{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T14:28:24Z","timestamp":1784125704225,"version":"3.55.0"},"reference-count":48,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2023,5,4]],"date-time":"2023-05-04T00:00:00Z","timestamp":1683158400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Guangxi Science and Technology Bureau, China","award":["No. AB21238003"],"award-info":[{"award-number":["No. AB21238003"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Atypical porcine pestivirus (APPV), a newly discovered virus, is associated with the type A-II congenital tremor (CT) in neonatal piglets. APPV distributes throughout the world and causes certain economic losses to the swine industry. The specific primers and probe were designed targeting the 5\u2032 untranslated region (UTR) of APPV to amplify a 90 bp fragment, and the recombinant standard plasmid was constructed. After optimizing the concentrations of primers and probe, annealing temperature, and reaction cycles, a crystal digital RT-PCR (cdRT-PCR) and real-time quantitative RT-PCR (qRT-PCR) were successfully established. The results showed that the standard curves of the qRT-PCR and the cdRT-PCR had R2 values of 0.999 and 0.9998, respectively. Both methods could specifically detect APPV, and no amplification signal was obtained from other swine viruses. The limit of detection (LOD) of the cdRT-PCR was 0.1 copies\/\u00b5L, and that of the qRT-PCR was 10 copies\/\u00b5L. The intra-assay and inter-assay coefficients of variation of repeatability and reproducibility were less than 0.90% for the qRT-PCR and less than 5.27% for the cdRT-PCR. The 60 clinical tissue samples were analyzed using both methods, and the positivity rates of APPV were 23.33% by the qRT-PCR and 25% by the cdRT-PCR, with a coincidence rate of 98.33%. The results indicated that the cdRT-PCR and the qRT-PCR developed here are highly specific, sensitive methods for the rapid and accurate detection of APPV.<\/jats:p>","DOI":"10.3390\/vetsci10050330","type":"journal-article","created":{"date-parts":[[2023,5,5]],"date-time":"2023-05-05T02:08:42Z","timestamp":1683252522000},"page":"330","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus"],"prefix":"10.3390","volume":"10","author":[{"given":"Huixin","family":"Liu","sequence":"first","affiliation":[{"name":"College of Animal Science and Technology, Guangxi University, Nanning 530005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1023-9635","authenticated-orcid":false,"given":"Kaichuang","family":"Shi","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Guangxi University, Nanning 530005, China"},{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuping","family":"Feng","sequence":"additional","affiliation":[{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanwen","family":"Yin","sequence":"additional","affiliation":[{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9625-0527","authenticated-orcid":false,"given":"Feng","family":"Long","sequence":"additional","affiliation":[{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0416-6150","authenticated-orcid":false,"given":"Hongbin","family":"Si","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Guangxi University, Nanning 530005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,5,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2106","DOI":"10.1099\/jgv.0.000873","article-title":"Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae","volume":"98","author":"Smith","year":"2017","journal-title":"J. Gen. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2994","DOI":"10.1099\/jgv.0.000251","article-title":"Discovery of a novel putative atypical porcine pestivirus in pigs in the USA","volume":"96","author":"Hause","year":"2015","journal-title":"J. Gen. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Arruda, B.L., Arruda, P.H., Magstadt, D.R., Schwartz, K.J., Dohlman, T., Schleining, J.A., Patterson, A.R., Visek, C.A., and Victoria, J.G. (2016). Identification of a divergent lineage porcine pestivirus in nursing piglets with congenital tremors and reproduction of disease following experimental inoculation. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0150104"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1020","DOI":"10.1111\/tbed.12659","article-title":"Identification of atypical porcine pestivirus infection in swine herds in China","volume":"64","author":"Zhang","year":"2017","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2104","DOI":"10.3201\/eid2312.170951","article-title":"High abundance and genetic variability of atypical porcine pestivirus in pigs from Europe and Asia","volume":"23","author":"Postel","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e22","DOI":"10.1111\/tbed.12532","article-title":"High prevalence of highly variable atypical porcine pestiviruses found in Germany","volume":"64","author":"Beer","year":"2017","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1645","DOI":"10.1111\/tbed.12699","article-title":"First report of the novel atypical porcine pestivirus in Spain and a retrospective study","volume":"64","author":"Canturri","year":"2017","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2039","DOI":"10.1111\/tbed.12981","article-title":"Detection and phylogenetic characterization of atypical porcine pestivirus strains in Hungary","volume":"65","author":"Biksi","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1111\/tbed.12753","article-title":"Presence of atypical porcine pestivirus (APPV) in Brazilian