{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T18:54:30Z","timestamp":1793300070255,"version":"4.5.0"},"reference-count":41,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2025YFD1800101"],"award-info":[{"award-number":["2025YFD1800101"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vet. Sci.","Veterinary Sciences"],"abstract":"<jats:p>To achieve rapid, accurate, and quantitative detection of peste des petits ruminants virus (PPRV), we aligned the available PPRV genomic sequences, screened for conserved regions, and designed specific primers and probes accordingly. An RT-droplet digital PCR (RT-ddPCR) method was established, and a series of reaction conditions were optimized. Subsequently, the specificity, sensitivity, and repeatability of the method were subsequently evaluated. The results demonstrated that the RT-ddPCR method exhibited good specificity, with positive results obtained exclusively for PPRV and negative results for all other goat-derived viral or bacterial biological products and seed viruses tested. The method showed promising analytical sensitivity, with a LOD of 1.0 copies\/\u03bcL for gene copy number. The method also demonstrated good repeatability, with coefficients of variation in all groups not exceeding 5%. In a preliminary clinical evaluation using 150 field samples, RT-ddPCR identified 113 positive cases, compared to 105 by RT-qPCR, with 8 additional low-copy-number positives detected exclusively by RT-ddPCR, and although the sample size is limited for precise diagnostic performance estimation, the assay showed 100% relative diagnostic sensitivity (105\/105) and 82.2% relative diagnostic specificity (37\/45), with almost perfect agreement between the two methods (\u03ba = 0.87). As the positive signals in these discordant samples were too weak for independent confirmation by Sanger sequencing, they should be interpreted as \u201cpresumptive positives\u201d requiring further investigation. The RT-ddPCR method established in this study enables sensitive and rapid detection of PPRV nucleic acid. However, detection of low-copy-number targets should be interpreted with caution, as it does not necessarily indicate active infection or epidemiologically significant virus circulation. This assay shows promise as a valuable research and surveillance tool for PPRV nucleic acid detection, particularly for confirming low-level viral RNA in samples near the detection limit of conventional methods, and could complement existing RT-qPCR-based approaches in PPR eradication programs.<\/jats:p>","DOI":"10.3390\/vetsci13080841","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T16:05:19Z","timestamp":1787241919000},"page":"841","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Validation of an RT-Droplet Digital PCR Assay for Detection of Peste des Petits Ruminants"],"prefix":"10.3390","volume":"13","author":[{"given":"Rima","family":"Si","sequence":"first","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"},{"name":"College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8707-312X","authenticated-orcid":false,"given":"Jiao","family":"Xu","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaohua","family":"Wang","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yumeng","family":"Liu","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linlin","family":"Fang","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiani","family":"Li","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266105, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingli","family":"Wang","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiarong","family":"Yu","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Zhai","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"},{"name":"College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qinghua","family":"Wang","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2241-8507","authenticated-orcid":false,"given":"Jingyue","family":"Bao","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiliang","family":"Wang","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lin","family":"Yang","sequence":"additional","affiliation":[{"name":"Reference Laboratory for Peste des Petits Ruminants, China Animal Health and Epidemiology Center, Qingdao 266000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1080\/01652176.2020.1714096","article-title":"Peste des petits ruminants in large ruminants, camels and unusual hosts","volume":"40","author":"Rahman","year":"2020","journal-title":"Vet. Q."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Saad, Z., Muhammad, S.S., Sultan, A., Laiba, S., Muhammad, S.M., Aman, U.K., and Muhammad, M.A. (2024). Recapitulation of Peste des Petits Ruminants (PPR) Prevalence in Small Ruminant Populations of Pakistan from 2004 to 2023: A Systematic Review and Meta-Analysis. Vet. Sci., 11.","DOI":"10.3390\/vetsci11060280"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e70182","DOI":"10.1002\/vms3.70182","article-title":"Structure, Attachment and Transmembrane Internalisation of Peste Des Petits Ruminants Virus","volume":"11","author":"Zou","year":"2025","journal-title":"Vet. Med. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kutumbetov, L., Myrzakhmetova, B., Tussipova, A., Zhapparova, G., Bissenbayeva, K., Tlenchiyeva, T., Nurabayev, S., Kerimbayev, A., Orynbayev, M., and Makhashov, E. (2025). Susceptibility and Transmission Dynamics of Peste des Petits Ruminants Virus in Domestic and Wild Ruminants: Experimental Insights from Kazakhstan. Viruses, 17.","DOI":"10.3390\/v17091231"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1837","DOI":"10.3855\/jidc.19577","article-title":"Peste des petits ruminants: Past, present, and future scope","volume":"18","author":"Fayyad","year":"2024","journal-title":"J. Infect. Dev. Ctries."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"105774","DOI":"10.1016\/j.meegid.2025.105774","article-title":"Genomic analysis of peste des petits ruminants virus in Europe: Common origin for emergence in Greece, Romania, and Bulgarias","volume":"132","author":"Guendouz","year":"2025","journal-title":"Infect. Genet. Evol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"299","DOI":"10.3201\/eid1502.080817","article-title":"Peste des petits ruminants virus in Tibet, China","volume":"15","author":"Wang","year":"2009","journal-title":"Emerg. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.virol.2017.07.018","article-title":"Evolutionary dynamics of recent peste des petits ruminants virus epidemic in China during 2013\u20132014","volume":"510","author":"Bao","year":"2017","journal-title":"Virology"},{"key":"ref_9","unstructured":"FAO (2025). WOAH and FAO Call on Members to Strengthen Global Efforts to Eradicate PPR, FAO."},{"key":"ref_10","unstructured":"FAO (2025). PPR Global Eradication Programme (GEP), FAO."},{"key":"ref_11","unstructured":"FAO (2025). PPR Monitoring and Assessment Tool (PMAT), FAO, World Organisation for Animal Health."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1007\/s11250-010-9527-0","article-title":"Isolation and identification of virulent peste des petits ruminants viruses from PPR outbreaks in India","volume":"42","author":"Balamurugan","year":"2010","journal-title":"Trop. Anim. Health Prod."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/s12575-024-00249-y","article-title":"Molecular Characterization of Lineage-IV Peste Des Petits Ruminants Virus and the Development of In-House Indirect Enzyme-Linked Immunosorbent Assay (IELISA) for its Rapid Detection","volume":"26","author":"Kamal","year":"2024","journal-title":"Biol. Proced. Online"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2881","DOI":"10.1111\/tbed.13660","article-title":"Comparative evaluation of different antigen detection methods for the detection of peste des petits ruminants virus","volume":"67","author":"Halecker","year":"2020","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Soverini, S., Bernardi, S., and Galimberti, S. (2020). Molecular Testing in CML between Old and New Methods: Are We at a Turning Point?. J. Clin. Med., 9.","DOI":"10.3390\/jcm9123865"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"104111","DOI":"10.1063\/5.0191132","article-title":"Assumption-free analysis for amplification-based quantitative nucleic acid detection","volume":"160","author":"Fu","year":"2024","journal-title":"J. Chem. Phys."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1007\/s00216-012-6445-1","article-title":"Characterization of in vitro transcription amplification linearity and variability in the low copy number regime using External RNA Control Consortium (ERCC) spike-ins","volume":"405","author":"Kralj","year":"2013","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_18","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_19","doi-asserted-by":"crossref","first-page":"9744","DOI":"10.1038\/s41598-025-94115-w","article-title":"Absolute quantification of rare gene targets in limited samples using crude lysate and ddPCR","volume":"15","author":"Kaur","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5601","DOI":"10.1021\/acs.est.5b00253","article-title":"Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species","volume":"49","author":"Doi","year":"2015","journal-title":"Environ. Sci. Technol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1621","DOI":"10.1128\/JCM.00211-17","article-title":"Applications of Digital PCR for Clinical Microbiology","volume":"55","author":"Kuypers","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Njogu, A.K., Logozzo, F., Conner, W.R., and Shropshire, J.D. (2024). Counting rare Wolbachia endosymbionts using digital droplet PCR. bioRxiv.","DOI":"10.1101\/2024.12.10.627731"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Yu, Z., Chen, L., Cui, Q., Yan, H., Li, J., Luo, X., Li, Y., Ju, Z., Yong, Y., and Zhao, N. (2025). Development and application of reverse transcriptase droplet digital PCR technology for sensitive detection of BVDV-1 and BTV in bovine semen. BMC Vet. Res., 21.","DOI":"10.1186\/s12917-025-04506-4"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Yu, J., Zhu, L., Zuo, Y., Gao, S., Wang, Q., Xu, J., Fang, L., Liu, Y., Liu, S., and Wang, X. (2026). Development and validation of a novel triplex droplet digital PCR assay for simultaneous detection of African swine fever virus, pseudorabies virus, and porcine parvovirus. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1710807"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"veae012","DOI":"10.1093\/ve\/veae012","article-title":"Comparative evolutionary analyses of peste des petits ruminants virus genetic lineages","volume":"10","author":"Courcells","year":"2024","journal-title":"Virus Evol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Xu, J., Li, J., Wang, Q., Zhou, J., Liu, S., Wang, Y., Yu, J., Bao, J., and Yang, L. (2026). Establishment of a Quadruplex RT-qPCR Method for the Detection of All Lineages of PPRV. Animals, 16.","DOI":"10.3390\/ani16091397"},{"key":"ref_27","unstructured":"WOAH (2025). PPR: A Threat to Economic Resilience, WOAH."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"106633","DOI":"10.1016\/j.prevetmed.2025.106633","article-title":"When farmers\u2019 knowledge matters: Improving epidemiological understanding of Peste des petits ruminants in northern Nigeria","volume":"244","author":"Kodo","year":"2025","journal-title":"Prev. Vet. Med."},{"key":"ref_29","unstructured":"WOAH (2025). PPR: A Continuing Threat to Farmers\u2019 Livelihood, WOAH."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1111\/tbed.14282","article-title":"Impact of Peste des petits ruminants for sub-Saharan African farmers: A bioeconomic household production model","volume":"69","author":"Lhermie","year":"2022","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Aboah, J., Apolloni, A., Duboz, R., Wieland, B., Kotchofa, P., Okoth, E., and Dione, M. (2023). Ex-ante impact of pest des petits ruminant control on micro and macro socioeconomic indicators in Senegal: A system dynamics modelling approach. PLoS ONE, 18.","DOI":"10.1371\/journal.pone.0287386"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Mahapatra, M., Howson, E., Fowler, V., Batten, C., Flannery, J., Selvaraj, M., and Parida, S. (2019). Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme. Viruses, 11.","DOI":"10.3390\/v11080699"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"17760","DOI":"10.1038\/s41598-018-35636-5","article-title":"Development of real-time reverse transcription recombinase polymerase amplification (RPA) for rapid detection of peste des petits ruminants virus in clinical samples and its comparison with real-time PCR test","volume":"8","author":"Li","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"114971","DOI":"10.1016\/j.jviromet.2024.114971","article-title":"Establishment of a RAA-CRISPR Cas12a based diagnostic method for peste des petits ruminants virus N gene and M gene","volume":"329","author":"Xu","year":"2024","journal-title":"J. Virol. Methods"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1007\/s00216-016-9972-3","article-title":"A new immunoassay of serum antibodies against Peste des petits ruminants virus using quantum dots and a lateral-flow test strip","volume":"409","author":"Cheng","year":"2017","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1016\/j.jviromet.2010.01.014","article-title":"Quantitative one-step real-time RT-PCR for the fast detection of the four genotypes of PPRV","volume":"165","author":"Kwiatek","year":"2010","journal-title":"J. Virol. Methods"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"113735","DOI":"10.1016\/j.jviromet.2019.113735","article-title":"Improved PCR diagnostics using up-to-date in silico validation: An F-gene RT-qPCR assay for the detection of all four lineages of peste des petits ruminants virus","volume":"274","author":"Flannery","year":"2019","journal-title":"J. Virol. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.jviromet.2016.05.019","article-title":"Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR","volume":"235","author":"Li","year":"2016","journal-title":"J. Virol. Methods"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1016\/j.jviromet.2010.11.022","article-title":"A real time RT-PCR assay for the specific detection of Peste des petits ruminants virus","volume":"171","author":"Batten","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1642","DOI":"10.1021\/acs.analchem.6b03746","article-title":"Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases","volume":"89","author":"Sidstedi","year":"2017","journal-title":"Anal. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1186\/s13007-014-0042-6","article-title":"Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples","volume":"10","author":"Dreo","year":"2014","journal-title":"Plant Methods"}],"container-title":["Veterinary Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-7381\/13\/8\/841\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T04:26:33Z","timestamp":1787372793000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-7381\/13\/8\/841"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,20]]},"references-count":41,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["vetsci13080841"],"URL":"https:\/\/doi.org\/10.3390\/vetsci13080841","relation":{},"ISSN":["2306-7381"],"issn-type":[{"value":"2306-7381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,20]]}}}