.J. Towner, Evaluation of a real-time PCR hybridization assay for rapid detection of Legionella pneumophila in hospital and environmental water samples, Clin. Microbiol. Infect. 9 (2003) 754–758. [10] N. Wellinghausen, C. Frost, R. Marre, Detection of legionellae in hospital water samples by quantitative real-time Light Cycler PCR, Appl. Environ. Microbiol. 67 (2001) 3985–3993. [11] M.A. Yanez, C. Carrasco-Serrano, V.M. Barbera, V. Catalan, Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene, Appl. Environ. Microbiol. 71 (2005) 3433–3441. [12] L. Gutierrez, M. Mauriat, J. Pelloux, C. Bellini, O. Van Wuytswinkel, Towards a systematic validation of references in real-time RT-PCR, Plant Cell 20 (2008) 1734–1735. [13] M.J. Cavaluzzi, P.N. Borer, Revised UV extinction coefficients for nucleoside-50 monophosphates and unpaired DNA and RNA, Nucleic Acids Res. 32 (2004) 13. [14] P.G. Held, Measure your purity: assessment of nucleic acid purity via UV absorbance, GIT Lab. J. 5–6 (2008) (2008) 9–11. [15] C.A. English, S. Merson, J.T. Keer, Use of elemental analysis to determine comparative performance of established DNA quantification methods, Anal. Chem. 78 (2006) 4630–4633. [16] M. Holden, R. Haynes, S. Rabb, N.K. Satija, J. Yang, Basic factors affecting quantification of total DNA by UV spectroscopy and PicoGreen fluorescence, J. Agric. Food Chem. 57 (2009) 7221–7226.

