{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T20:17:51Z","timestamp":1774383471093,"version":"3.50.1"},"reference-count":23,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T00:00:00Z","timestamp":1774310400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Project for Basic Research in Jinhua Central Hospital","award":["JY2022-6-03"],"award-info":[{"award-number":["JY2022-6-03"]}]},{"name":"Jinhua Municipal Science and Technology Plan Project Social Development Major Projects","award":["2023-3-067"],"award-info":[{"award-number":["2023-3-067"]}]},{"name":"Jinhua Municipal Science and Technology Project Public Welfare Project","award":["2022-4-088"],"award-info":[{"award-number":["2022-4-088"]}]},{"name":"Jinhua Municipal Science and Technology Plan Project Social Development Key Project","award":["2024-3-069"],"award-info":[{"award-number":["2024-3-069"]}]},{"name":"Jinhua Municipal Science and Technology Plan Project Social Development Key Project","award":["2023-3-105"],"award-info":[{"award-number":["2023-3-105"]}]},{"name":"Public Welfare Fund Research Project Analysis Test Project","award":["LTGC24H200004"],"award-info":[{"award-number":["LTGC24H200004"]}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>\n                      Current\n                      <jats:italic>Toxoplasma gondii<\/jats:italic>\n                      (TOX), Epstein\u2013Barr virus (EBV), rubella virus, human cytomegalovirus (HCMV), and herpes simplex virus types I and II (HSV I and II) (TORCHes) diagnostic methods are limited by challenges, such as multi-step workflows and cold-chain dependency. This study aimed to develop an integrated \u201csample-in-result-out\u201d system combining rapid nucleic acid release, room-temperature-stable multiplex polymerase chain reaction (PCR), and automated interpretation to improve diagnostic accuracy.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We utilized a self-prepared lysis buffer for fast nucleic acid extraction from diverse sample types. Primers, probes, and reaction components for six TORCHes pathogens were fabricated into freeze-dried microspheres via vacuum freeze-drying, enabling single-tube multicolor melting curve analysis (MMCA). The companion software, TORCHes-MCAv1.0, was used to automate pathogen typing and result reporting. Evaluation metrics encompassed sensitivity, specificity, precision, stability, and anti-interference ability. The system was validated using 210 clinical samples compared to commercial single-target qPCR, with Sanger sequencing resolving any discrepancies.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Nucleic acid extraction and release were completed within 1\u202fmin. Freeze-dried microspheres demonstrated exceptional repeatability, with\n                      <jats:italic>T<\/jats:italic>\n                      <jats:sub>m<\/jats:sub>\n                      intra- and inter-batch CVs of 0.03\u20130.18%. Limits of detection (LoD) were 200 copies\/mL for HSV-I\/II, HCMV, and TOX, and 500 copies\/mL for EBV and RV. Specificity analysis showed no cross-reactivity with non-target pathogens. Stability testing confirmed the microspheres could be stored stably at room temperature for 1\u202fyear. Clinical validation showed near-perfect agreement with qPCR (Kappa\u202f=\u202f0.965\u20131.000), with all seven discrepant results confirmed correct via sequencing. The TORCHes-MCAv1.0 software achieved 100% concordance with manual interpretation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>We developed a robust, streamlined integrated TORCHes detection system. Its simplicity, stability, and high-throughput nature make it a valuable complementary tool for confirming active infection and differentiating pathogen subtypes in complex clinical scenarios.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1788209","type":"journal-article","created":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T16:43:08Z","timestamp":1774370588000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["A streamlined integrated system integrating lysate release, freeze-dried reagents for multiplex polymerase chain reaction, and intelligent analysis for TORCHes pathogen identification"],"prefix":"10.3389","volume":"17","author":[{"given":"Miao","family":"Fu","sequence":"first","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Siying","family":"He","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yingchao","family":"Wu","sequence":"additional","affiliation":[{"name":"Shanghai Pudong New Area People\u2019s Hospital","place":["Shanghai, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sisi","family":"Mao","sequence":"additional","affiliation":[{"name":"Lanxi Traditional Chinese Medicine Hospital","place":["Lanxi, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Na","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinhua","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lingzhi","family":"Zhou","sequence":"additional","affiliation":[{"name":"School of Laboratory Medicine and Life Sciences, Wenzhou Medical University","place":["Wenzhou, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiang","family":"Xi","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Junqi","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huabin","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yongjun","family":"Ma","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine","place":["Jinhua, Zhejiang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,24]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"122233","DOI":"10.1016\/j.ijpharm.2022.122233","article-title":"Freeze-drying: a flourishing strategy to fabricate stable pharmaceutical and biological products","volume":"628","author":"Abla","year":"2022","journal-title":"Int. 