Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases
Identifieur interne : 000947 ( Pmc/Corpus ); précédent : 000946; suivant : 000948Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases
Auteurs : Fujiko Sunaga ; Shinobu Tsuchiaka ; Mai Kishimoto ; Hiroshi Aoki ; Mari Kakinoki ; Katsumasa Kure ; Hanako Okumura ; Maho Okumura ; Atsushi Okumura ; Makoto Nagai ; Tsutomu Omatsu ; Tetsuya MizutaniSource :
- The Journal of Veterinary Medical Science [ 0916-7250 ] ; 2019.
Abstract
The etiology of Porcine respiratory disease complex is complicated by infections with
multiple pathogens, and multiple infections increase the difficulty in identifying the
causal pathogen. In this present study, we developed a detection system of microbes from
porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a
broad range of pathogens associated with porcine respiratory diseases in a single run. We
selected 17 porcine respiratory pathogens (
Url:
DOI: 10.1292/jvms.19-0063
PubMed: 31866601
PubMed Central: 7041981
Links to Exploration step
PMC:7041981Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Development of a one-run real-time PCR detection system for pathogens
associated with porcine respiratory diseases</title><author><name sortKey="Sunaga, Fujiko" sort="Sunaga, Fujiko" uniqKey="Sunaga F" first="Fujiko" last="Sunaga">Fujiko Sunaga</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Tsuchiaka, Shinobu" sort="Tsuchiaka, Shinobu" uniqKey="Tsuchiaka S" first="Shinobu" last="Tsuchiaka">Shinobu Tsuchiaka</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Kishimoto, Mai" sort="Kishimoto, Mai" uniqKey="Kishimoto M" first="Mai" last="Kishimoto">Mai Kishimoto</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Aoki, Hiroshi" sort="Aoki, Hiroshi" uniqKey="Aoki H" first="Hiroshi" last="Aoki">Hiroshi Aoki</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Kakinoki, Mari" sort="Kakinoki, Mari" uniqKey="Kakinoki M" first="Mari" last="Kakinoki">Mari Kakinoki</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Kure, Katsumasa" sort="Kure, Katsumasa" uniqKey="Kure K" first="Katsumasa" last="Kure">Katsumasa Kure</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Okumura, Hanako" sort="Okumura, Hanako" uniqKey="Okumura H" first="Hanako" last="Okumura">Hanako Okumura</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Okumura, Maho" sort="Okumura, Maho" uniqKey="Okumura M" first="Maho" last="Okumura">Maho Okumura</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Okumura, Atsushi" sort="Okumura, Atsushi" uniqKey="Okumura A" first="Atsushi" last="Okumura">Atsushi Okumura</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Nagai, Makoto" sort="Nagai, Makoto" uniqKey="Nagai M" first="Makoto" last="Nagai">Makoto Nagai</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Omatsu, Tsutomu" sort="Omatsu, Tsutomu" uniqKey="Omatsu T" first="Tsutomu" last="Omatsu">Tsutomu Omatsu</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Mizutani, Tetsuya" sort="Mizutani, Tetsuya" uniqKey="Mizutani T" first="Tetsuya" last="Mizutani">Tetsuya Mizutani</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31866601</idno><idno type="pmc">7041981</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041981</idno><idno type="RBID">PMC:7041981</idno><idno type="doi">10.1292/jvms.19-0063</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000947</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000947</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Development of a one-run real-time PCR detection system for pathogens
associated with porcine respiratory diseases</title><author><name sortKey="Sunaga, Fujiko" sort="Sunaga, Fujiko" uniqKey="Sunaga F" first="Fujiko" last="Sunaga">Fujiko Sunaga</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Tsuchiaka, Shinobu" sort="Tsuchiaka, Shinobu" uniqKey="Tsuchiaka S" first="Shinobu" last="Tsuchiaka">Shinobu Tsuchiaka</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Kishimoto, Mai" sort="Kishimoto, Mai" uniqKey="Kishimoto M" first="Mai" last="Kishimoto">Mai Kishimoto</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Aoki, Hiroshi" sort="Aoki, Hiroshi" uniqKey="Aoki H" first="Hiroshi" last="Aoki">Hiroshi Aoki</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Kakinoki, Mari" sort="Kakinoki, Mari" uniqKey="Kakinoki M" first="Mari" last="Kakinoki">Mari Kakinoki</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Kure, Katsumasa" sort="Kure, Katsumasa" uniqKey="Kure K" first="Katsumasa" last="Kure">Katsumasa Kure</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Okumura, Hanako" sort="Okumura, Hanako" uniqKey="Okumura H" first="Hanako" last="Okumura">Hanako Okumura</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Okumura, Maho" sort="Okumura, Maho" uniqKey="Okumura M" first="Maho" last="Okumura">Maho Okumura</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Okumura, Atsushi" sort="Okumura, Atsushi" uniqKey="Okumura A" first="Atsushi" last="Okumura">Atsushi Okumura</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Nagai, Makoto" sort="Nagai, Makoto" uniqKey="Nagai M" first="Makoto" last="Nagai">Makoto Nagai</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Omatsu, Tsutomu" sort="Omatsu, Tsutomu" uniqKey="Omatsu T" first="Tsutomu" last="Omatsu">Tsutomu Omatsu</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Mizutani, Tetsuya" sort="Mizutani, Tetsuya" uniqKey="Mizutani T" first="Tetsuya" last="Mizutani">Tetsuya Mizutani</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Veterinary Medical Science</title><idno type="ISSN">0916-7250</idno><idno type="eISSN">1347-7439</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The etiology of Porcine respiratory disease complex is complicated by infections with
multiple pathogens, and multiple infections increase the difficulty in identifying the
causal pathogen. In this present study, we developed a detection system of microbes from
porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a
broad range of pathogens associated with porcine respiratory diseases in a single run. We
selected 17 porcine respiratory pathogens (<italic>Actinobacillus
pleuropneumoniae</italic>, <italic>Boldetella bronchiseptica</italic>,
<italic>Haemophilus parasuis</italic>, <italic>Pasteurella multocida</italic>,
<italic>Pasteurella multocid</italic>a toxin, <italic>Streptococcus suis</italic>,
<italic>Mycoplasma hyopneumoniae</italic>, <italic>Mycoplasma hyorhinis</italic>,
<italic>Mycoplasma hyosynovie</italic>, porcine circovirus 2, pseudorabies virus,
porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory
virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating
encephalomyelitis virus) as detection targets and designed novel specific primer-probe
sets for seven of them. In sensitivity test by using standard curves from synthesized DNA,
all primer-probe sets showed high sensitivity. However, porcine reproductive and
respiratory virus is known to have a high frequency of genetic mutations, and the primer
and probe sequences will need to be checked at a considerable frequency when performing
Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory
symptoms on six farms were tested by the Dempo-PCR to validate the assay’s clinical
performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and
porcine reproductive and respiratory virus US strain, <italic>Mycoplasma
hyorhinis</italic>, <italic>Haemophilus parasuis</italic>, and porcine cytomegalovirus
were detected at high frequency. These results suggest that Dempo-PCR assay can be applied
as a screening system with wide detection targets.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Angen, O" uniqKey="Angen O">O. Angen</name></author><author><name sortKey="Jensen, J" uniqKey="Jensen J">J. Jensen</name></author><author><name sortKey="Lavritsen, D T" uniqKey="Lavritsen D">D. T. Lavritsen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bonifait, L" uniqKey="Bonifait L">L. Bonifait</name></author><author><name sortKey="Veillette, M" uniqKey="Veillette M">M. Veillette</name></author><author><name sortKey="Letourneau, V" uniqKey="Letourneau V">V. Létourneau</name></author><author><name sortKey="Grenier, D" uniqKey="Grenier D">D. Grenier</name></author><author><name sortKey="Duchaine, C" uniqKey="Duchaine C">C. Duchaine</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Edington, N" uniqKey="Edington N">N. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Vet Med Sci</journal-id><journal-id journal-id-type="iso-abbrev">J. Vet. Med. Sci</journal-id><journal-id journal-id-type="publisher-id">JVMS</journal-id><journal-title-group><journal-title>The Journal of Veterinary Medical Science</journal-title></journal-title-group><issn pub-type="ppub">0916-7250</issn><issn pub-type="epub">1347-7439</issn><publisher><publisher-name>The Japanese Society of Veterinary Science</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31866601</article-id><article-id pub-id-type="pmc">7041981</article-id><article-id pub-id-type="publisher-id">19-0063</article-id><article-id pub-id-type="doi">10.1292/jvms.19-0063</article-id><article-categories><subj-group subj-group-type="heading"><subject>Virology</subject><subj-group><subject>Full Paper</subject></subj-group></subj-group></article-categories><title-group><article-title>Development of a one-run real-time PCR detection system for pathogens
associated with porcine respiratory diseases</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>SUNAGA</surname><given-names>Fujiko</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref rid="cor1" ref-type="corresp"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>TSUCHIAKA</surname><given-names>Shinobu</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>KISHIMOTO</surname><given-names>Mai</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>AOKI</surname><given-names>Hiroshi</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>KAKINOKI</surname><given-names>Mari</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>KURE</surname><given-names>Katsumasa</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>OKUMURA</surname><given-names>Hanako</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>OKUMURA</surname><given-names>Maho</given-names></name><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>OKUMURA</surname><given-names>Atsushi</given-names></name><xref ref-type="aff" rid="aff7"><sup>7</sup></xref></contrib><contrib contrib-type="author"><name><surname>NAGAI</surname><given-names>Makoto</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>OMATSU</surname><given-names>Tsutomu</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>MIZUTANI</surname><given-names>Tetsuya</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><aff id="aff1"><label>1)</label>Laboratory of Infectious Disease, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan</aff><aff id="aff2"><label>2)</label>Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai, Fuchu, Tokyo 183–8509, Japan</aff><aff id="aff3"><label>3)</label>The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501-1193, Japan</aff><aff id="aff4"><label>4)</label>Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan</aff><aff id="aff5"><label>5)</label>Value Farm Consulting Co., Ltd., 1704-3 Nishi Oi, Tsukuba, Ibaraki 300-1260, Japan</aff><aff id="aff6"><label>6)</label>Drexel University Dornsife School of Public Health, Philadelphia PA 19104, USA</aff><aff id="aff7"><label>7)</label>Centre for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY 10032, USA</aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>Correspondence to: Sunaga, F.: <email xlink:href="sunaga@azabu-u.ac.jp">sunaga@azabu-u.ac.jp</email></corresp></author-notes><pub-date pub-type="epub"><day>23</day><month>12</month><year>2019</year></pub-date><pub-date pub-type="ppub"><month>2</month><year>2020</year></pub-date><volume>82</volume><issue>2</issue><fpage>217</fpage><lpage>223</lpage><history><date date-type="received"><day>29</day><month>1</month><year>2019</year></date><date date-type="accepted"><day>12</day><month>12</month><year>2019</year></date></history><permissions><copyright-statement>©2020 The Japanese Society of Veterinary Science</copyright-statement><copyright-year>2020</copyright-year><license license-type="open-access"><license-p>This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0:
<ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by-nc-nd/4.0/">https://creativecommons.org/licenses/by-nc-nd/4.0/</ext-link>)</license-p></license></permissions><abstract><p>The etiology of Porcine respiratory disease complex is complicated by infections with
multiple pathogens, and multiple infections increase the difficulty in identifying the
causal pathogen. In this present study, we developed a detection system of microbes from
porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a
broad range of pathogens associated with porcine respiratory diseases in a single run. We
selected 17 porcine respiratory pathogens (<italic>Actinobacillus
pleuropneumoniae</italic>, <italic>Boldetella bronchiseptica</italic>,
<italic>Haemophilus parasuis</italic>, <italic>Pasteurella multocida</italic>,
<italic>Pasteurella multocid</italic>a toxin, <italic>Streptococcus suis</italic>,
