{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T06:48:17Z","timestamp":1782197297237,"version":"3.54.5"},"reference-count":42,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>\n                    Diarrhea caused by bacterial and viral pathogens remains a major cause of childhood morbidity and mortality, leading to dehydration, malnutrition, and substantial healthcare burdens; rapid and accurate diagnosis is therefore essential for timely treatment. In clinical practice, detection still commonly relies on traditional methods such as stool culture and enzyme-linked immunosorbent assays. These approaches are not only time-consuming and lack sensitivity but also typically require separate, pathogen-specific tests, complicating comprehensive diagnosis. While quantitative PCR (qPCR) offers speed and sensitivity, dye-based methods lack specificity, and fluorescent probe-based assays are costly. To address these limitations, we developed an integrated assay that combines PCR with a sequence-specific invasive reaction (Invader) in a single closed tube. This method detects six major pediatric enteric pathogens:\n                    <jats:italic>Shigella<\/jats:italic>\n                    spp.,\n                    <jats:italic>Vibrio cholerae<\/jats:italic>\n                    , non-typhoidal\n                    <jats:italic>Salmonella<\/jats:italic>\n                    ,\n                    <jats:italic>Campylobacter jejuni<\/jats:italic>\n                    , rotavirus, and enteric adenovirus (types 40\/41). The assay demonstrated high sensitivity, with detection limits as low as 2 copies per reaction for each target and exhibited no cross-reactivity. The entire process was completed within 45 minutes. By employing universal Invader probes, we reduced the cost per test to approximately 2 RMB. Validation using both simulated and clinical stool samples suggested the assay\u2019s potential reliability and consistency with standard methods. This closed-tube PCR-Invader assay provides a rapid, cost-effective, and unified platform for the parallel detection of six major diarrheal pathogens in a single test run. It shows promise as a practical alternative to current disparate and pathogen-specific testing strategies, with considerable potential for use in primary healthcare settings and large-scale screening to guide the management of childhood infectious diarrhea.\n                  <\/jats:p>","DOI":"10.3389\/fcimb.2026.1841155","type":"journal-article","created":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T06:18:44Z","timestamp":1782195524000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid and low-cost detection of six major pediatric enteric pathogens using a closed-tube PCR-invasive reaction assay"],"prefix":"10.3389","volume":"16","author":[{"given":"Nan","family":"Yang","sequence":"first","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Han","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yihsuan","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guiping","family":"Kong","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yucan","family":"Zheng","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuo","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Shen","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiu","family":"Han","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Faculty of Medicine, Yangzhou University","place":["Yangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhifeng","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology, Children\u2019s Hospital of Nanjing Medical University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,6,23]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2023.e18049","article-title":"Evaluation of antibiotic utilization pattern in the treatment of acute diarrheal diseases at Debre Tabor comprehensive specialized hospital, Debre Tabor, Ethiopia: A retrospective cross-sectional study","volume":"9","author":"Addis","year":"2023","journal-title":"Heliyon"},{"key":"B2","doi-asserted-by":"publisher","first-page":"BSR20211790","DOI":"10.1042\/bsr20211790","article-title":"Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia)","volume":"41","author":"Alarjani","year":"2021","journal-title":"Biosci. Rep."},{"key":"B3","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1586\/14737159.5.2.209","article-title":"Basic principles of real-time quantitative PCR","volume":"5","author":"Arya","year":"2005","journal-title":"Expert Rev. Mol. Diagn."},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.7759\/cureus.50546","article-title":"Shigella as a cause of diarrhea hospitalization in children under five: Evaluation by conventional and molecular methods","volume":"15","author":"Bansal","year":"2023","journal-title":"Cureus"},{"key":"B5","doi-asserted-by":"publisher","first-page":"1223","DOI":"10.1093\/cid\/cir730","article-title":"Quantitative real-time polymerase chain reaction for enteropathogenic Escherichia coli: a tool for investigation of asymptomatic versus symptomatic infections","volume":"53","author":"Barletta","year":"2011","journal-title":"Clin. Infect. Dis."},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0298603","article-title":"Strengthening laboratories in response to outbreaks in humanitarian emergencies and conflict settings: Results, challenges and lessons from expanding PCR diagnostic capacities for COVID-19 testing in Yemen","volume":"19","author":"Bashir","year":"2024","journal-title":"PloS One"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02790-23","article-title":"Gut microbiome and antibiotic resistance effects during travelers' diarrhea treatment and prevention","volume":"15","author":"Blake","year":"2024","journal-title":"mBio"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1186\/s13756-019-0659-3","article-title":"Implementation and impact of pediatric antimicrobial stewardship programs: a systematic scoping review","volume":"9","author":"Don\u00e0","year":"2020","journal-title":"Antimicrob. Resist. Infect. Ctrl."