FIELD: biotechnology.
SUBSTANCE: production of vaccines against infectious feline rhinotracheitis FHV. A method is described for indirectly determining the titer of infectious activity of the causative agent of infectious feline rhinotracheitis in non-inactivated raw materials for vaccines using polymerase chain reaction and quantitative hybridization-fluorescence detection in real time. The method includes the following stages: DNA isolation of feline infectious rhinotracheitis virus using 5 M guanidine isothiocyanate and 90% propanol-2; carrying out polymerase chain reaction and quantitative hybridization-fluorescent detection in real time using developed original oligonucleotides: FHV-TKG-TF with design 5'-CGTGGTGAATTATCAGCTGA-3', FHV-TKG-TR with design 5'-CTGTTATTGTGGATAGTCTGG-3' and FHV-TKG-TP with design 5'-Su 5.5 new - CAAGATTTGCCGCACCATACCT-BHQ-3-3' , which are designed for the TKG gene region of the DNA of the feline infectious rhinotracheitis virus; calculation of quantification cycle values based on real-time PCR data; determination of the titer of infectious activity of cultured feline infectious rhinotracheitis virus in non-inactivated raw materials for vaccines based on the developed logarithmic regression function: lg TFHV = -0.2998×Cq+9.3735 with an approximation confidence of 0.9967 and an amplification efficiency of 99.43%. The main advantages of the proposed invention are the ability to reduce the time of analysis of virus-containing raw materials for a vaccine to 2.5 hours; to increase the specificity and sensitivity of the analysis for determining the titer of infectious activity of the feline infectious rhinotracheitis virus.
EFFECT: determining with a high degree of reliability the titer of infectious activity of the causative agent of infectious feline rhinotracheitis in non-inactivated raw materials for a vaccine based on real-time PCR using original specific oligonucleotides for the TKG gene region of the DNA of the FHV virus and a developed logarithmic model.
2 cl, 4 dwg, 6 tbl, 5 ex
| Title | Year | Author | Number |
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| METHOD FOR INDIRECT DETERMINATION OF INFECTIOUS ACTIVITY TITRE OF CAUSATIVE AGENT OF INFECTIOUS FELINE RHINOTRACHEITIS IN RAW MATERIAL FOR VACCINE USING SECOND-ORDER DIFFERENTIAL OF POINT OF INTERSECTION OF LOGISTIC CURVE OF AMPLIFICATION REACTION OF UL35 GENE REGION | 2023 |
|
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