<italic>Mycoplasma hyopneumoniae</italic>, <italic>Mycoplasma hyorhinis</italic>,
<italic>Mycoplasma hyosynovie</italic>, porcine circovirus 2, pseudorabies virus,
porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory
virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating
encephalomyelitis virus) as detection targets and designed novel specific primer-probe
sets for seven of them. In sensitivity test by using standard curves from synthesized DNA,
all primer-probe sets showed high sensitivity. However, porcine reproductive and
respiratory virus is known to have a high frequency of genetic mutations, and the primer
and probe sequences will need to be checked at a considerable frequency when performing
Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory
symptoms on six farms were tested by the Dempo-PCR to validate the assay’s clinical
performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and
porcine reproductive and respiratory virus US strain, <italic>Mycoplasma
hyorhinis</italic>, <italic>Haemophilus parasuis</italic>, and porcine cytomegalovirus
were detected at high frequency. These results suggest that Dempo-PCR assay can be applied
as a screening system with wide detection targets.</p></abstract><kwd-group><kwd>diagnosis</kwd><kwd>porcine</kwd><kwd>respiratory disease</kwd><kwd>TaqMan real-time PCR</kwd></kwd-group></article-meta></front><body><p>Respiratory infections constitute some of the most important diseases of growing pigs and
result in substantial economic losses [<xref rid="r17" ref-type="bibr">17</xref>]. Multiple
pathogens contribute to a polymicrobial infection known as Porcine Respiratory Disease Complex
(PRDC) [<xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r9" ref-type="bibr">9</xref>,
<xref rid="r21" ref-type="bibr">21</xref>]. The most commonly isolated pathogens are porcine
reproductive and respiratory virus (PRRSV), swine influenza A virus (SIV), porcine circovirus
2 (PCV2), and <italic>Mycoplasma hyopneumoniae</italic>. The other pathogens associated with
PRDC are <italic>Streptococcus</italic> suis, <italic>Actinobacillus</italic>pleuropneumoniae, <italic>Pasteurella multocida</italic>, <italic>Pasteurella</italic>multocida toxin, <italic>Boldetella bronchiseptica</italic>, <italic>Haemophilus</italic>parasuis, <italic>Mycoplasma hyorhinis</italic>, <italic>Mycoplasma hyosynovie,</italic>pseudorabies virus (PRV), porcine respiratory corona virus (PRCV), Porcine cytomegalovirus
(PCMV) [<xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r8" ref-type="bibr">8</xref>,
<xref rid="r15" ref-type="bibr">15</xref>, <xref rid="r20" ref-type="bibr">20</xref>].
Infection with each single pathogen does not necessarily result in appearance of symptoms, but
complex infections with a variety of pathogens, including the indigenous agents, develop
severe conditions. Infections with such multiple pathogens make it difficult to rapidly
identify the etiology of PRDC. To adopt appropriate measures, such as vaccination or hygiene
management, and to minimize the economic loss of PRDC, it is necessary to quickly, accurately
and comprehensively detect multiple pathogens present in varying proportions in each herd.
Serological tests [<xref rid="r13" ref-type="bibr">13</xref>], pathogen isolation [<xref rid="r22" ref-type="bibr">22</xref>] and PCR-based tests [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r11" ref-type="bibr">11</xref>] are currently available to diagnose
PRDC in laboratories. Most tests are based on a one assay-one pathogen approach, and they are
not enough for diagnosis of PRDC in terms of comprehensiveness and rapidity. Tsuchiaka
<italic>et al</italic>. previously developed a system to detect microbes in bovine diarrhea
by TaqMan real-time PCR, permitting the simultaneous screening of 19 pathogens associated with
diarrhea [<xref rid="r26" ref-type="bibr">26</xref>]. TaqMan real-time PCR possesses the
advantages of high sensitivity, high specificity, and simple operation.</p><p>The objective of this study is to develop a system based on TaqMan real-time PCR that can
detect 17 pathogens, including viruses and bacteria, associated with porcine respiratory
diseases in one run.</p><sec sec-type="materials|methods" id="s1"><title>MATERIALS AND METHODS</title><sec><title>Primer and probe design</title><p>A total of 17 primer-probe sets were used to detect pathogens that certainly or possibly
cause respiratory diseases on porcine. Each primer-probe set was designed to detect a
single target pathogen. New primer-probe sets were designed for <italic>Pasteurella
multocida</italic> and toxin, <italic>M. hyosynovie</italic>, PCV2, PCMV, SIV and PHEV
using the PrimerQuest software (Integrated DNA Technologies, Inc., Coralville, IA, USA)
based on consensus sequences of each pathogen obtained from the GenBank database. Primer
and probe information and their target pathogens are summarized in <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title>The nucleotide information of the primer-probe sets used for Dempo-PCR</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" rowspan="1" colspan="1">Target pathogen</th><th align="center" rowspan="1" colspan="1">Target gene</th><th align="left" rowspan="1" colspan="1"></th><th align="center" rowspan="1" colspan="1">Primer/Probe (FAM/TAMRA) sequence 5′-3′</th><th align="center" rowspan="1" colspan="1">Reference No.</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Actinobacillus
pleuropneumoniae</italic></td><td align="left" valign="top" rowspan="3" colspan="1">omlA</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">GGGGACGTAACTCGGTGATT</td><td align="center" rowspan="1" colspan="1">[<xref rid="r1" ref-type="bibr">1</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">GCTCACCAACGTTTGCTCAT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">CGGTGCGGACACCTATATCT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Boldetella
bronchiseptica</italic></td><td align="left" valign="top" rowspan="3" colspan="1">Fla2</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">AGGCTCCCAAGAGAGAAAGGCTT</td><td align="center" rowspan="1" colspan="1">[<xref rid="r24" ref-type="bibr">24</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">AAACCTGCCGTAATCCAGGC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">ACCGGGCAGCTAGGCCGC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Haemophilus
parasuis</italic></td><td align="left" valign="top" rowspan="3" colspan="1">CTinfF1</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">CGACTTACTTGAAGCCATTCTTCTT</td><td align="center" rowspan="1" colspan="1">[<xref rid="r27" ref-type="bibr">27</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">CCGCTTGCCATACCCTCTT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">ATCGGAAGTATTAGAATTAAGTGC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Pasteurella