},{"key":"B9","doi-asserted-by":"publisher","first-page":"916","DOI":"10.1128\/jcm.02697-13","article-title":"Real-time PCR threshold cycle cutoffs help to identify agents causing acute childhood diarrhea in Zanzibar","volume":"52","author":"Elfving","year":"2014","journal-title":"J. Clin. Microbiol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"1211","DOI":"10.1016\/s1473-3099(18)30362-1","article-title":"Estimates of the global, regional, and national morbidity, mortality, and aetiologies of diarrhoea in 195 countries: a systematic analysis for the Global Burden of Disease Study 2016","volume":"18","year":"2018","journal-title":"Lancet Infect. Dis."},{"key":"B11","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1016\/s1473-3099(19)30401-3","article-title":"Quantifying risks and interventions that have affected the burden of diarrhoea among children younger than 5 years: an analysis of the Global Burden of Disease Study 2017","volume":"20","year":"2020","journal-title":"Lancet Infect. Dis."},{"key":"B12","doi-asserted-by":"publisher","first-page":"519","DOI":"10.1016\/s1473-3099(24)00691-1","article-title":"Global, regional, and national age-sex-specific burden of diarrhoeal diseases, their risk factors, and aetiologies 1990-2021, for 204 countries and territories: a systematic analysis for the Global Burden of Disease Study 2021","volume":"25","year":"2025","journal-title":"Lancet Infect. Dis."},{"key":"B13","doi-asserted-by":"publisher","first-page":"2645","DOI":"10.2147\/ijn.S132670","article-title":"Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor","volume":"12","author":"Ge","year":"2017","journal-title":"Int. J. Nanomed."},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0206316","article-title":"Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR","volume":"13","author":"Heymans","year":"2018","journal-title":"PloS One"},{"key":"B15","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1002\/med4.88","article-title":"Emergence and detection of gastro-enteric virulence genes in non-pathogenic Vibrio cholerae: Implications for public health and water safety in African developmental regions","volume":"2","author":"Igere","year":"2024","journal-title":"Med. Adv."},{"key":"B16","doi-asserted-by":"publisher","first-page":"258","DOI":"10.37201\/req\/026.2025","article-title":"Acute infectious gastroenteritis in childhood: the role of rapid multiplex molecular syndromic panels in diagnosis and clinical management","volume":"38","author":"Jim\u00e9nez-Jim\u00e9nez","year":"2025","journal-title":"Rev. Esp. Quimioter."},{"key":"B17","doi-asserted-by":"publisher","first-page":"311","DOI":"10.1038\/s41746-024-01319-x","article-title":"The adaptation of a single institution diabetes care platform into a nationally available turnkey solution","volume":"7","author":"Kim","year":"2024","journal-title":"NPJ Dig. Med."},{"key":"B18","doi-asserted-by":"publisher","first-page":"95","DOI":"10.1016\/j.mam.2005.12.007","article-title":"The real-time polymerase chain reaction","volume":"27","author":"Kubista","year":"2006","journal-title":"Mol. Aspects. Med."},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0159027","article-title":"Peptide-recombinant VP6 protein based enzyme immunoassay for the detection of Group A rotaviruses in multiple host species","volume":"11","author":"Kumar","year":"2016","journal-title":"PloS One"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1155\/2015\/142912","article-title":"Genetic diversity of human adenovirus in children with acute gastroenteritis, Albania 2013-2015","volume":"2015","author":"La Rosa","year":"2015","journal-title":"BioMed. Res. Int."},{"key":"B21","doi-asserted-by":"publisher","first-page":"e204","DOI":"10.1016\/s2214-109x(19)30541-8","article-title":"Diarrhoeal disease and subsequent risk of death in infants and children residing in low-income and middle-income countries: analysis of the GEMS case-control study and 12-month GEMS-1A follow-on study","volume":"8","author":"Levine","year":"2020","journal-title":"Lancet Glob. Health"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-024-01881-2","article-title":"Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples","volume":"12","author":"Li","year":"2024","journal-title":"Microbiome"},{"key":"B23","doi-asserted-by":"publisher","first-page":"358","DOI":"10.1186\/s12916-020-01820-6","article-title":"The dynamic wound microbiome","volume":"18","author":"Liu","year":"2020","journal-title":"BMC Med."},{"key":"B24","doi-asserted-by":"publisher","first-page":"552","DOI":"10.15403\/jgld-5653","article-title":"Efficacy of molecular methods for the diagnosis of enteropathogenic microorganisms associated with diarrhoea: A systematic review","volume":"33","author":"Lopez Mu\u00f1oz","year":"2024","journal-title":"J. Gastrointestin. Liv. Dis."},{"key":"B25","doi-asserted-by":"publisher","first-page":"388","DOI":"10.1016\/j.bios.2016.12.015","article-title":"Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles","volume":"90","author":"Lu","year":"2017","journal-title":"Biosens. Bioelectron."