{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,6]],"date-time":"2026-04-06T18:44:19Z","timestamp":1775501059760,"version":"3.50.1"},"reference-count":25,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T00:00:00Z","timestamp":1668643200000},"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. Microbiol."],"abstract":"<jats:p><jats:italic>Haemophilus influenzae<\/jats:italic> is a main human pathogen that results in a series of diseases in children and adults, such as pneumonia, bacteremia, and meningitis. Although there are many detection methods, they cannot meet the requirements of an early diagnosis. For the prevention and control of <jats:italic>H. influenzae<\/jats:italic> infection, quick, sensitive, and particular diagnostics are crucial. Loop-mediated isothermal amplification (LAMP) coupled with restricted endonuclease digestion and real-time fluorescence (<jats:italic>H. influenzae<\/jats:italic>-ERT-LAMP) detection was employed to diagnose <jats:italic>H. influenzae<\/jats:italic>. <jats:italic>H. influenzae<\/jats:italic>-ERT-LAMP combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence identification into a single-pot reaction, allowing for the rapid identification of <jats:italic>H. influenzae<\/jats:italic> in 40 min. The outer membrane protein (OMP) P6 gene of <jats:italic>H. influenzae<\/jats:italic> was employed to build a sequence of <jats:italic>H. influenzae<\/jats:italic>-ERT-LAMP primers. The limit of detection (LoD) of <jats:italic>H. influenzae<\/jats:italic>-ERT-LAMP test was 40\u2009fg of genomic DNA per reaction, and the non-<jats:italic>H. influenzae<\/jats:italic> templates did not provide positive outcomes. To investigate the applicability of <jats:italic>H. influenzae<\/jats:italic>-ERT-LAMP method in clinical sample detection, 30 sputum specimens were obtained from individuals suspected of being infected with <jats:italic>H. influenzae<\/jats:italic>. <jats:italic>H. influenzae<\/jats:italic>-ERT-LAMP outcomes were in total agreement with LAMP-LFB and PCR. The <jats:italic>H. influenzae<\/jats:italic>-ERT-LAMP assay provides rapid, accurate, and sensitive detection making it a promising screening strategy in clinical and basic lab settings.<\/jats:p>","DOI":"10.3389\/fmicb.2022.1037343","type":"journal-article","created":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T05:08:08Z","timestamp":1668661688000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Development and clinical application of a endonuclease restriction real-time loop-mediated isothermal amplification (ERT-LAMP) assay for rapid detection of Haemophilus influenzae"],"prefix":"10.3389","volume":"13","author":[{"given":"Jinzhi","family":"Cheng","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuhong","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xue","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jingrun","family":"Lu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiahong","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2022,11,17]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1016\/j.exppara.2009.12.023","article-title":"A single fluorescence-based LAMP reaction for identifying multiple parasites in mosquitoes","volume":"125","author":"Aonuma","year":"2010","journal-title":"Exp. Parasitol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1186\/s12866-022-02547-5","article-title":"Detection of Haemophilus influenzae by loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay","volume":"22","author":"Cao","year":"2022","journal-title":"BMC Microbiol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"1553","DOI":"10.1128\/JCM.39.4.1553-1558.2001","article-title":"Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR","volume":"39","author":"Corless","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"103274","DOI":"10.1016\/j.ebiom.2021.103274","article-title":"Molecular diagnostic assays for the detection of common bacterial meningitis pathogens: a narrative review","volume":"65","author":"Diallo","year":"2021","journal-title":"EBioMedicine"},{"key":"ref5","doi-asserted-by":"publisher","first-page":"2382","DOI":"10.1128\/jcm.32.10.2382-2386.1994","article-title":"PCR for capsular typing of Haemophilus influenzae","volume":"32","author":"Falla","year":"1994","journal-title":"J. Clin. Microbiol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1471","DOI":"10.1007\/s10096-020-03865-0","article-title":"Molecular epidemiology of invasive Haemophilus influenzae disease in Portugal: an update of the post-vaccine period, 2011-2018","volume":"39","author":"Heliodoro","year":"2020","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1016\/j.mimet.2007.09.019","article-title":"Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites","volume":"71","author":"Iseki","year":"2007","journal-title":"J. Microbiol. Methods"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1111\/j.1574-695X.1999.tb01384.x","article-title":"Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae","volume":"26","author":"Karalus","year":"1999","journal-title":"FEMS