multocida</italic></td><td align="left" valign="top" rowspan="3" colspan="1">Kmt1</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">GGGCTTGTCGGTAGTCTTT</td><td align="center" rowspan="1" colspan="1">This study</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">CGGCAAATAACAATAAGCTGAGTA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">CGGCGCAACTGATTGGACGTTATT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Pasteurella multocida
toxin</italic></td><td align="left" valign="top" rowspan="3" colspan="1">toxA</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">GATACAGTAATTTCAGCGCCTTT</td><td align="center" rowspan="1" colspan="1">This study</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">GCAGGAAGTTCCCAGTAATTTG</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">TGGTGCGATTCCAGAGGCAATAGA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Streptococcus suis</italic></td><td align="left" valign="top" rowspan="3" colspan="1">16S RNA gene</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">AGAAGAGTGGAAAGTTTCTCA</td><td align="center" rowspan="1" colspan="1">[<xref rid="r2" ref-type="bibr">2</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">TCACAGTTTCCAAAGCGT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">CAAACCGCCTGCGCTCGCTTTACG</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Mycoplasma
hyopneumoniae</italic></td><td align="left" valign="top" rowspan="3" colspan="1"><italic>p102</italic></td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">GTCAAAGTCAAAGTCAGCAAAC</td><td align="center" rowspan="1" colspan="1">[<xref rid="r18" ref-type="bibr">18</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">AGCTGTTCAAATGCTTGTCC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">ACCAGTTTCCACTTCATCGCCTCA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Mycoplasma
hyorhinis</italic></td><td align="left" valign="top" rowspan="3" colspan="1"><italic>p37</italic></td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">TATCTCATTGACCTTGACTAAC</td><td align="center" rowspan="1" colspan="1">[<xref rid="r25" ref-type="bibr">25</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">ATTTTCGCCAATAGCATTTG</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">CATCCTCTTGCTTGACTACTCCTG</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1"><italic>Mycoplasma
hyosynovie</italic></td><td align="left" valign="top" rowspan="3" colspan="1">rpoB</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">GCTGATATTCCTAACGCATCAAAC</td><td align="center" rowspan="1" colspan="1">This study</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">CACCTTTAGGGCTAGTTCTTCC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">TGACCAAGGAATTGTTAGAGTTGGATCTGA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Porcine circovirus 2</td><td align="left" valign="top" rowspan="3" colspan="1">ORF2 (capsid protein)</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">CCATCTTGGCCAGATCCTC</td><td align="center" rowspan="1" colspan="1">This study</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">AGGCGGGTGTTGAAGATG</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">CACCGTTACCGCTGGAGAAGGAAA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Pseudorabies virus</td><td align="left" valign="top" rowspan="3" colspan="1">gE</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">CTTCCACTCGCAGCTCTTCTC</td><td align="center" rowspan="1" colspan="1">[<xref rid="r16" ref-type="bibr">16</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">GTRAAGTTCTCGCGCGAGT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">TTCGACCTGATGCCGC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Porcine cytomegalovirus</td><td align="left" valign="top" rowspan="3" colspan="1">gB</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">CTCTCAAGAAGATGCCGTCTG</td><td align="center" rowspan="1" colspan="1">This study</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">CTGCTGATATTCCAAGTGACGTA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">ACAAAGCCTAGCCCGAGCGTATT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Swine influenza A virus</td><td align="left" valign="top" rowspan="3" colspan="1">matrix (M) gene</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">GGCTCTCATGGAATGGCTAAA</td><td align="center" rowspan="1" colspan="1">This study</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">TGCAGTCCTCGCTCACT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">TTTGTGTTCACGCTCACCGTGC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Porcine reproductive and respiratory
virus US strain</td><td align="left" valign="top" rowspan="3" colspan="1">3′UTR</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">ATRATGRGCTGGCATTC</td><td align="center" rowspan="1" colspan="1">[<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r28" ref-type="bibr">28</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">ACACGGTCGCCCTAATTG</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">TGTGGTGAATGGCACTGATTGACA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Porcine reproductive and respiratory
virus EU strain</td><td align="left" valign="top" rowspan="3" colspan="1">ORF7</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">GCACCACCTCACCCAGAC</td><td align="center" rowspan="1" colspan="1">[<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r28" ref-type="bibr">28</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">CAGTTCCTGCGCCTTGAT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">CCTCTGYYTGCAATCGATCCAGAC</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Porcine respiratory coronavirus</td><td align="left" valign="top" rowspan="3" colspan="1">Nucleocapsid</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">AGCTATTGGACTTCAAAGGAAGATG</td><td align="center" rowspan="1" colspan="1">[<xref rid="r19" ref-type="bibr">19</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">CATAGGCATTAATCTGCTGAAGGAA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">TCACGTTCACACACAAATACCACTTGCCA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">Porcine hemagglutinating
