FIELD: biotechnology.
SUBSTANCE: described is a method for indirect determination of the concentration of virions of the causative agent of canine parvoviral enteritis in raw material for vaccines. Method involves: recovering DNA of the causative agent of canine parvoviral enteritis by a solid-phase method; conducting an amplification reaction in real time with detection of a fluorescent sigmoid signal for the analysed samples using oligonucleotides designed for the target region of the NS1 gene of the causative agent of canine parvoviral enteritis in range of 381…638 bp; calculation of the maximum on the graph of the second derivative of the sigmoid function for the products of the amplification reaction; determination of the infectious activity titre of the causative agent of canine parvoviral enteritis in the raw material for the vaccine based on the developed mathematical function CCPV = -0.3012 × Cp-NS1 + 9.0213 with high accuracy of approximation R2 = 0.9944 and amplification efficiency of 99.74%. In the invention, dependence of the concentration of CPV virions and values of points Cp-NS1 are established with high accuracy of approximation (R2 = 0.9944) and amplification efficiency of 99.74%, which enables to indirectly determine the concentration of virions of the causative agent of canine parvoviral enteritis in the raw material for vaccines using the amplification of the NS1-gene of viral DNA.
EFFECT: main advantages of the invention are the possibility of eliminating the probability of contamination during the reaction and subjectivity when taking into account the analysis results; possibility to reduce time of analysis of virus-containing raw material for vaccines to 2 hours; possibility to increase specificity and sensitivity of analysis.
2 cl, 4 dwg, 3 tbl, 4 ex
