{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,6]],"date-time":"2026-06-06T15:16:49Z","timestamp":1780759009227,"version":"3.54.1"},"reference-count":45,"publisher":"Microbiology Society","issue":"12","funder":[{"DOI":"10.13039\/501100007403","name":"N\u00e1rodn\u00ed Agentura pro Zem\u011bd\u011blsk\u00fd V\u00fdzkum","doi-asserted-by":"crossref","award":["MZe-RO0518"],"award-info":[{"award-number":["MZe-RO0518"]}],"id":[{"id":"10.13039\/501100007403","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,12,13]]},"abstract":"<jats:p>\n                  <jats:bold>Introduction<\/jats:bold>. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the <jats:italic>\n                     <jats:named-content content-type=\"species\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.10879\" xlink:type=\"simple\">Mycobacterium avium<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic> complex (MAC) and <jats:italic>\n                     <jats:named-content content-type=\"species\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.6313\" xlink:type=\"simple\">Mycobacterium abscessus<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic> complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.<\/jats:p>\n               <jats:p>\n                  <jats:bold>Gap statement<\/jats:bold>. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.<\/jats:p>\n               <jats:p>\n                  <jats:bold>Aim<\/jats:bold>. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.<\/jats:p>\n               <jats:p>\n                  <jats:bold>Methodology<\/jats:bold>. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of <jats:italic>\n                     <jats:named-content content-type=\"subspecies\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.29179\" xlink:type=\"simple\">M. abscessus<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic> subsp. <jats:italic>\n                     <jats:named-content content-type=\"subspecies\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.29179\" xlink:type=\"simple\">massiliense<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic> (MAM).<\/jats:p>\n               <jats:p>\n                  <jats:bold>Results<\/jats:bold>. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between <jats:italic>\n                     <jats:named-content content-type=\"subspecies\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.22536\" xlink:type=\"simple\">M. abscessus<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic> subsp. <jats:italic>\n                     <jats:named-content content-type=\"subspecies\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.22536\" xlink:type=\"simple\">abscessus<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic>\/subsp. <jats:italic>bolletii<\/jats:italic> (MAAb\/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5\u200a% MAAb\/MAB, 14.7\u200a% MAM and 26.2\u200a% MAC. The LOD was determined to be 1\u200a490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4\u200a%. Verification of the qPCR results with sequencing showed 100\u200a% homology.<\/jats:p>\n               <jats:p>\n                  <jats:bold>Conclusions<\/jats:bold>. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients\u2019 sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.<\/jats:p>","DOI":"10.1099\/jmm.0.001611","type":"journal-article","created":{"date-parts":[[2022,12,13]],"date-time":"2022-12-13T17:18:59Z","timestamp":1670951939000},"source":"Crossref","is-referenced-by-count":2,"title":["Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR"],"prefix":"10.1099","volume":"71","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7203-5569","authenticated-orcid":false,"given":"Radka","family":"Dziedzinska","sequence":"first","affiliation":[{"name":"Laboratory of Neurobiology and Pathological Physiology, Institute of Animal Physiology and Genetics, Czech 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