encephalomyelitis virus</td><td align="left" valign="top" rowspan="3" colspan="1">Spike protein</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">CAACCAGATCCTTCCACATATAAAG</td><td align="center" rowspan="1" colspan="1">This study</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">GAGCAATCATCCTCCACAAGA</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">ATACAACCAGGTCAGCATTGCCCT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="3" colspan="1">β-actin</td><td align="left" valign="top" rowspan="3" colspan="1">Actin</td><td align="center" rowspan="1" colspan="1">F</td><td align="left" rowspan="1" colspan="1">AGCGCAAGTACTCCGTGTG</td><td align="center" rowspan="1" colspan="1">[<xref rid="r29" ref-type="bibr">29</xref>]</td></tr><tr><td align="center" rowspan="1" colspan="1">R</td><td align="left" rowspan="1" colspan="1">CGGACTCATCGTACTCCTGCTT</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">P</td><td align="left" rowspan="1" colspan="1">TCGCTGTCCACCTTCCAGCAGATGT</td><td rowspan="1" colspan="1"></td></tr></tbody></table><table-wrap-foot><p>F, Forward primer; R, Reverse primer; P, Probe.</p></table-wrap-foot></table-wrap>. GenBank accession numbers, the reference sequence, country, host and first
deposited year used for primers and probes design of each pathogen were shown in <xref ref-type="supplementary-material" rid="pdf_001">Supplemental Table 1</xref>. Previously reported qPCR
assays were used for 10 pathogen species, including RNA, DNA viruses and bacteria [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>, <xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r16" ref-type="bibr">16</xref>, <xref rid="r18" ref-type="bibr">18</xref>, <xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r25" ref-type="bibr">25</xref>, <xref rid="r27" ref-type="bibr">27</xref>, <xref rid="r28" ref-type="bibr">28</xref>]. Furthermore, as an internal control within the
Dempo-PCR reaction, primer-probe sets for β-actin were synthesized as previously reported
[<xref rid="r29" ref-type="bibr">29</xref>]. All hydrolysis probes were labeled with the
reporter dye FAM (6-carboxyfluorecein) at the 5′ end and the fluorescent dye TAMRA
(6-carboxytetramethylrhodamine) at the 3′ end. Primers and probes were purchased from
Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA), and probes containing the mixed base
were produced at Integrated DNA Technologies (Integrated DNA Technologies, Inc.).</p></sec><sec><title>Real-time PCR</title><p>A One Step PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa Bio, Kusatsu, Japan) was
used to detect viral RNA, and Premix Ex Taq (Perfect Real Time) (TaKaRa Bio) was used to
detect viral and bacterial DNA. All reactions were performed in a total volume of 20
<italic>µl</italic>, which contained the sample nucleic acid, primers, probes (the final
concentration of all primers and probes was 0.2 <italic>µ</italic>M) and all other
components included in the kits, according to the manufactures’ protocols. Thermal cycling
conditions were as follows: 45°C for 5 min and 95°C for 30 sec, followed by 40 cycles of
95°C for 10 sec, 55°C for 20 sec, and 72°C for 20 sec [<xref rid="r26" ref-type="bibr">26</xref>]. Fluorescent signal data were analyzed using an automatic quantification
algorithm in LightCycler Nano Software 1.1 (Roche Diagnostics GmbH), and the parameters of
analysis were as follows: exclude early cycle=7, minimum relative amplifications=0, and
minimum amplification quality=5.</p></sec><sec><title>Validation of real-time PCR performance using synthesized DNA</title><p>To verify the sensitivity, linearity, and efficiency of the real-time PCR assay, the
limit of detection (LOD), correlation coefficient (R<sup>2</sup>) and PCR efficiency (E)
were determined from standard curves. Standard curves were obtained, and the LOD,
R<sup>2</sup> and E were calculated as described previously [<xref rid="r11" ref-type="bibr">11</xref>, <xref rid="r26" ref-type="bibr">26</xref>].</p></sec><sec><title>Evaluation of real-time PCR performance using synthesized DNA</title><p>For the purpose of validation, real-time PCR reliability, sensitivity, and linearity of
standard curves were verified by testing tenfold serial dilutions of synthesized DNA,
including each target genome sequence (1 × 10<sup>0</sup> to 1 × 10<sup>6</sup>copies/reaction). The synthesized DNA was purchased from Integrated DNA Technologies
(Integrated DNA Technologies, Inc.). Pathogen dilutions were repeated twice in separate
runs, and a standard curve was constructed from the Cq values. The PCR efficiency (E) was
calculated using the standard curve slope according to the following formula:
E=(10<sup>−1/slope (−1)</sup>). The correlation co- efficient (R<sup>2</sup>) was also
calculated. The limit of detection (LOD) was defined as the lowest concentration at which
a fluorescent signal could be detected in all reactions. Reproducibility (inter-assay
variance) was assessed using the coefficient value (CV) calculated based on quantification
cycle (Cq) values.</p></sec><sec><title>Clinical samples and DNA and RNA extraction</title><p>The assay was applied to test clinical samples. A total of 30 samples of porcine lung
tissue submitted in 2016–2018 to Azabu University for diagnosis of porcine respiratory
pathogens were used to test. These pigs were 48 to 135 days old and belonged to 6 farms (A
to F), all showing respiratory symptoms (<xref ref-type="supplementary-material" rid="pdf_001">Supplementary Table 3</xref>). Lung tissues were minced by scissors, diluted 1:10 in
phosphate buffered saline (PBS, pH 7.4), homogenized for 20 sec at 3,200 rpm with the
presence of three stainless steel beads (φ4 mm) by using the bead crusher
<italic>µ</italic>T-12 (TAITEC, Inc.), and centrifuged at 12,000 g for 5 min to obtain
the supernatant. Bacteria nucleic acids, viral DNA, and viral RNA were extracted from the
supernatant using a QIAamp<sup>®</sup> cador<sup>®</sup> Pathogen Kit (Qiagen, Hilden,
Germany) with a sample volume of 200 <italic>µl</italic> and elution volume of 50
<italic>µl</italic>, as described by the manufacturer. The extracted DNA and RNA were
stored at −80°C until examination. The extracted nucleic acids were evaluated in
triplicated by targeting respiratory disease complex pathogens in a single run of
Dempo-PCR. When the Cq values were calculated by algorithm described above in more than
two out of three runs, the samples were considered positive. In order to compare Dempo-PCR
assay with the classical method, the conventional PCR (cPCR) was performed under each
condition using conventional primers (<xref ref-type="supplementary-material" rid="pdf_001">Supplementary
Table 2</xref>). A PrimeScript<sup>TM</sup> RT Master Mix (TaKaRa Bio) and
GoTaq<sup>®</sup> Green Master Mix (Promega) was used. All reactions were performed in a
total volume of 25 <italic>µl</italic>, which contained the sample nucleic acid, primers
