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

Abstract

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.

Nested quantitative PCR approach for urinary cell‐free EZH2 mRNA and its potential clinical application in bladder cancer

“…In addition, affected by low abundance as whole urine, VEGF mRNA frequently found in tissues of renal cancer was at most detected in 62% (31/50) urine supernatant samples of patients with same disease . Some studies found a nested real time PCR method used in virus/bacteria detection could increase the clinical sensitivity . This, this study developed a nqPCR method by incorporating a conventional RT‐PCR for initial amplification of cf‐mRNAs in urine supernatant to improve the detection sensitivity and reduce false negatives.…”

Advances in canine distemper virus pathogenesis research: a wildlife perspective

“…Fischer et al [112] took it one step further by developing a technique of reverse transcription followed by a nested real-time PCR. The technique was performed on several clinical samples and proved to be two orders of magnitude more sensitive than RT-PCR.…”

“…Diagnosis is made by demonstration of typical histopathological lesions including the presence of viral inclusion bodies in lymphoid tissue, respiratory, urinary and gastro-intestinal tract epithelium and brain; by the presence of distinctive virions in negatively stained electron-microscopic preparations of faeces; and through the detection of viral antigen in tissue by immunofluorescence or immunohistochemistry [54,104]. Immunofluorescence has routinely been used as a diagnostic test; however, it is not sensitive and can detect CDV antigens only when the virus is still present in the epithelial cells [55,107] with false-negative results under certain clinical conditions [112,123].…”

Canine distemper virus detection by different methods of One-Step RT-qPCR

“…RT-qPCR assays have been applied to detect and quantify canine distemper virus in clinical specimens of naturally infected dogs or to distinguish vaccine strains from wild strains, using two-step and real-time probes chemistry (ELIA et al, 2006;SCAGLIARINI et al, 2007;FISCHER et al, 2013;WILkES et al, 2014). Recently, surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS) and conjugated gold nanoparticles methodologies were also developed for CDV detection (BASSO et al, 2013;2015a;2015b).…”