442 443 444 445 446 447 448 449 450 451 452 453 454

[17] L.K. Zhang, M.L. Gross, Matrix-assisted laser desorption/ionization mass spectrometry methods for oligodeoxynucleotides: improvements in matrix, detection limits, quantification and sequencing, J. Am. Soc. Mass Spectrosc. 11 (2000) 854–865. [18] B.A. Bruenner, T.T. Yip, T.W. Hutchens, Quantitative analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry, Rapid Commun. Mass Spectrosc. 10 (1996) 1797–1801. [19] S. Lustig, D. Lampaert, K. De Cremer, J. De Kimpe, R. Cornelis, P. Schram, Capability of flatbed electrophoresis (IEF and native PAGE) combined with sector field ICP–MS and autoradiography for the speciation of Cr, Ga, In, Pt and V in incubated serum samples, J. Anal. At. Spectrom. 14 (1999) 1357–1362. [20] J.S. Becker, S.F. Boulyga, J.S. Becker, C. Pickhardt-Damoc, M. Przybylski, Structural identification and quantification of protein phosphorylations after gel electrophoretic separation using Fourier transform ion cyclotron resonance mass spectrometry and laser ablation inductively coupled plasma mass spectrometry, Int. J. Mass Spectrom. 228 (2003) 985–997. [21] J.S. Becker, M. Zoriy, J.S. Becker, C. Pickhardt, M. Przybylski, Determination of phosphorus and metals in human brain proteins after isolation by gel electrophoresis by laser ablation inductively coupled plasma source mass spectrometry, J. Anal. At. Spectrom. 19 (2004) 149–152. [22] C.-F. Yeh, S. Jiang, Determination of monophosphate nucleotides by capillary electrophoresis inductively coupled plasma mass spectrometry, Analyst 127 (2002) 1324–1327. [23] C.L. Camp, B.L. Sharp, H.J. Reid, J. Entwisle, H. Goenaga-Infante, Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma– mass spectrometry, Anal. Bioanal. Chem. Chem. 402 (2012) 367–372. [24] M. Wind, I. Feldmann, N. Jakubowski, W.D. Lehmann, Spotting and quantification of phosphoproteins purified by gel electrophoresis by laser ablation element mass spectrometry with phosphorus-31 detection, Electrophoresis 24 (2003) 1276–1280. [25] M. Edler, N. Jakubowski, M. Linscheid, Quantitation of melphalan DANN adducts using HPLC–ICP MS, J. Mass Spectrom. 41 (2006) 507–516. [26] M. Edler, N. Jakubowski, M. Linscheid, Styrene oxide DNA adducts: quantitative determination using 31P monitoring, Anal. Bioanal. Chem. 381 (2005) 205–211. [27] G. O’Connor, C. Dawson, A. Woolford, K.S. Webb, T. Catterick, Quantitation of oligonucleotides by phosphodiesterase digestion followed by isotope dilution mass spectrometry: proof of concept, Anal. Chem. 74 (2002) 3670–3676. [28] C.D. Medley, B.K. Muralidhara, S. Chico, S. Durban, P. Mehelic, C. Demarest, Quantification of plasmid DNA deposited on gold particles for particlemediated epidermal delivery using ICP–MS, Anal. Bioanal. Chem. 398 (2010) 527–535. [29] I. Yang, M.S. Han, Y.H. Yim, E. Hwang, S.R. Park, A strategy for establishing accurate quantitation standards of oligonucleotides: quantitation of phosphorus of DNA phosphodiester bonds using inductively coupled plasma–optical emission spectroscopy, Anal. Biochem. 335 (2004) 150–161. [30] M.J. Holden, S.A. Rabb, Y.B. Tewari, M.R. Winchester, Traceable phosphorus measurements by ICP–OES and HPLC for the quantitation of DNA, Anal. Chem. 79 (2007) 1536–1541. [31] C.E. Donald, P. Stokes, G. O’Connor, A. Woolford, A comparison of enzymatic digestion for the quantitation of an oligonucleotide by liquid chromatography–isotope dilution mass spectrometry, J. Chromatogr. B 817 (2005) 173–182. [32] R.G. Brennan, S.A. Rabb, M.J. Holden, M.R. Winchester, A. Montaser, Potential primary measurement tool for the quantification of DNA, Anal. Chem. 81 (2009) 3414–3420. [33] International Vocabulary of Metrology—Basic and General Concepts and Associated Terms (VIM). JCGM 200 (2008). [34] The International System of Units. Available from: www.bipm.org/en/si/. [35] Evaluation of Measurement Data—Guide of the Expression of Uncertainty in Measurement (GUM). JCGM 100 (2008). [36] S. Bhat, N. Curac, T. Mostyn, G.S. Bains, K.R. Griffiths, K.R. Emslie, Comparison of methods for accurate quantification of DNA mass concentration with traceability to the International System of Units, Anal. Chem. 82 (2010) 7185– 7192. [37] T.A. Skvortsov, T.L. Azhikina, A review of the transcriptome analysis of bacterial pathogens in vivo: problems and solutions, Russ. J. Bioorg. Chem. 36 (2010) 550–559. [38] M. Chien, I. Morozova, S. Shi, H. Sheng, J. Chen, S. Gomez, G. Asamani, K. Hill, J. Nuara, M. Feder, J. Rineer, J.J. Greenberg, V. Steshenko, S.H. Park, B. Zhao, E. Teplitskaya, J.R. Edwards, S. Pampou, A. Georghiou, I.C. Chou, W. Iannuccilli, M.E. Ulz, D.H. Kim, A. Geringer-Sameth, C. Goldsberry, P. Morozov, S.G. Fischer, G. Segal, X. Qu, A. Rzhetsky, P. Zhang, E. Cayanis, P.J. De Jong, J. Ju, S. Kalachikov, H.A. Shuman, J.J. Russo, The genomic sequence of the accidental pathogen Legionella pneumophila, Science 305 (2004) 1966–1968.

Please cite this article in press as: O. Leclerc et al., Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry, Anal. Biochem. (2013), http://dx.doi.org/10.1016/j.ab.2012.12.023

Q2

509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585