(the final concentration of all primers was 0.4 <italic>µ</italic>M) and all other
components included in the kits, according to the manufactures’ protocols. Amplicons were
detected by electrophoresing. The samples which showed the results of the Dempo-PCR assay
is not consistent with the cPCR assay did not match, were confirmed by direct sequencing
of amplification products.</p><p>All the experiments were conducted in accordance with the Guidelines for the Care and Use
of Laboratory Animals of Azabu University.</p></sec></sec><sec sec-type="results" id="s2"><title>RESULTS</title><sec><title>Sensitivity, linearity, and efficiency evaluated with standard curves from
synthesized DNA</title><p>To evaluate the sensitivity, linearity, and efficiency of the PCR, 10-fold serial
dilutions of synthesized DNA were tested by real- time PCR. Standard curves were
constructed from Cq values, then the LOD, R<sup>2</sup>, and E were evaluated (<xref ref-type="supplementary-material" rid="pdf_001">Supplementary Fig. 1</xref>). <xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title>Performance of sensitivity tests</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" rowspan="1" colspan="1">Type of materials</th><th align="center" valign="middle" rowspan="1" colspan="1">Pathogens</th><th align="center" valign="middle" rowspan="1" colspan="1">LOD<break></break>(/reaction)</th><th align="center" valign="middle" rowspan="1" colspan="1">ReproducibilityCV<break></break>(%)</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="17" colspan="1">DNA (copy number)</td><td align="left" rowspan="1" colspan="1"><italic>Actinobacillus pleuropneumoniae</italic></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.27–1.67</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Boldetella bronchiseptica</italic></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.00–1.10</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Haemophilus parasuis</italic></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.10–0.91</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Pasteurella multocida</italic></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.07–0.83</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Pasteurella multocida</italic> toxin</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.01–0.71</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Streptococcus suis</italic></td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">0.02–0.51</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Mycoplasma hyopneumoniae</italic></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.08–1.51</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Mycoplasma hyorhinis</italic></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.25–1.60</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Mycoplasma hyosynoviae</italic></td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.04–0.37</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine circovirus 2</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">0.18–2.43</td></tr><tr><td align="left" rowspan="1" colspan="1">Pseudorabies virus</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">0.56–1.47</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine cytomegalovirus</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.12–0.53</td></tr><tr><td align="left" rowspan="1" colspan="1">Swine influenza A virus</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.22–1.52</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine reproductive and respiratory virus US strain</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">0.73–1.96</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine reproductive and respiratory virus EU strain</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">0.06–1.11</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine respiratory coronavirus</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.05–2.62</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine hemagglutinating encephalomyelitis virus</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.03–0.63</td></tr></tbody></table><table-wrap-foot><p>LOD, Limit of detection; CV, Coefficient of variation.</p></table-wrap-foot></table-wrap> shows the results for LOD number and CVs of run-to-run variants. The LOD,
based on DNA copy number, was ≤100 copies/reaction for all primer-probe sets. The CVs were
at most 2.62%; this reproducibility was observed with PRCV testing. In addition, the
calibration curves of all assays covered a linear dynamic range of more than five orders
of magnitude and showed R<sup>2</sup> values of at least 0.9922. Although the PCR
efficiency for PRRSV US strain and PRV was slightly low (81.6% and 88.6%, respectively),
the PCR efficiency in all detection assays was more than 80%, which was enough to quantify
the target copy number.</p></sec><sec><title>Dempo-PCR performance in clinical sample testing</title><p>A total of 30 lungs from different affected animals on six farms with respiratory disease
outbreaks were applied to Dempo-PCR. In addition, cPCR assay were also performed to
compare the sensitivities of Dempo-PCR assay. As the results, there were samples detected
by Dempo-PCR but not detected by cPCR. The sequences of these samples proved to be
identical to the sequence of the target pathogens by direct sequencing of amplification
products. To the contrary, there were no samples detected by cPCR but not detected by
Dempo-PCR (<xref ref-type="supplementary-material" rid="pdf_001">Supplementary Table 3</xref>).</p><p>The results are presented as the number and percentage of positive samples from each farm
(<xref rid="tbl_003" ref-type="table">Table 3</xref><table-wrap id="tbl_003" orientation="portrait" position="float"><label>Table 3.