{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T07:45:17Z","timestamp":1793259917942,"version":"4.5.0"},"reference-count":37,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2021,10,26]],"date-time":"2021-10-26T00:00:00Z","timestamp":1635206400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/pages\/standard-publication-reuse-rights"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81830071"],"award-info":[{"award-number":["81830071"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Zhejiang Provincial Natural Science Foundation of China","award":["LR20H200001"],"award-info":[{"award-number":["LR20H200001"]}]},{"name":"Wenzhou Science and Technology Bureau Project","award":["Y20170029"],"award-info":[{"award-number":["Y20170029"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,5,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:sec>\n                  <jats:title>Objectives<\/jats:title>\n                  <jats:p>Determining mitochondrial DNA (mtDNA) A-to-G substitution at nucleotide 3243 (m.3243A&amp;gt;G) heteroplasmy is essential for both precision diagnosis of m.3243A&amp;gt;G\u2013associated mitochondrial disease and genetic counseling. Precise determination of m.3243A&amp;gt;G heteroplasmy is challenging, however, without appropriate strategies to accommodate heteroplasmic levels ranging from 1% to 100% in samples carrying thousands to millions of mtDNA copies.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>We used a combined strategy of amplification-refractory mutation system\u2013quantitative polymerase chain reaction (ARMS-qPCR) and droplet digital PCR (ddPCR) to determine m.3243A&amp;gt;G heteroplasmy. Primers were specifically designed and screened for both ARMS-qPCR and ddPCR to determine m.3243A&amp;gt;G heteroplasmy. An optimized ARMS-qPCR\u2013ddPCR\u2013based strategy was established using artificial standards, with different mixtures of m.3243A-containing and m.3243G-containing plasmids and further tested using clinical samples containing the m.3243A&amp;gt;G mutation.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>One of 20 primer pairs designed in the study was omitted for ARMS-qPCR\u2013ddPCR strategy application according to criteria of 85% to 110%, R2&amp;gt; 0.98 amplification efficiency, melt curve with a single clear peak, and specificity for m.3243A and m.3243G artificial standards (|CtWt-CtMut|max). Using plasmid standards with various m.3243A&amp;gt;G heteroplasmy (1%-100%) at low, mid, and high copy numbers (3,000, 104, and 105-107, respectively) and DNA from the blood of 20 patients carrying m.3243A&amp;gt;G with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, we found that ARMS-qPCR was reliable for determining m.3243A&amp;gt;G at 3% to 100% for low copy number and 1% to 100% for mid to high copy number samples. Meanwhile, ddPCR was reliable for determining m.3243A&amp;gt;G at 1% to 100% at low to mid copy number samples.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>An ARMS-qPCR\u2013ddPCR\u2013based strategy was successfully established for precise determination of m.3243A&amp;gt;G heteroplasmy in complex clinical samples.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1093\/ajcp\/aqab174","type":"journal-article","created":{"date-parts":[[2021,9,8]],"date-time":"2021-09-08T11:21:22Z","timestamp":1631100082000},"page":"664-677","source":"Crossref","is-referenced-by-count":3,"title":["Determining Mitochondrial 3243A&amp;gt;G Heteroplasmy Using an ARMS-ddPCR Strategy"],"prefix":"10.1093","volume":"157","author":[{"given":"Pu","family":"Xu","sequence":"first","affiliation":[{"name":"Laboratory Medicine, The First Affiliated Hospital of Xi\u2019an Medical University , Xi\u2019an , 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Medical University , Wenzhou , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuohua","family":"Zhou","sequence":"additional","affiliation":[{"name":"School of Laboratory Medicine and Life Sciences, Wenzhou Medical University , Wenzhou , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hezhi","family":"Fang","sequence":"additional","affiliation":[{"name":"School of Laboratory Medicine and Life Sciences, Wenzhou Medical University , Wenzhou , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng","family":"Gao","sequence":"additional","affiliation":[{"name":"Zhejiang Provincial People\u2019s Hospital, Affiliated People\u2019s Hospital of Hangzhou Medical College , Hangzhou , China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lijun","family":"Shen","sequence":"additional","affiliation":[{"name":"School of Laboratory Medicine and Life Sciences, Wenzhou Medical University , Wenzhou , 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