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In this study, we developed a highly sensitive qPCR assay for specific detection of\n                      <jats:italic>Burkholderia pseudomallei<\/jats:italic>\n                      and melioidosis disease diagnosis based on a novel target sequence.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      An extensive\n                      <jats:italic>in-silico<\/jats:italic>\n                      analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of\u00a0\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      . The applicability of the assay for\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      detection in clinical and environmental matrices was evaluated by spiking\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      cells in the blood, urine, soil, and water along with suitable internal controls.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      A novel 85-nucleotide-long sequence was identified using\n                      <jats:italic>in-silico<\/jats:italic>\n                      tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      . The assay was found to be highly sensitive, with a detection limit of 3\u00a0\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 \u00d7 10\n                      <jats:sup>2<\/jats:sup>\n                      CFU\/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 \u00d7 10\n                      <jats:sup>1<\/jats:sup>\n                      CFU\/mL of river water and 2 \u00d7 10\n                      <jats:sup>3<\/jats:sup>\n                      CFU\/gm of paddy field soil.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of\n                      <jats:italic>B. pseudomallei<\/jats:italic>\n                      in various clinical and environmental matrices.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12941-024-00693-4","type":"journal-article","created":{"date-parts":[[2024,4,10]],"date-time":"2024-04-10T13:01:44Z","timestamp":1712754104000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices"],"prefix":"10.1186","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4398-9838","authenticated-orcid":false,"given":"Pranjal Kumar","family":"Yadav","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4122-6129","authenticated-orcid":false,"given":"Suchetna","family":"Singh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-2752-4340","authenticated-orcid":false,"given":"Moumita","family":"Paul","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-4233-3506","authenticated-orcid":false,"given":"Sanjay","family":"Kumar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-9813-7373","authenticated-orcid":false,"given":"S.","family":"Ponmariappan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0046-9426","authenticated-orcid":false,"given":"Duraipandian","family":"Thavaselvam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,4,10]]},"reference":[{"issue":"2","key":"693_CR1","doi-asserted-by":"publisher","first-page":"383","DOI":"10.1128\/CMR.18.2.383-416.2005","volume":"18","author":"AC Cheng","year":"2005","unstructured":"Cheng AC, Currie BJ. 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