Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution - 10(0.7) TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains.

译文

:犬瘟热病毒(CDV)是导致犬种严重和高度传染性疾病的原因。需要根据临床体征和实验室检查对犬瘟热进行实际诊断,以确认CDV感染。本研究旨在开发一种分子检测方法,以检测和区分野外和疫苗的CDV毒株。开发了逆转录,随后进行了巢式实时聚合酶链反应(RT-nqPCR),该方法具有分析特异性(未正确检测出健康犬和其他犬类传染病菌的所有样品)和灵敏度(疫苗株的所有重复均为阳性)稀释至3125倍-10(0.7)TCID50)。验证RT-nqPCR可在103例怀疑患有脾热的动物的不同临床样品(血液,尿液,直肠和结膜拭子)上进行CDV检测。基于RT-nqPCR,在至少一个临床样品中发现总共53只动物是阳性的。血液产生更多阳性样本(53个中有50个,占94.3%),其次是尿液(44 / 53,83.0%),直肠(38 / 53,71%)和结膜(27 / 53,50.9%)拭子。商业免疫色谱法(IC)分析仅在这些阳性犬的30个结膜样本中检测到CDV。对25个样品进行的核蛋白(NC)基因测序表明,其中23个更接近其他巴西田间毒株,其余两个更接近疫苗株。通过限制性片段长度多态性(RFLP)分析,使用产生Msp I限制性酶消化的单核苷酸序列差异来区分田间和疫苗CDV菌株。完整的检测方法比IC检测CDV更为灵敏。血液是更常见的阳性标本,加上限制性内切酶步骤可以区分疫苗和巴西田间菌株。

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