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Here, we introduce color cycle multiplex amplification (CCMA) to significantly increase the number of detectable DNA targets in a single qPCR reaction using standard instrumentation. In CCMA, presence of one DNA target species results in a pre-programmed pattern of fluorescence increases. This pattern is distinguished by cycle thresholds (Cts) through rationally designed delays in amplification. For example, we design an assay wherein Staphylococcus aureus sequentially induces FAM, then Cy5.5, then ROX fluorescence increases with more than 3 cycles between each signal. CCMA offers notably higher potential for multiplexing because it uses fluorescence permutation rather than combination. With 4 distinct fluorescence colors, CCMA theoretically allows the detection of up to 136 distinct DNA target sequences using fluorescence permutation. Experimentally, we demonstrated a single-tube qPCR assay screening 21 sepsis-related bacterial DNA targets in samples of blood, sputum, pleural effusion and bronchoalveolar lavage fluid, with 89% clinical sensitivity and 100% clinical specificity, showing its potential as a powerful tool for advanced quantitative screening in molecular diagnostics.<\/jats:p>","DOI":"10.1093\/nar\/gkae683","type":"journal-article","created":{"date-parts":[[2024,8,9]],"date-time":"2024-08-09T14:13:29Z","timestamp":1723212809000},"page":"e81-e81","source":"Crossref","is-referenced-by-count":27,"title":["Advancing quantitative PCR with color cycle multiplex amplification"],"prefix":"10.1093","volume":"52","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9278-1259","authenticated-orcid":false,"given":"Wei","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6161-5012","authenticated-orcid":false,"given":"Kerou","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Zhongshan Hospital, Fudan University , Shanghai , Shanghai\u00a0 200032 , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University , Shanghai 200433, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George\u00a0C","family":"Waldren","sequence":"additional","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sherry\u00a0X","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bonnie","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Zhongshan Hospital, Fudan University , Shanghai , Shanghai\u00a0 200032 , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0213-7663","authenticated-orcid":false,"given":"David\u00a0Y","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3104-8678","authenticated-orcid":false,"given":"Jinny\u00a0X","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Innovation, NuProbe USA , Houston , TX\u00a0 77054 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,8,9]]},"reference":[{"key":"2024092307442503600_B1","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1126\/scitranslmed.3003726","article-title":"Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA","volume":"4","author":"Forshew","year":"2012","journal-title":"Sci. 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