A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys
Identifieur interne : 001475 ( Pmc/Curation ); précédent : 001474; suivant : 001476A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys
Auteurs : Andrea Balboni [Italie] ; Laura Gallina [Italie] ; Alessandra Palladini [Italie] ; Santino Prosperi [Italie] ; Mara Battilani [Italie]Source :
- The Scientific World Journal [ 2356-6140 ] ; 2011.
Abstract
Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (
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DOI: 10.1100/2012/989514
PubMed: 22654650
PubMed Central: 3353321
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Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys</title>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and
Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys</title>
<author><name sortKey="Balboni, Andrea" sort="Balboni, Andrea" uniqKey="Balboni A" first="Andrea" last="Balboni">Andrea Balboni</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum-Università di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Italy</nlm:aff>
<country xml:lang="fr">Italie</country>
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<author><name sortKey="Gallina, Laura" sort="Gallina, Laura" uniqKey="Gallina L" first="Laura" last="Gallina">Laura Gallina</name>
<affiliation wicri:level="1"><nlm:aff id="I1">Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum-Università di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Italy</nlm:aff>
<country xml:lang="fr">Italie</country>
<wicri:regionArea>Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum-Università di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia</wicri:regionArea>
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<author><name sortKey="Palladini, Alessandra" sort="Palladini, Alessandra" uniqKey="Palladini A" first="Alessandra" last="Palladini">Alessandra Palladini</name>
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<author><name sortKey="Battilani, Mara" sort="Battilani, Mara" uniqKey="Battilani M" first="Mara" last="Battilani">Mara Battilani</name>
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<front><div type="abstract" xml:lang="en"><p>Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (<italic>Rhinolophus ferrumequinum</italic>
) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.</p>
</div>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">ScientificWorldJournal</journal-id>
<journal-id journal-id-type="iso-abbrev">ScientificWorldJournal</journal-id>
<journal-id journal-id-type="publisher-id">TSWJ</journal-id>
<journal-title-group><journal-title>The Scientific World Journal</journal-title>
</journal-title-group>
<issn pub-type="ppub">2356-6140</issn>
<issn pub-type="epub">1537-744X</issn>
<publisher><publisher-name>The Scientific World Journal</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">22654650</article-id>
<article-id pub-id-type="pmc">3353321</article-id>
<article-id pub-id-type="doi">10.1100/2012/989514</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and
Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Balboni</surname>
<given-names>Andrea</given-names>
</name>
<xref ref-type="aff" rid="I1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Gallina</surname>
<given-names>Laura</given-names>
</name>
<xref ref-type="aff" rid="I1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Palladini</surname>
<given-names>Alessandra</given-names>
</name>
<xref ref-type="aff" rid="I2"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Prosperi</surname>
<given-names>Santino</given-names>
</name>
<xref ref-type="aff" rid="I1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Battilani</surname>
<given-names>Mara</given-names>
</name>
<xref ref-type="aff" rid="I1"><sup>1</sup>
</xref>
<xref ref-type="corresp" rid="cor1">*</xref>
</contrib>
</contrib-group>
<aff id="I1"><sup>1</sup>
Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum-Università di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Italy</aff>
<aff id="I2"><sup>2</sup>
