{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T06:01:12Z","timestamp":1781157672116,"version":"3.54.1"},"reference-count":25,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T00:00:00Z","timestamp":1781136000000},"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:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>PRRSV-2 prevalent strains mainly include C-PRRSV, HP-PRRSV, and NADC30-like, with the latter being the current dominant lineage. Due to the high recombination and genetic variation of PRRSV, existing RT-qPCR assays face an increasing risk of false negatives. Therefore, based on the current prevalent strain sequences, it is of great significance to update and establish a one-step multiplex RT-qPCR method that can simultaneously detect C-PRRSV, HP-PRRSV and NADC30-like.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      By downloading the latest prevalent strain full genome sequences from NCBI and isolating them in our laboratory, the conserved and type-specific target regions of the three strains were screened in the high-variable region of the\n                      <jats:italic>nsp2<\/jats:italic>\n                      gene. TaqMan MGB probes and primers were designed. After optimizing the reaction conditions, the standard curve, amplification efficiency, sensitivity, specificity, repeatability and clinical application effect of this method were evaluated.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The established standard curve showed a good linear relationship within the range of 1 \u00d7 108 to 1 \u00d7 10\n                      <jats:sup>3<\/jats:sup>\n                      copies\/\u03bcL, with a correlation coefficient R\n                      <jats:sup>2<\/jats:sup>\n                      of 0.998 for all. The amplification efficiency ranged from 97.58 to 103.53%. The minimum detection limits for C-PRRSV, HP-PRRSV and NADC30-like were 10.128 copies, 8.998 copies and 8.458 copies, respectively. This method showed no cross-reaction with common porcine pathogens such as PRRSV-1, PCV2 and CSFV, and the intra-batch and inter-batch coefficient of variation was less than 2%. The positive rate of PRRSV in 588 samples was 15.5% (91\/588), which was higher than 13.94% (82\/588) of the reported methods. The consistency Kappa of the two methods was 0.87.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>This study successfully established a one-step multiplex RT-qPCR method based on current prevalent strain sequences, which offers high sensitivity, strong specificity, and good repeatability, and can be used for rapid differential diagnosis of the three PRRSV subtypes in clinical samples, thereby supporting precise diagnosis, epidemiological monitoring, and prevention and control of PRRSV in China.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1850527","type":"journal-article","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T05:47:02Z","timestamp":1781156822000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["The updated one-step multiplex RT-qPCR method for PRRSV classical strains, highly pathogenic strains and NADC30-like strains"],"prefix":"10.3389","volume":"17","author":[{"given":"Zengliang","family":"Guo","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Nanjing Agricultural University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"He","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences","place":["Harbin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Bai","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Nanjing Agricultural University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changyou","family":"Xia","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences","place":["Harbin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Jiang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Nanjing Agricultural University","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,6,11]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1080\/21505594.2025.2504124","article-title":"Genetic evolution and alterations in pathogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus.","volume":"16","author":"Li","year":"2025","journal-title":"Virulence"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.3390\/v17030379","article-title":"Genome and pathogenicity analysis of an NADC30-like PRRSV Strain in China\u2019s Xinjiang Province.","volume":"17","author":"Li","year":"2025","journal-title":"Viruses-Basel"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-9-203","article-title":"Chinese highly pathogenic porcine reproductive and respiratory syndrome virus exhibits more extensive tissue tropism for pigs.","volume":"9","author":"Li","year":"2012","journal-title":"Virology Virol. 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