pigs","volume":"65","author":"Weber","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Pedersen, K., Kristensen, C.S., Strandbygaard, B., B\u00f8tner, A., and Rasmussen, T.B. (2021). Detection of atypical porcine pestivirus in piglets from Danish sow herds. Viruses, 13.","DOI":"10.3390\/v13050717"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1761","DOI":"10.1111\/tbed.14149","article-title":"Detection and genetic analysis of a novel atypical porcine pestivirus from piglets with congenital tremor in Japan","volume":"69","author":"Kagawa","year":"2022","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1111\/tbed.13029","article-title":"Molecular characterization of two novel atypical porcine pestivirus (APPV) strains from piglets with congenital tremor in China","volume":"66","author":"Pan","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"468","DOI":"10.4142\/jvs.2018.19.3.468","article-title":"Identification and characterization of atypical porcine pestivirus genomes in newborn piglets with congenital tremor in China","volume":"19","author":"Shen","year":"2018","journal-title":"J. Vet. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.virol.2019.04.010","article-title":"12 novel atypical porcine pestivirus genomes from neonatal piglets with congenital tremors: A newly emerging branch and high prevalence in China","volume":"533","author":"Yan","year":"2019","journal-title":"Virology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1099\/jgv.0.001188","article-title":"Prevalence and genome characteristics of atypical porcine pestivirus in southwest China","volume":"100","author":"Zhou","year":"2019","journal-title":"J. Gen. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1002\/vms3.407","article-title":"Evolution and genetic diversity of atypical porcine pestivirus (APPV) from piglets with congenital tremor in Guangxi Province, Southern China","volume":"7","author":"Shi","year":"2021","journal-title":"Vet. Med. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"105282","DOI":"10.1016\/j.micpath.2021.105282","article-title":"Genetic characterization of atypical porcine pestivirus (APPV) in China and the successful isolation of a novel APPV strain within genotype 2","volume":"161","author":"Zhang","year":"2021","journal-title":"Microb. Pathog."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Dall Agnol, A.M., Alfieri, A.F., and Alfieri, A.A. (2020). Pestivirus K (atypical porcine pestivirus): Update on the virus, viral infection, and the association with congenital tremor in newborn piglets. Viruses, 12.","DOI":"10.3390\/v12080903"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Stenberg, H., Hellman, S., Lindstr\u00f6m, L., Jacobson, M., Fossum, C., Hayer, J., and Malmberg, M. (2022). Congenital tremor and splay leg in piglets\u2014Insights into the virome, local cytokine response, and histology. BMC Vet. Res., 18.","DOI":"10.1186\/s12917-022-03443-w"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13567-016-0406-1","article-title":"Congenital infection with atypical porcine pestivirus (APPV) is associated with disease and viral persistence","volume":"48","author":"Schwarz","year":"2017","journal-title":"Vet. Res."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Folgueiras-Gonz\u00e1lez, A., van den Braak, R., Simmelink, B., Deijs, M., van der Hoek, L., and de Groof, A. (2020). Atypical porcine pestivirus circulation and molecular evolution within an affected swine herd. Viruses, 12.","DOI":"10.3390\/v12101080"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"952","DOI":"10.1177\/10406387211022683","article-title":"Distribution and persistence of atypical porcine pestivirus in experimentally inoculated pigs","volume":"33","author":"Buckley","year":"2021","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1111\/tbed.12854","article-title":"Frequent infection of wild boar with atypical porcine pestivirus (APPV)","volume":"65","author":"Cagatay","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Sozzi, E., Salogni, C., Lelli, D., Barbieri, I., Moreno, A., Alborali, G.L., and Lavazza, A. (2019). Molecular survey and phylogenetic analysis of atypical porcine pestivirus (APPV) identified in swine and wild boar from Northern Italy. Viruses, 11.","DOI":"10.3390\/v11121142"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1111\/tbed.12824","article-title":"Detection of atypical porcine pestivirus in Brazil in the central nervous system of suckling piglets with congenital tremor","volume":"65","author":"Gatto","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2519","DOI":"10.1007\/s00705-019-04345-3","article-title":"Viral load and histological distribution of atypical porcine pestivirus in different tissues of naturally infected piglets","volume":"164","author":"Liu","year":"2019","journal-title":"Arch. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"De Groof, A., Deijs, M., Guelen, L., van Grinsven, L., van Os-Galdos, L., Vogels, W., Derks, C., Cruijsen, T., Geurts, V., and Vrijenhoek, M. (2016). Atypical porcine pestivirus: A possible cause of congenital tremor type A-II in newborn piglets. Viruses, 8.","DOI":"10.3390\/v8100271"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4093","DOI":"10.1093\/jas\/skz267","article-title":"Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality","volume":"97","author":"Sutton","year":"2019","journal-title":"J. Anim. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"198151","DOI":"10.1016\/j.virusres.2020.198151","article-title":"Classical swine fever virus: The past, present and future","volume":"289","author":"Ganges","year":"2020","journal-title":"Virus Res."