Dipartimento di Biologia Animale, Università degli Studi di Pavia, Via Taramelli 24, 27100 Pavia, Italy</aff>
<author-notes><corresp id="cor1">*Mara Battilani: <email>mara.battilani@unibo.it</email>
</corresp>
<fn fn-type="other"><p>Academic Editors: R. Marquet, M. McCrae, and S. Y. Morozov</p>
</fn>
</author-notes>
<pub-date pub-type="collection"><year>2012</year>
</pub-date>
<pub-date pub-type="epub"><day>22</day>
<month>11</month>
<year>2011</year>
</pub-date>
<volume>2012</volume>
<elocation-id>989514</elocation-id>
<history><date date-type="received"><day>5</day>
<month>10</month>
<year>2011</year>
</date>
<date date-type="accepted"><day>16</day>
<month>11</month>
<year>2011</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2012 Andrea Balboni et al.</copyright-statement>
<copyright-year>2012</copyright-year>
<license xlink:href="https://creativecommons.org/licenses/by/3.0/"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
</license>
</permissions>
<abstract><p>Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (<italic>Rhinolophus ferrumequinum</italic>
) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.</p>
</abstract>
</article-meta>
</front>
<floats-group><fig id="fig1" position="float"><label>Figure 1</label>
<caption><p>Distribution of bat sampling in Italy. Sites of bat sampling from August to November 2009. For correspondence between letters and places, see <xref ref-type="table" rid="tab1">Table 1</xref>
.</p>
</caption>
<graphic xlink:href="TSWJ2012-989514.001"></graphic>
</fig>
<fig id="fig2" position="float"><label>Figure 2</label>
<caption><p>Multiple sequence alignment of primer binding sites. The alignments include 10 SARS-related coronavirus reference strains: SBRp3 (Bat SARS CoV Rp3/2004, DQ071615, identified from <italic>Rhinolophus pearsoni</italic>
), SBRs (SARS coronavirus Rs_672/2006, FJ588686, identified from <italic>Rhinolophus sinicus</italic>
), SHTor2 (SARS coronavirus Tor2, AY274119, identified from Human), SC (Civet SARS CoV SZ16/2003, AY304488, identified from<italic> Paguma larvata</italic>
), SB (SARS coronavirus isolate CFB/SZ/94/03, AY545919, identified from<italic> Melogale moschata</italic>
), SBRf1 (Bat SARS CoV Rf1/2004, DQ412042, identified from <italic>Rhinolophus ferrumequinum</italic>
), SBRm1 (Bat SARS CoV Rm1/2004, DQ412043, identified from <italic>Rhinolophus macrotis</italic>
), SBHKU3-1 (Bat coronavirus HKU3, DQ022305, identified from <italic>Rhinolophus sinicus</italic>
), SB273 (Bat CoV 273/2005, DQ648856, identified from <italic>Rhinolophus ferrumequinum</italic>
), and SB279 (Bat CoV 279/2005, DQ648857, identified from <italic>Rhinolophus macrotis</italic>
).</p>
</caption>
<graphic xlink:href="TSWJ2012-989514.002"></graphic>
</fig>
<fig id="fig3" position="float"><label>Figure 3</label>
<caption><p>Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10<sup>−1</sup>
copies/<italic>μ</italic>
L. For recombinant plasmid dilution with a concentration of 1 × 10<sup>−1</sup>
copies/<italic>μ</italic>
L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10<sup>3</sup>
, 1 × 10<sup>2</sup>
, 1 × 10<sup>1</sup>
, 1 × 10<sup>0</sup>
, and 1 × 10<sup>−1</sup>
copies/<italic>μ</italic>
L, respectively. W: no template control (water).</p>
</caption>
<graphic xlink:href="TSWJ2012-989514.003"></graphic>
</fig>
<fig id="fig4" position="float"><label>Figure 4</label>
<caption><p>Melting curve analysis of standard plasmid dilutions and sample 893/09-11. In gray: signals obtained from the standard plasmid dilutions; in black: signals obtained from the sample 893.09-11; derivative −<italic>dF</italic>
/<italic>dT</italic>
where <italic>F</italic>
is fluorescence and <italic>T</italic>
is time; °C: temperature (centigrade).</p>
</caption>
<graphic xlink:href="TSWJ2012-989514.004"></graphic>
</fig>
<fig id="fig5" position="float"><label>Figure 5</label>
<caption><p>Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10<sup>4</sup>
copies/<italic>μ</italic>
L; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.</p>
</caption>
<graphic xlink:href="TSWJ2012-989514.005"></graphic>
</fig>