</label><caption><title>Detection of targets in lung tissue from clinical cases by Dempo-PCR</title></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="3" valign="middle" align="center" colspan="1">Pathogens</th><th colspan="7" valign="middle" align="center" rowspan="1">Positive samples in Dempo-PCR</th></tr><tr><th colspan="7" rowspan="1"><hr></hr></th></tr><tr><th align="center" valign="middle" rowspan="1" colspan="1">Farm A<break></break>N=4<break></break>n (%)</th><th align="center" valign="middle" rowspan="1" colspan="1">Farm B<break></break>N=8<break></break>n (%)</th><th align="center" valign="middle" rowspan="1" colspan="1">Farm C<break></break>N=7<break></break>n (%)</th><th align="center" valign="middle" rowspan="1" colspan="1">Farm D<break></break>N=2<break></break>n (%)</th><th align="center" valign="middle" rowspan="1" colspan="1">Farm E<break></break>N=5<break></break>n (%)</th><th align="center" valign="middle" rowspan="1" colspan="1">Farm F<break></break>N=4<break></break>n (%)</th><th align="center" valign="middle" rowspan="1" colspan="1">Total<break></break>N=30<break></break>n (%)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>Actinobacillus pleuropneumoniae</italic></td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">1 (50.0)</td><td align="center" rowspan="1" colspan="1">2 (40.0)</td><td align="center" rowspan="1" colspan="1">2 (50.0)</td><td align="center" rowspan="1" colspan="1">5 (16.7)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Boldetella bronchiseptica</italic></td><td align="center" rowspan="1" colspan="1">1 (25.0)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">1 (50.0)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">1 (25.0)</td><td align="center" rowspan="1" colspan="1">3 (10.0)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Haemophilus parasuis</italic></td><td align="center" rowspan="1" colspan="1">4 (100)</td><td align="center" rowspan="1" colspan="1">4 (50.0)</td><td align="center" rowspan="1" colspan="1">6 (85.7)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">14 (46.7)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Pasteurella multocida</italic></td><td align="center" rowspan="1" colspan="1">3 (75.0)</td><td align="center" rowspan="1" colspan="1">1 (12.5)</td><td align="center" rowspan="1" colspan="1">1 (14.3)</td><td align="center" rowspan="1" colspan="1">2 (100)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">1 (25.0)</td><td align="center" rowspan="1" colspan="1">8 (26.7)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Pasteurella multocida</italic> toxin</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Streptococcus suis</italic></td><td align="center" rowspan="1" colspan="1">1 (25.0)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">2 (40.0)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">3 (10.0)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Mycoplasma hyopneumoniae</italic></td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">2 (25.0)</td><td align="center" rowspan="1" colspan="1">6 (85.7)</td><td align="center" rowspan="1" colspan="1">2 (100)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">2 (50.0)</td><td align="center" rowspan="1" colspan="1">12 (40.0)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Mycoplasma hyorhinis</italic></td><td align="center" rowspan="1" colspan="1">4 (100)</td><td align="center" rowspan="1" colspan="1">6 (75.0)</td><td align="center" rowspan="1" colspan="1">7 (100)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">2 (50.0)</td><td align="center" rowspan="1" colspan="1">19 (63.3)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Mycoplasma hyosynoviae</italic></td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine circovirus 2</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">7 (100)</td><td align="center" rowspan="1" colspan="1">2 (100)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">2 (50)</td><td align="center" rowspan="1" colspan="1">11 (36.6)</td></tr><tr><td align="left" rowspan="1" colspan="1">Pseudorabies virus</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine cytomegalovirus</td><td align="center" rowspan="1" colspan="1">4 (100)</td><td align="center" rowspan="1" colspan="1">5 (62.5)</td><td align="center" rowspan="1" colspan="1">5 (71.4)</td><td align="center" rowspan="1" colspan="1">1 (50.0)</td><td align="center" rowspan="1" colspan="1">3 (60.0)</td><td align="center" rowspan="1" colspan="1">4 (100)</td><td align="center" rowspan="1" colspan="1">22 (73.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">Swine influenza A virus</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">3 (37.5)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">3 (10.0)</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine reproductive and respiratory virus US strain</td><td align="center" rowspan="1" colspan="1">3 (75.0)</td><td align="center" rowspan="1" colspan="1">7 (87.5)</td><td align="center" rowspan="1" colspan="1">7 (100)</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">2 (50.0)</td><td align="center" rowspan="1" colspan="1">19 (63.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine reproductive and respiratory virus EU strain</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine respiratory coronavirus</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">2 (50.0)</td><td align="center" rowspan="1" colspan="1">2 (6.7)</td></tr><tr><td align="left" rowspan="1" colspan="1">Porcine hemagglutinating encephalomyelitis virus</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr></tbody></table></table-wrap>). In samples from farm C and F, both viral and bacterial pathogens,
including PCV2 (100% and 50%, respectively), PRRSV US strain (100% and 50%, respectively)
and <italic>M. hyopneumoniae</italic> (85.7% and 50%, respectively), were detected at high
frequency, whereas mainly bacterial pathogens, including <italic>B.
bronchiseptica</italic>, <italic>H. parasuis</italic>, <italic>P. multocida</italic>,
<italic>S. suis</italic> and <italic>M. hyorhinis</italic> were prevailed in farm A, and
<italic>A. pleuropneumoniae</italic> and <italic>S. suis</italic> were prevailed in farm
E. In samples from farms B, mixed infections of PRRSV US strain, SIV and bacterial
pathogens; <italic>H. parasuis</italic>, <italic>P. multocida</italic>, <italic>M.
hyorhinis</italic>, and <italic>M. hyopneumoniae</italic> were detected. PCMV was
detected at high frequency from all farms, whereas <italic>P. multocida</italic> toxin,
<italic>M. hyosynooviae</italic>, PRV, PRRSV EU strain, and PHEV were not detected.</p></sec></sec><sec sec-type="discussion" id="s3"><title>DISCUSSION</title><p>PRDC is one of the most important health concerns for pig producers and involves multiple
viral and bacterial pathogens. PRDC is multifactorial, with both infectious and
non-infectious factors contributing to respiratory disease seen in pigs between the ages of
3 and 6 months. The interaction of viral and bacterial pathogens, environmental factors,
pig-specific factors and management conditions all contribute to the development and impact
the severity of PRDC [<xref rid="r20" ref-type="bibr">20</xref>]. The most commonly isolated
pathogens are PRRSV, SIV, PCV2, and <italic>M. hyopneumoniae</italic>. The other pathogens
associated with PRDC are <italic>S. suis, A. pleuropneumoniae</italic>, <italic>P.
multocida, P. multocida</italic> toxin, <italic>B. bronchiseptica</italic>, <italic>H.
