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
| Autor: | Cláudio Wageck Canal, Mariangela da Costa Allgayer, Cristine Dossin Bastos Fischer, Vagner Ricardo Lunge, André Salvador Kazantzi Fonseca, Nilo Ikuta, Aline Makiejczuk, Fernanda Kieling Moreira Lehmann, Cristine Hoffmeister Cardoso |
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| Rok vydání: | 2013 |
| Předmět: |
Veterinary Medicine
Canine distemper virus animal diseases RT-PCR Urine Biology Real-Time Polymerase Chain Reaction Polymerase Chain Reaction Sensitivity and Specificity Article Virus Dogs Virology Diagnosis Dog medicine Animals Dog Diseases Distemper Distemper Virus Canine Canine distemper virus diseases Viral Vaccines Reverse Transcription medicine.disease Reverse transcriptase Nucleoprotein Detection Restriction enzyme Real-time polymerase chain reaction Restriction fragment length polymorphism Brazil Polymorphism Restriction Fragment Length |
| Zdroj: | Journal of Virological Methods |
| ISSN: | 0166-0934 |
| Popis: | Highlights • 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. |
| Databáze: | OpenAIRE |
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