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However, to the best of our knowledge, only a few laboratories worldwide conduct tests for the identification of this group of viruses. Therefore, in this study, we aimed to develop and validate a real-time RT-PCR assay for the identification of PeV-As.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      To design and validate a real-time PCR primer\u2013probe targeting the\n                      <jats:italic>5\u2032-UTR<\/jats:italic>\n                      region of PeV-As, the 5\u2032-\n                      <jats:italic>UTR<\/jats:italic>\n                      sequences of PeV-As available in GenBank were aligned using the MUSCLE algorithm in MEGA v7.0. Thereafter, the highly conserved 5\u2032-\n                      <jats:italic>UTR<\/jats:italic>\n                      region was selected, and its primer\u2013probe sequence was designed using Primer Premier v5.0. This primer\u2013probe sequence was then evaluated for specificity, sensitivity, and repeatability, and for its validation, it was tested using fecal samples from 728 healthy children living in Beijing (China).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The PeV-A real-time RT-PCR assay detected only the RNA-positive standards of PeV-A genotypes (1\u20138, 14, 17, and 18), whereas 72 serotypes of non-PeV-A EV viruses were undetected. In addition, the\n                      <jats:italic>VP1<\/jats:italic>\n                      region of these 11 PeV-A genotypes that tested positive were amplified using the primers designed in this study. Typing results indicated that eight, one, and two strains of the 11 were PeV-A1, PeV-A4, and PeV-A6, respectively. We also determined and presented the genetic characterization and phylogenetic analyses results corresponding to these 11\n                      <jats:italic>VP1<\/jats:italic>\n                      region sequences. Furthermore, real-time RT-PCR assay showed good sensitivity with\n                      <jats:italic>LOD<\/jats:italic>\n                      of 10\n                      <jats:sup>2<\/jats:sup>\n                      copies\/\u03bcL. Positive results in eight parallel experiments at each concentration gradient from 10\n                      <jats:sup>7<\/jats:sup>\n                      copies\/\u03bcL to 10\n                      <jats:sup>2<\/jats:sup>\n                      copies\/\u03bcL, indicating good repeatability.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>Our findings suggested that the real-time RT-PCR assay developed in this study can be applied for routine PeV-A identification. We detected PeV-A1, 4 and 6 genotypes in the 728 faecal samples using this method. Additionally, we believe that our results will serve as a foundation for further studies on PeV-As and facilitate the expansion of the gene sequence information available in GenBank.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12985-021-01689-8","type":"journal-article","created":{"date-parts":[[2021,11,20]],"date-time":"2021-11-20T12:10:23Z","timestamp":1637410223000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Development of a real-time RT-PCR assay for the detection of pan-human parechoviruses"],"prefix":"10.1186","volume":"18","author":[{"given":"Huanhuan","family":"Lu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinbo","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Keyi","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhenzhi","family":"Han","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yang","family":"Song","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongyan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tianjiao","family":"Ji","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongmei","family":"Yan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuangli","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenbo","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2692-5437","authenticated-orcid":false,"given":"Yong","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2021,11,20]]},"reference":[{"issue":"1","key":"1689_CR1","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1097\/01.inf.0000148931.05186.99","volume":"24","author":"JW Tang","year":"2005","unstructured":"Tang JW, Bendig JW, Ossuetta I. 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The study was approved by the Second Ethics Review Committee of the National Institute for Viral Diseases Control and Prevention (IVDC), Chinese Center for Diseases Control and Prevention, all experimental protocols were approved by IVDC and the methods were carried out in accordance with the approved guidelines.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and content to participate:"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"227"}}