Immunol. Med. Microbiol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"3621","DOI":"10.1128\/JCM.00515-11","article-title":"Loop-mediated isothermal amplification assay for detection of Haemophilus influenzae type b in cerebrospinal fluid","volume":"49","author":"Kim","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"95","DOI":"10.3389\/fpubh.2020.00095","article-title":"Molecular epidemiology and antimicrobial resistance of Haemophilus influenzae in adult patients in Shanghai, China","volume":"8","author":"Li","year":"2020","journal-title":"Front. Public Health"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"3758","DOI":"10.1021\/ac3003825","article-title":"Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing","volume":"84","author":"Liang","year":"2012","journal-title":"Anal. Chem."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"774","DOI":"10.1128\/iai.54.3.774-779.1986","article-title":"Antigenic characterization of the P6 protein of nontypable Haemophilus influenzae","volume":"54","author":"Murphy","year":"1986","journal-title":"Infect. Immun."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"2658","DOI":"10.1128\/iai.59.8.2658-2663.1991","article-title":"Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms","volume":"59","author":"Nelson","year":"1991","journal-title":"Infect. Immun."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1080\/21645515.2017.1385687","article-title":"Cost-effectiveness of the Haemophilus influenzae type b vaccine for infants in mainland China","volume":"14","author":"Ning","year":"2018","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"E63","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"96","DOI":"10.1080\/20009666.2020.1835213","article-title":"Haemophilus influenzae purulent pericarditis in an immunocompetent individual","volume":"11","author":"Shah","year":"2021","journal-title":"J. Commun. Hosp. Intern. Med. Perspect."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"19823","DOI":"10.1038\/s41598-019-56225-0","article-title":"Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification","volume":"9","author":"Takano","year":"2019","journal-title":"Sci. Rep."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"367","DOI":"10.3967\/0895-3988.2012.03.016","article-title":"Rapid detection of Haemophilus influenzae and Haemophilus parainfluenzae in nasopharyngeal swabs by multiplex PCR","volume":"25","author":"Tian","year":"2012","journal-title":"Biomed. Environ. Sci."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"368","DOI":"10.1128\/CMR.00040-06","article-title":"Antimicrobial resistance in Haemophilus influenzae","volume":"20","author":"Tristram","year":"2007","journal-title":"Clin. Microbiol. Rev."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1099\/00222615-33-4-271","article-title":"Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification","volume":"33","author":"Van Ketel","year":"1990","journal-title":"J. Med. Microbiol."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"192","DOI":"10.3389\/fmicb.2017.00192","article-title":"Loop-mediated isothermal amplification label-based gold nanoparticles lateral flow biosensor for detection of Enterococcus faecalis and Staphylococcus aureus","volume":"8","author":"Wang","year":"2017","journal-title":"Front. Microbiol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"748746","DOI":"10.3389\/fbioe.2021.748746","article-title":"A novel real-time reverse transcription loop-mediated isothermal amplification detection platform: application to diagnosis of COVID-19","volume":"9","author":"Wang","year":"2021","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1104","DOI":"10.3389\/fmicb.2016.01104","article-title":"Endonuclease restriction-mediated real-time polymerase chain reaction: a novel technique for rapid, sensitive and quantitative detection of nucleic-acid sequence","volume":"7","author":"Wang","year":"2016","journal-title":"Front. Microbiol."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"457","DOI":"10.1002\/ppul.20789","article-title":"Antimicrobial susceptibility of Haemophilus influenzae strains and antibiotics usage patterns in pediatric outpatients: results from a children's hospital in China (2000\u20132004)","volume":"43","author":"Wang","year":"2008","journal-title":"Pediatr. Pulmonol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"104205","DOI":"10.1016\/j.meegid.2020.104205","article-title":"Molecular epidemiology and evolution of Haemophilus influenzae","volume":"80","author":"Wen","year":"2020","journal-title":"Infect. Genet. Evol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2022.1037343\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T05:08:12Z","timestamp":1668661692000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2022.1037343\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,17]]},"references-count":25,"alternative-id":["10.3389\/fmicb.2022.1037343"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2022.1037343","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,17]]},"article-number":"1037343"}}