Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.
Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.
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DOI：
10.1016/j.jviromet.2013.08.002
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发表时间：
2013-12
影响因子：
3.1
通讯作者：
Lunge VR
中科院分区：
文献类型：
--
作者：
Fischer CD;Ikuta N;Canal CW;Makiejczuk A;Allgayer Mda C;Cardoso CH;Lehmann FK;Fonseca AS;Lunge VR
这是第一个反转录-巢式真实的时间PCR检测犬瘟热病毒的报告。该技术能够从疫苗株和野毒株中检测到CDV RNA。RT-nqPCR技术具有敏感性和特异性。该方法可用于不同临床样品的分析。该方法可作为犬瘟热诊断的重要手段。犬瘟热病毒（CDV）是一种严重的和高度传染性的疾病，狗的原因。犬瘟热的临床症状和实验室检查的基础上实际诊断需要确认犬瘟热病毒感染。本研究的目的是建立一种分子检测和区分CDV野毒株和疫苗株的方法。开发了逆转录后巢式真实的时间聚合酶链反应（RT-nqPCR），其表现出分析特异性（来自健康犬和其他犬传染性病原体的所有样品均未被错误检测）和灵敏度（疫苗株的所有重复样品在高达3125倍稀释度-100.7 TCID 50时均为阳性）。对103只疑似犬瘟热动物的不同临床样本（血液、尿液、直肠和结膜拭子）进行了RT-nqPCR检测验证。基于RT-nqPCR，在至少一个临床样品中发现总共53只动物为阳性。血液样本的阳性率较高（50/53，94.3%），其次是尿液样本（44/53，83.0%）、直肠样本（38/53，71%）和结膜样本（27/53，50.9%）。商业免疫层析（IC）检测仅在这些阳性犬的30个结膜样本中检测到CDV。核蛋白（NC）基因测序的25个样品表明，其中23个更接近其他巴西野外菌株，其余两个疫苗株。通过限制性片段长度多态性（RFLP）分析，使用产生Msp I限制性酶切的单核苷酸序列差异来区分田间和疫苗CDV毒株。完全法比IC法检测CDV的灵敏度高。血液是更常见的阳性标本，增加限制性内切酶步骤可以区分疫苗和巴西田间菌株。
This is the first report of a reverse transcription-nested real time PCR for CDV detection. The technique was able to detect CDV RNA from vaccine and field strains. This new technique (RT-nqPCR) was sensitive and specific. Different clinical samples could be analysed by the method. The method here described should be an important tool for diagnosis of canine distemper. 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 – 100.7 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.
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影响因子：
5
作者：
Pinto LD;Streck AF;Gonçalves KR;Souza CK;Corbellini ÂO;Corbellini LG;Canal CW
通讯作者：
Canal CW
DOI：
10.1590/s0102-09352007000300016
发表时间：
2007-06-01
期刊：
Arquivo Brasileiro de Medicina Veterinária e Zootecnia
影响因子：
--
作者：
Castilho, J.G.;Brandão, P.E.;Kotait, I.
通讯作者：
Kotait, I.
影响因子：
3.1
作者：
Decaro N;Martella V;Ricci D;Elia G;Desario C;Campolo M;Cavaliere N;Di Trani L;Tempesta M;Buonavoglia C
通讯作者：
Buonavoglia C
影响因子：
9.4
作者：
Mochizuki, M;Hashimoto, M;Ishiguro, S
通讯作者：
Ishiguro, S
影响因子：
2.2
作者：
Scagliarini, A.;Dal Pozzo, F.;Morganti, L.
通讯作者：
Morganti, L.