},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2022.858555","article-title":"A regional pooling intervention in a high-throughput COVID-19 diagnostic laboratory to enhance throughput, save resources and time over a period of 6 months","volume":"13","author":"Mandhan","year":"2022","journal-title":"Front. Microbiol."},{"key":"B27","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1016\/j.mrfmmm.2004.08.016","article-title":"The Invader assay for SNP genotyping","volume":"573","author":"Olivier","year":"2005","journal-title":"Mutat. Res."},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1002\/mbo3.70104","article-title":"Next-generation sequencing in infectious-disease diagnostics: Economic, regulatory, and clinical pathways to adoption","volume":"14","author":"Osei Sekyere","year":"2025","journal-title":"Microbiol. Open"},{"key":"B29","doi-asserted-by":"publisher","first-page":"432","DOI":"10.7883\/yoken.JJID.2014.450","article-title":"Loop-mediated isothermal amplification (LAMP) for detection of Campylobacter jejuni and C. coli in Thai children with diarrhea","volume":"68","author":"Pham","year":"2015","journal-title":"Jpn. J. Infect. Dis."},{"key":"B30","doi-asserted-by":"publisher","first-page":"315","DOI":"10.3390\/pathogens12020315","article-title":"High burden of co-infection with multiple enteric pathogens in children suffering with diarrhoea from rural and peri-urban communities in South Africa","volume":"12","author":"Potgieter","year":"2023","journal-title":"Pathogens"},{"key":"B31","doi-asserted-by":"publisher","first-page":"1044","DOI":"10.3390\/diagnostics15081044","article-title":"Impact of multiplex PCR on diagnosis of bacterial and fungal infections and choice of appropriate antimicrobial therapy","volume":"15","author":"Serapide","year":"2025","journal-title":"Diagn. (Basel)."},{"key":"B32","doi-asserted-by":"publisher","first-page":"995","DOI":"10.1039\/d2an01679f","article-title":"Visualized RNA detection of SARS-CoV-2 in a closed tube by coupling RT-PCR with nested invasive reaction","volume":"148","author":"Shan","year":"2023","journal-title":"Analyst"},{"key":"B33","doi-asserted-by":"publisher","first-page":"5775","DOI":"10.1039\/c9an00970a","article-title":"Sequence-encoded quantitative invader assay enables highly sensitive hepatitis B virus DNA quantification in a single tube without the use of a calibration curve","volume":"144","author":"Sheng","year":"2019","journal-title":"Analyst"},{"key":"B34","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1007\/s10620-021-07330-6","article-title":"Is the medium still the message? Culture-independent diagnosis of gastrointestinal infections","volume":"67","author":"Sood","year":"2022","journal-title":"Dig. Dis. Sci."},{"key":"B35","doi-asserted-by":"publisher","first-page":"393","DOI":"10.46234\/ccdcw2025.065","article-title":"A quadruplex digital PCR assay for the simultaneous detection of four intestinal bacterial pathogens and its application in wastewater samples","volume":"7","author":"Sun","year":"2025","journal-title":"China CDC. Wkly."},{"key":"B36","doi-asserted-by":"publisher","first-page":"2687","DOI":"10.1128\/jcm.27.12.2687-2691.1989","article-title":"Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli","volume":"27","author":"Venkatesan","year":"1989","journal-title":"J. Clin. Microbiol."},{"key":"B37","doi-asserted-by":"publisher","first-page":"2464","DOI":"10.1038\/s41467-021-22551-z","article-title":"Etiological, epidemiological, and clinical features of acute diarrhea in China","volume":"12","author":"Wang","year":"2021","journal-title":"Nat. Commun."},{"key":"B38","doi-asserted-by":"publisher","first-page":"76","DOI":"10.1016\/j.mimet.2018.06.006","article-title":"Simultaneous detection and quantification of 19 diarrhea-related pathogens with a quantitative real-time PCR panel assay","volume":"151","author":"Wongboot","year":"2018","journal-title":"J. Microbiol. Methods"},{"key":"B39","doi-asserted-by":"publisher","first-page":"6751","DOI":"10.1007\/s00216-018-1316-z","article-title":"Highly sensitive and specific real-time PCR by employing serial invasive reaction as a sequence identifier for quantifying EGFR mutation abundance in cfDNA","volume":"410","author":"Xiang","year":"2018","journal-title":"Anal. Bioanal. Chem."},{"key":"B40","doi-asserted-by":"publisher","first-page":"16728","DOI":"10.1038\/s41598-025-96960-1","article-title":"A TaqMan qPCR for precise detection and quantification of diarrheagenic Escherichia coli","volume":"15","author":"Xue","year":"2025","journal-title":"Sci. Rep."},{"key":"B41","doi-asserted-by":"publisher","first-page":"122","DOI":"10.1186\/s40249-025-01392-8","article-title":"Co-infection and interaction of enteric pathogens in acute diarrhea among children under five years: a large-scale multicenter observational study from China","volume":"14","author":"Zhang","year":"2025","journal-title":"Infect. Dis. Poverty."},{"key":"B42","doi-asserted-by":"publisher","first-page":"7395","DOI":"10.1002\/anie.201001529","article-title":"Ultrasensitive DNA detection by cascade enzymatic signal amplification based on Afu flap endonuclease coupled with nicking endonuclease","volume":"50","author":"Zou","year":"2011","journal-title":"Angew. Chem. Int. Ed."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2026.1841155\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T06:18:45Z","timestamp":1782195525000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2026.1841155\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,23]]},"references-count":42,"alternative-id":["10.3389\/fcimb.2026.1841155"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2026.1841155","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,23]]},"article-number":"1841155"}}