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Virol."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"438","DOI":"10.1177\/1040638718763474","article-title":"Excess EDTA interferes with cortisol measurement using a solid-phase, chemiluminescent enzyme immunoassay","volume":"30","author":"Kemppainen","year":"2018","journal-title":"J. Vet. Diagn. Invest."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"l6507","DOI":"10.1136\/bmj.l6507","article-title":"Screening for cytomegalovirus infection in pregnancy","volume":"367","author":"Kilby","year":"2019","journal-title":"BMJ"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1186\/s12985-022-01789-z","article-title":"Multiplex detection of eight different viral enteropathogens in clinical samples, combining RT-PCR technology with melting curve analysis","volume":"19","author":"Li","year":"2022","journal-title":"Virol. J."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"94","DOI":"10.1093\/jpids\/piab105","article-title":"Neonatal herpes simplex virus infection: epidemiology and outcomes in the modern era","volume":"11","author":"Melvin","year":"2022","journal-title":"J. Pediatric Infect. Dis. Soc."},{"key":"ref11","first-page":"230","article-title":"Role of virus infection as etiological factors of miscarriage","volume":"67","author":"Mitsoda","year":"2014","journal-title":"Wiad. Lek."},{"key":"ref12","volume-title":"Diagnosis of Toxoplasmosis","year":"2015"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1016\/j.clp.2014.11.001","article-title":"TORCHes infections","volume":"42","author":"Neu","year":"2015","journal-title":"Clin. Perinatol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"e687","DOI":"10.1016\/j.jogc.2018.05.036","article-title":"No. 285-toxoplasmosis in pregnancy: prevention, screening, and treatment","volume":"40","author":"Paquet","year":"2018","journal-title":"J. Obstet. Gynaecol. Can."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"693","DOI":"10.1128\/JCM.01868-16","article-title":"Testing for cytomegalovirus in pregnancy","volume":"55","author":"Saldan","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref16","volume-title":"Herpes Simplex Virus Type I and II Nucleic Acid Genotyping Detection Kit (PCR-Fluorescent probe Method)","year":"2023"},{"key":"ref17","doi-asserted-by":"publisher","first-page":"8482","DOI":"10.1080\/14767058.2021.1983537","article-title":"Viral prevalence by gestational age and season in a large neonatal cord blood cohort","volume":"35","author":"Sloan","year":"2022","journal-title":"J. Matern. Fetal Neonatal Med."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"e24051","DOI":"10.1002\/jcla.24051","article-title":"Rapid and sensitive detection of L-FABP for prediction and diagnosis of acute kidney injury in critically ill patients by chemiluminescent immunoassay","volume":"35","author":"Sun","year":"2021","journal-title":"J. Clin. Lab. Anal."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1128\/JCM.01535-14","article-title":"Advances in laboratory methods for detection and typing of norovirus","volume":"53","author":"Vinj\u00e9","year":"2015","journal-title":"J. Clin. Microbiol."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/j.jviromet.2011.04.001","article-title":"Diagnosis of recent primary rubella virus infections: significance of glycoprotein-based IgM serology, IgG avidity and immunoblot analysis","volume":"174","author":"Wandinger","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"e23208","DOI":"10.1002\/jcla.23208","article-title":"Evaluation of a commercial latex agglutination test for detecting rotavirus A and human adenovirus in children\u2019s stool specimens","volume":"34","author":"Xiang","year":"2020","journal-title":"J. Clin. Lab. Anal."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"116234","DOI":"10.1016\/j.diagmicrobio.2024.116234","article-title":"A novel multiplex real-time PCR assay for the detection of cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1\/2 and strategies for application to blood screening","volume":"109","author":"Xu","year":"2024","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"553","DOI":"10.3760\/cma.j.cn114452-20191020-00597","article-title":"Expert consensus on standardized TORCH laboratory detection and clinical application","volume":"43","author":"Zhu","year":"2020","journal-title":"Chin. J. Lab. 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