parasuis</italic>, <italic>M. hyorhinis, M. hyosynovie,</italic> PRV, PRCV, PCMV [<xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r8" ref-type="bibr">8</xref>, <xref rid="r15" ref-type="bibr">15</xref>, <xref rid="r20" ref-type="bibr">20</xref>]. However,
no single-reaction diagnostic test currently exists for the simultaneous detection of major
pathogens commonly associated with PRDC. Routine diagnostic methods for detection of viruses
implicated in PRDC include virus isolation in cell culture, antigen detection by direct
fluorescent antibody staining, and enzyme immunoassay [<xref rid="r5" ref-type="bibr">5</xref>] and culture-based methods for bacteria [<xref rid="r23" ref-type="bibr">23</xref>]. These methods are time-consuming and require independent tests for each
pathogen. Furthermore, the detection of bacterial pathogens typically depends on
culture-based methods that can take several days to obtain results. Due to their high
sensitivity and ease of use, PCR and real-time PCR tests have been developed for several
agents implicated in the PRDC; however, these tests typically target single pathogens [<xref rid="r24" ref-type="bibr">24</xref>]. A multiplex PCR assay capable of detecting five
porcine viruses including two porcine respiratory viruses was developed [<xref rid="r4" ref-type="bibr">4</xref>]. However, to date, there are no diagnostic tests
capable of simultaneous detection of multiple major viral and bacterial porcine respiratory
pathogens in a single reaction.</p><p>Recently Lung <italic>et al</italic>. [<xref rid="r15" ref-type="bibr">15</xref>] reported
a novel prototype automated microarray that integrates and automates all steps of post-PCR
microarray processing for the simultaneous detection and typing of four bacteria (<italic>M.
hyopneumoniae, P. multocida, S. enterica serovar Choleraesuis, S. suis</italic>) and four
viruses (PRRSV, SIV, PCV2, PRCV) differentiation of the two PRRSV genotypes and pathogenic
versus non-pathogenic <italic>P. multocida</italic> strains. This electronic microarray
assay can be completed in less than 4 hr with little user handling plus approximately 1.5 hr
for the RT-PCR. These methods are highly specific and sensitive, and easy to operate, but
these are expensive to run and requires expensive equipment. On the other hand, Dempo-PCR
assay can be completed in less than 3 hr, and easy operate.</p><p>In this study, Dempo-PCR has been developed, following the methods of diagnosis of bovine
diarrhea developed by Tsuchiaka <italic>et al.</italic> [<xref rid="r26" ref-type="bibr">26</xref>]. Since all primer-probe sets were optimized in the same temperature
conditions, Dempo-PCR can detect a total of 17 pathogens, including 8 viruses, 8 bacteria,
and 1 toxin, in a single run of TaqMan real-time PCR. In sensitivity test by using standard
curves from synthesized DNA, all primer-probe sets showed high sensitivity. Furthermore, the
results of detection of target pathogens from clinical samples using this method showed
similar results to the respective conventional PCR method. However, PRRS virus is known to
have a high frequency of genetic mutations, and the primer and probe sequences will need to
be checked at a considerable frequency when performing Dempo-PCR from field samples. The
type of pathogens involved in PRDC is specific to the regions and countries where production
occurs [<xref rid="r20" ref-type="bibr">20</xref>]. Therefore, it may be necessary to change
the inspect pathogens according to the regions. However, Dempo-PCR is possible to detect
many types of pathogens simultaneously.</p><p>By Dempo-PCR assay, multiple PRDC pathogens can be detected comprehensively and
simultaneously. This assay can quickly elucidate existence of pathogens in a sample. In this
study, Multiple viral and bacterial porcine respiratory pathogens were detected from pigs of
all farms examined. Especially, five bacteria pathogens (<italic>A.
pleuropneumoniae</italic>, <italic>B. bronchiseptica</italic>, <italic>P.
multocida</italic>, <italic>M. hyopneumoniae</italic>, <italic>M. hyorhinis</italic>) and
four viruses (PCV2, PCMV, PRRS US strain, PRCV) were detected on pigs of farm F. In this
study, PCMV was detected in high proportion from pigs of all farms, and mixed infection with
multiple pathogens was observed. PCMV has been documented worldwide, and shows high
infection rates on pig farms in Japan, Europe, North America, and China [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r6" ref-type="bibr">6</xref>]. Hansen
<italic>et al.</italic>, reported that a significant association between PCMV and PCV2 was
only found in the cases of PRDC, and the role of PCMV in PRDC needs to be elucidated [<xref rid="r7" ref-type="bibr">7</xref>]. It is necessary to elucidate the combination of
multiple pathogens for the elucidation of the etiology of PRDC, and Dempo -PCR will be a
useful tool for that. PHEV is a subclinical infection, but its role as a respiratory
pathogen was suggested since it was isolated from the acute respiratory disease in pigs in
Michigan in 2015 [<xref rid="r14" ref-type="bibr">14</xref>]. The swine serological survey
of PHEV also showed that it is widely and highly distributed in Japan [<xref rid="r10" ref-type="bibr">10</xref>]. Therefore, PHEV was added to the target pathogens of Dempo-PCR, but
it was not detected from these samples.</p><p>In conclusion, Dempo-PCR can identify a wider range of existing pathogens quickly and
easily compared to one assay-one pathogen test. Considering multiple etiology of PRDC,
screening by Dempo-PCR would help us determine treatment and prevention measures. This
detection system may provide an alternative testing method that is simpler, faster, and more
comprehensive than existing assays.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="pdf_001"><caption><title>Supplement figure and table</title></caption><media mimetype="application" mime-subtype="pdf" xlink:href="jvms-82-217-s001.pdf" orientation="portrait" xlink:type="simple" id="d35e1708" position="anchor"></media></supplementary-material></sec></body><back><ack><p>This work was partially supported by The Ministry of Agriculture, Forestry
and Fisheries of Japan, Award Number: the Research Project for Improving Food Safety and
Animal Health of the Ministry of Agriculture, Forestry and Fisheries of Japan
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