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Zheng, H.H., Fu, P.F., Chen, H.Y., and Wang, Z.Y. (2022). Pseudorabies virus: From pathogenesis to prevention strategies. Viruses, 14.","DOI":"10.3390\/v14081638"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1406","DOI":"10.3389\/fmicb.2019.01406","article-title":"Detection and genetic characterization of atypical porcine pestivirus in piglets with congenital tremors in Southern China","volume":"10","author":"Xie","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"114220","DOI":"10.1016\/j.jviromet.2021.114220","article-title":"Development of a quantitative real time RT-PCR assay for sensitive and rapid detection of emerging atypical porcine pestivirus associated with congenital tremor in pigs","volume":"296","author":"Yuan","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"114006","DOI":"10.1016\/j.jviromet.2020.114006","article-title":"Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus","volume":"287","author":"Liu","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Liu, H., Shi, K., Zhao, J., Yin, Y., Chen, Y., Si, H., Qu, S., Long, F., and Lu, W. (2022). Development of a one-step multiplex qRT-PCR assay for the detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus. BMC Vet. Res., 18.","DOI":"10.1186\/s12917-022-03144-4"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4182","DOI":"10.1002\/jmv.26846","article-title":"Droplet digital PCR of viral DNA\/RNA, current progress, challenges, and future perspectives","volume":"93","author":"Kojabad","year":"2021","journal-title":"J. Med. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1080\/07388551.2022.2037503","article-title":"Current commercial dPCR platforms: Technology and market review","volume":"43","author":"Tan","year":"2023","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1038\/nmeth.2633","article-title":"Absolute quantification by droplet digital PCR versus analog real-time PCR","volume":"10","author":"Hindson","year":"2013","journal-title":"Nat. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1007\/s42770-022-00728-y","article-title":"Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection","volume":"53","author":"Deng","year":"2022","journal-title":"Braz. J. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Choe, S., Park, G.N., Cha, R.M., Hyun, B.H., Park, B.K., and An, D.J. (2020). Prevalence and genetic diversity of atypical porcine pestivirus (APPV) detected in South Korean wild boars. Viruses, 12.","DOI":"10.3390\/v12060680"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1111\/tbed.12807","article-title":"Viruses associated with congenital tremor and high lethality in piglets","volume":"65","author":"Possatti","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1128\/CMR.19.1.165-256.2006","article-title":"Real-time PCR in clinical microbiology: Applications for routine laboratory testing","volume":"19","author":"Espy","year":"2006","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1373\/clinchem.2019.304048","article-title":"Digital PCR-An emerging technology with broad applications in microbiology","volume":"66","author":"Salipante","year":"2020","journal-title":"Clin. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"27735","DOI":"10.1038\/srep27735","article-title":"Presence of atypical porcine pestivirus (APPV) genomes in newborn piglets correlates with congenital tremor","volume":"6","author":"Postel","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_44","first-page":"429","article-title":"First report of atypical porcine pestivirus in piglets with congenital tremor in Canada","volume":"59","author":"Dessureault","year":"2018","journal-title":"Can. Vet. J."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Kaufmann, C., Stalder, H., Sidler, X., Renzullo, S., Gurtner, C., Grahofer, A., and Schweizer, M. (2019). Long-term circulation of atypical porcine pestivirus (APPV) within Switzerland. Viruses, 11.","DOI":"10.3390\/v11070653"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1111\/tbed.14046","article-title":"Genetic diversity and prevalence of atypical porcine pestivirus in the midwest of US swine herds during 2016-2018","volume":"69","author":"Yuan","year":"2022","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Yuan, J., Han, Z., Li, J., Huang, Y., Yang, J., Ding, H., Zhang, J., Zhu, M., Zhang, Y., and Liao, J. (2017). Atypical porcine pestivirus as a novel type of pestivirus in pigs in China. Front. Microbiol., 8.","DOI":"10.3389\/fmicb.2017.00862"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Ma, H., Li, W., Zhang, M., Yang, Z., Lin, L., Ghonaim, A.H., and He, Q. (2022). The diversity and spatiotemporally evolutionary dynamic of atypical porcine pestivirus in China. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.937918"}],"container-title":["Veterinary Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-7381\/10\/5\/330\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:29:04Z","timestamp":1760124544000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-7381\/10\/5\/330"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,4]]},"references-count":48,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2023,5]]}},"alternative-id":["vetsci10050330"],"URL":"https:\/\/doi.org\/10.3390\/vetsci10050330","relation":{},"ISSN":["2306-7381"],"issn-type":[{"value":"2306-7381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,5,4]]}}}