{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T23:59:55Z","timestamp":1789084795936,"version":"build-2803163510"},"reference-count":26,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,2,23]],"date-time":"2022-02-23T00:00:00Z","timestamp":1645574400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Fundamental Research Grant Scheme,Malaysian Ministry of Higher Education","award":["Fundamental Research Grant Scheme (FRGS) No. 203.PPSP.617150"],"award-info":[{"award-number":["Fundamental Research Grant Scheme (FRGS) No. 203.PPSP.617150"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Despite the advanced understanding of the disease, melioidosis, an infection caused by Burkholderia pseudomallei, continues to be of global interest. The bacterial virulence factor, type six secretion system-5 (T6SS-5), in particular, is an essential factor for B. pseudomallei that is associated with internalization and intracellular survival of the pathogen. To detect the virulence gene cluster, this study has successfully developed a novel seven-gene (tssC-5, tagD-5, tssA-5, hcp-5, tssB-5, tssF-5, and vgrG-5) multiplex PCR assay. The optimum annealing temperature for this assay ranged between 59 and 62 \u00b0C. The limit of detection for this assay was 103 CFU\/mL for all genes, excluding tssF-5, which was found at 105 CFU\/mL of the bacterial concentration. In sensitivity and specificity tests, this multiplex assay was able to amplify all of the seven target genes from 93.8% (n = 33\/35) clinical and 100% (n = 2\/2) environmental isolates of B. pseudomallei. Whereas only four genes (tssC-5, tagD-5, tssF-5, and vgrG-5) were amplified from Bukholderia thailandesis, two genes (tagD-5 and tssB-5) were amplified from Bukholderia stagnalis, and zero target genes were amplified from Bukholderia ubonensis. No amplification of any genes was obtained when tested against isolated DNA from non-Bukholderia species (n = 20), which include Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, and others. In conclusion, this multiplex PCR assay is sensitive, species-specific, rapid, and reliable to detect the virulent gene cluster T6SS-5 of B. pseudomallei.<\/jats:p>","DOI":"10.3390\/diagnostics12030562","type":"journal-article","created":{"date-parts":[[2022,2,22]],"date-time":"2022-02-22T22:34:30Z","timestamp":1645569270000},"page":"562","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of Multiplex PCR Assay for Screening of T6SS-5 Gene Cluster: The Burkholderia pseudomallei Virulence Factor"],"prefix":"10.3390","volume":"12","author":[{"given":"Noreafifah","family":"Semail","sequence":"first","affiliation":[{"name":"School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Azian","family":"Harun","sequence":"additional","affiliation":[{"name":"School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7850-6405","authenticated-orcid":false,"given":"Ismail","family":"Aziah","sequence":"additional","affiliation":[{"name":"Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0009-2772","authenticated-orcid":false,"given":"Nik Mohd Noor","family":"Nik Zuraina","sequence":"additional","affiliation":[{"name":"School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3344-1518","authenticated-orcid":false,"given":"Zakuan Zainy","family":"Deris","sequence":"additional","affiliation":[{"name":"School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Seng, R., Saiprom, N., Phunpang, R., Baltazar, C.J., Boontawee, S., and Thodthasri, T. 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