A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus

Abstract

A minor groove binder (MGB) probe assay was developed to discriminate between type 2-based vaccines and field strains of canine parvovirus (CPV). Considering that most of the CPV vaccines contain the old type 2, no longer circulating in canine population, two MGB probes specific for CPV-2 and the antigenic variants (types 2a, 2b and 2c), respectively, were labeled with different fluorophores. The MGB probe assay was able to discriminate correctly between the old type and the variants, with a detection limit of 10(1) DNA copies and a good reproducibility. Quantitation of the viral DNA loads was accurate, as demonstrated by comparing the CPV DNA titres to those calculated by means of the TaqMan assay recognising all CPV types. This assay will ensure resolution of most diagnostic problems in dogs showing CPV disease shortly after CPV vaccination, although it does not discriminate between field strains and type 2b-based vaccines, recently licensed to market in some countries.

Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics

“…For nucleic acid detection, PCR amplification makes possible the detection of <10 copies per sample (Decaro et al, 2006;Hoffmann et al, 2006;Saiki et al, 1985). However, the use of PCR or other primer-based asymmetric amplification schemes introduces significant complexity to sample preparation requirements, complications with real world sample matrices, and difficulties in multiplexing, all of which limit the ability for use outside a controlled laboratory environment.…”

Isolation and Typing of Canine Parvovirus in CRFK Cell Line in Puducherry, South India

“…Of the 18 samples passaged in CRFK cells, 3 samples (1544, 1272 and 3367) which showed mild cytopathic effect also demonstrated high HA titres up to third passage levels. The HA titres at third passage level were 1:2 8 , 1:2 11 and 1:2 6 for samples 1544, 1272 and 3367, respectively. Out of the 18 samples passaged in CRFK cells, cryolysates of the samples 1544, 1272 and 3367 showed positive reactions in the form of distinct brown spots by dot-ELISA at third passage level (Fig.…”

“…Glu, which occurs in a residue of the capsid protein that is considered important for the antigenic properties of CPV-2. This variant (CPV-2/ Glu-426 mutant), currently named as CPV-2c, is distributed in Italy [5], Spain [6], United Kingdom [7], and very recently in Portugal [8]. The disease is reported to be prevalent among canine populations in Puducherry [9].…”

Canine Parvoviruslar Üzerine Güncelleme: Canine Konakçıyı Etkileyen Genetik Varyasyonlara Vurgu Yaparak Moleküler ve Genomik Yönler

“…The results revealed the high specificity and low sensitivity of the antigen-detection kits [91] . Molecular biology techniques, such as traditional polymerase chain reaction (PCR) and quantitative PCR (realtime PCR; qPCR) have been widely developed and used in the detection of CPV genetic materials from blood and fecal samples [7,41,43,48] . Because of the sensitivity, specificity, and reproducibility of PCR and real-time PCR in detection of CPV DNA, this method might replace traditional methods such as virus isolation and antibody detection.…”