兔出血癥病毒 RHDV1 和 RHDV2一步法雙重 Taq-Man探針熒光定量RT-PCR體系的建立和應用

打開文本圖片集

中圖分類號：S855.3 文獻標識碼：A 文章編號：1000-4440（2025）05-0937-06

Abstract：Rabbit hemorrhagic disease（RHD）caused by rabbit hemorrhagic disease virus（RHDV）isan acute ndhighlylethal infectiousdiseasethat posesaseriousthreattotherabitbreding industry.Inthisstudy，primersand

TaqMan probeswere designed based on the capsid protein （VP6O）gene sequences of RHDVto establish a dual TaqMan quantitativeRT-PCRdetectionsystem capableof simultaneously detecting both RHDV1 and RHDV2 strains. Thereaction system included 10.0μL of 2× One Step RTPCR Buffer Il ， 0.6μL of forward primer（10 μmol/L），

0.6μL of reverse primer （ 10μmol/L ， 0.4μL of ROX Reference Dye （ 50× ）， 0.8μL of each fluorescent probe（10 μmol/L ）， 2.0μL of test sample，and 4.8μLddH2O . The reaction program was as follows： 42ΩΩ for 5 min， 95‰ for 10s ， 959C for5s， 60YC for 30s ，and 4O cycles.Results showed that the system had strong specificity and no cross-reactions with rotavirus，Pasteurell multocida，Bordetella bronchiseptica，Pseudomonas aeruginosa，and Salmonella spp.，and high sensitivitywithaminimumdetectionlimitof1OOcopiesper microliter.Repeatabilityanalysis through intra-groupand inter-group repetitions showed that the coefficient of variation of Ct valueswas between 0.2% and 2.9% ，indicating good stabilityofthesystem.When testing112 clinicalsamples（6Olivertisuesand 52 nasal and anal swabs）usingthis system， the overall detection rate of RHDV reached 69 % ，while the detection rate of the conventional RT-PCR system was only 51% .The dual quantitative RT-PCR method established in this study has high sensitivity，strong specificity，and good repeatability，providing areliable technical support for therapid clinical diagnosisand quantitative analysis of RHDV.

Key words：rabbit hemorrhagic disease virus； fluorescence quantitative RT-PCR；TaqMan probe

兔病毒性出血癥（Rabbit hemorrhagicdisease，RHD），又稱兔出血癥或兔瘟，是由兔出血癥病毒（Rabbithemorrhagicdiseasevirus，RHDV）引起的一種急性、高度接觸傳染性、致死性疾病，主要感染家兔和野兔[1]。（剩余9483字）