<table-wrap id="tab1" position="float"><label>Table 1</label>
<caption><p>Bats tested for coronavirus infection using SYBR Green real-time PCR assay.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"></th>
<th align="left" rowspan="1" colspan="1">Location</th>
<th align="center" rowspan="1" colspan="1">Date</th>
<th align="center" rowspan="1" colspan="1">No. of bats</th>
<th align="center" rowspan="1" colspan="1">Positives</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">San Cesario sul Panaro, MO</td>
<td align="center" rowspan="1" colspan="1">16/08/2009</td>
<td align="center" rowspan="1" colspan="1">11</td>
<td align="center" rowspan="1" colspan="1">10</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">B</td>
<td align="left" rowspan="1" colspan="1">Sigillo, PG</td>
<td align="center" rowspan="1" colspan="1">18/09/2009</td>
<td align="center" rowspan="1" colspan="1">1</td>
<td align="center" rowspan="1" colspan="1">0</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">C</td>
<td align="left" rowspan="1" colspan="1">Monte Croara, S. Lazzaro, BO</td>
<td align="center" rowspan="1" colspan="1">05/10/2009</td>
<td align="center" rowspan="1" colspan="1">2</td>
<td align="center" rowspan="1" colspan="1">0</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">D</td>
<td align="left" rowspan="1" colspan="1">Piobesi d'Alba, CN</td>
<td align="center" rowspan="1" colspan="1">11/10/2009</td>
<td align="center" rowspan="1" colspan="1">10</td>
<td align="center" rowspan="1" colspan="1">0</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">E</td>
<td align="left" rowspan="1" colspan="1">Rossana, CN</td>
<td align="center" rowspan="1" colspan="1">11/10/2009</td>
<td align="center" rowspan="1" colspan="1">12</td>
<td align="center" rowspan="1" colspan="1">3</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">F</td>
<td align="left" rowspan="1" colspan="1">Giovo, SV</td>
<td align="center" rowspan="1" colspan="1">19/10/2009</td>
<td align="center" rowspan="1" colspan="1">3</td>
<td align="center" rowspan="1" colspan="1">3</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">G</td>
<td align="left" rowspan="1" colspan="1">Val di Trebbia, PC</td>
<td align="center" rowspan="1" colspan="1">20/10/2009</td>
<td align="center" rowspan="1" colspan="1">3</td>
<td align="center" rowspan="1" colspan="1">1</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">H</td>
<td align="left" rowspan="1" colspan="1">Castell'arquato, PC</td>
<td align="center" rowspan="1" colspan="1">04/11/2009</td>
<td align="center" rowspan="1" colspan="1">3</td>
<td align="center" rowspan="1" colspan="1">2</td>
</tr>
<tr><td align="left" colspan="5" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">Total</td>
<td align="center" rowspan="1" colspan="1"></td>
<td align="center" rowspan="1" colspan="1">45</td>
<td align="center" rowspan="1" colspan="1">19</td>
</tr>
</tbody>
</table>
</table-wrap>
<table-wrap id="tab2" position="float"><label>Table 2</label>
<caption><p>Intra- and interassay variability of the SYBR green real-time PCR method.</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" colspan="5" rowspan="1">Intra- and interassay variability</th>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1">Samples</td>
<td align="center" rowspan="1" colspan="1">Replicate (and assay) numbers</td>
<td align="center" rowspan="1" colspan="1">Mean (SD)</td>
<td align="center" rowspan="1" colspan="1">log<sub>10</sub>
mean (SD)</td>
<td align="center" rowspan="1" colspan="1">log<sub>10</sub>
CV%</td>
</tr>
<tr><td align="left" colspan="5" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" colspan="5" rowspan="1">Intraassay variability</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">A1</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">1,86<italic>E</italic>
+ 06 (2,35<italic>E</italic>
+ 05)</td>
<td align="center" rowspan="1" colspan="1">6,27 (0,05)</td>
<td align="center" rowspan="1" colspan="1">0,84</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">A2</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">2,03<italic>E</italic>
