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Species-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100\u00a0ng\/\u03bcL and, tenfold serially diluting it up to 10\u00a0pg\/\u03bcL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Two mPCR assays were developed. Optimal primer annealing temperature of 55\u00a0\u00b0C was established and utilized in the final amplification conditions. The assays detected all targeted bacteria, with a 100\u00a0pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4\u00a0h and 8\u00a0h of incubation, 41% (5\/12) and 100% (12\/12) of the bacteria were detected in culture fluids, respectively. The assays also identified\n                      <jats:italic>Salmonella<\/jats:italic>\n                      spp\n                      <jats:italic>.<\/jats:italic>\n                      and\n                      <jats:italic>Klebsiella pneumoniae<\/jats:italic>\n                      co-infections and extra pathogens (1\n                      <jats:italic>E. coli<\/jats:italic>\n                      and 2\n                      <jats:italic>K. pneumoniae<\/jats:italic>\n                      ) compared with culture. The sensitivity and specificity of the mPCR were 100.0% (71.7\u2013100.0) and 98.0% (90.7\u201399.0), respectively. The area under the ROC curve was 1.00 (1.00\u20131.00).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>The mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s41182-024-00606-3","type":"journal-article","created":{"date-parts":[[2024,6,5]],"date-time":"2024-06-05T03:01:52Z","timestamp":1717556512000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Development of two multiplex PCR assays for rapid detection of eleven Gram-negative bacteria in children with septicemia"],"prefix":"10.1186","volume":"52","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8672-594X","authenticated-orcid":false,"given":"Gabriel","family":"Miringu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abednego","family":"Musyoki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Betty","family":"Muriithi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ernest","family":"Wandera","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan","family":"Waithiru","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erick","family":"Odoyo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hisashi","family":"Shoji","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nelson","family":"Menza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshio","family":"Ichinose","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,6,5]]},"reference":[{"issue":"10","key":"606_CR1","doi-asserted-by":"publisher","DOI":"10.1097\/CCE.0000000000000775","volume":"4","author":"J Prest","year":"2022","unstructured":"Prest J, Nguyen T, Rajah T, Prest AB, Sathananthan M, Jeganathan N. 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