+ 05 (1,77<italic>E</italic>
+ 04)</td>
<td align="center" rowspan="1" colspan="1">5,31 (0,04)</td>
<td align="center" rowspan="1" colspan="1">0,71</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">A3</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">2,00<italic>E</italic>
+ 04 (8,96<italic>E</italic>
+ 02)</td>
<td align="center" rowspan="1" colspan="1">4,30 (0,02)</td>
<td align="center" rowspan="1" colspan="1">0,45</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">A4</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">2,26<italic>E</italic>
+ 03 (2,53<italic>E</italic>
+ 02)</td>
<td align="center" rowspan="1" colspan="1">3,35 (0,05)</td>
<td align="center" rowspan="1" colspan="1">1,41</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">A5</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">2,09<italic>E</italic>
+ 02 (8,22)</td>
<td align="center" rowspan="1" colspan="1">2,32 (0,02)</td>
<td align="center" rowspan="1" colspan="1">0,74</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">A6</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">1,98<italic>E</italic>
+ 01 (5,29)</td>
<td align="center" rowspan="1" colspan="1">1,28 (0,13)</td>
<td align="center" rowspan="1" colspan="1">9,99</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">A7</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">2,09<italic>E</italic>
+ 00 (0,44)</td>
<td align="center" rowspan="1" colspan="1">0,31 (0,09)</td>
<td align="center" rowspan="1" colspan="1">28,58</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">B1</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">5,79<italic>E</italic>
+ 02 (4,65<italic>E</italic>
+ 01)</td>
<td align="center" rowspan="1" colspan="1">2,76 (0,03)</td>
<td align="center" rowspan="1" colspan="1">1,27</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">B2</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">2,00<italic>E</italic>
+ 00 (0,10)</td>
<td align="center" rowspan="1" colspan="1">0,30 (0,02)</td>
<td align="center" rowspan="1" colspan="1">7,56</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">B3</td>
<td align="center" rowspan="1" colspan="1">3 (1)</td>
<td align="center" rowspan="1" colspan="1">4,01<italic>E</italic>
+ 00 (0,74)</td>
<td align="center" rowspan="1" colspan="1">0,60 (0,08)</td>
<td align="center" rowspan="1" colspan="1">13,68</td>
</tr>
<tr><td align="left" colspan="5" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" colspan="5" rowspan="1">Interassay variability</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">B1</td>
<td align="center" rowspan="1" colspan="1">3 (3)</td>
<td align="center" rowspan="1" colspan="1">6,08<italic>E</italic>
+ 02 (5,58<italic>E</italic>
+ 01)</td>
<td align="center" rowspan="1" colspan="1">2,78 (0,04)</td>
<td align="center" rowspan="1" colspan="1">1,38</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">B2</td>
<td align="center" rowspan="1" colspan="1">3 (3)</td>
<td align="center" rowspan="1" colspan="1">2,06<italic>E</italic>
+ 00 (0,34)</td>
<td align="center" rowspan="1" colspan="1">0,29 (0,07)</td>
<td align="center" rowspan="1" colspan="1">23,97</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">B3</td>
<td align="center" rowspan="1" colspan="1">3 (3)</td>
<td align="center" rowspan="1" colspan="1">3,11<italic>E</italic>
+ 00 (0,82)</td>
<td align="center" rowspan="1" colspan="1">0,46 (0,13)</td>
<td align="center" rowspan="1" colspan="1">28,79</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn><p>Successive 10-fold dilutions of recombinant plasmid: A1 (2 × 10<sup>6</sup>
), A2 (2 × 10<sup>5</sup>
), A3 (2 × 10<sup>4</sup>
), A4 (2 × 10<sup>3</sup>
), A5 (2 × 10<sup>2</sup>
), A6 (2 × 10<sup>1</sup>
), and A7 (2 × 10<sup>0</sup>
).</p>
</fn>
<fn><p>Three bat samples with different viral concentrations: B1 (10<sup>2</sup>
), B2 and B3 (10<sup>0</sup>
). </p>
</fn>
<fn><p>SD: standard deviation.</p>
</fn>
<fn><p>CV: coefficient of variation.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</floats-group>
</pmc>
</record>
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