Assessment of Polymerase Chain Reaction Sensitivity for the Detection of Chicken Anaemia Virus using Different Primers for Three Genes
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Date
2012
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Veterinarski Arhiv
Abstract
Chicken Anaemia Virus (CAV) genes cloned plasmid DNA templates (pCR4-CAV-VP1 and pCR4 -CAVVP2&3) were used in this study to develop a sensitive polymerase chain reaction (PCR) for CAV gene detection. A total of nine sets of primers, which include one set of published primers and two sets of designed primers for each gene of CAV, viz. VP1, VP2 and VP3, were used to assess the sensitivity of PCR. The PCR cycle
conditions were standardized with the designed primers to get a single, specific sized amplicon for each gene separately. PCR sensitivity assessment was done by making serial 10 fold dilutions of the cloned CAV plasmid templates and subjected to PCR with each set of primers for each gene of CAV. The highest dilution of the CAV
plasmid DNA showing a visible PCR amplicon was taken as the detection limit. The results showed that the designed primer VP1.2 was found to be more sensitive for the VP1 gene and the concentration of the plasmid DNA was 0.05 fg/µL or 8.6 x10^3 molecules/mL and VP 2.2 was found to be more sensitive for the VP2 gene and the concentration of the plasmid was 5 ag/µL or 9.9 x10^2 molecules/mL. In the case of VP3, the published primer VP 3.1 was found to be more sensitive for the VP3 gene and the concentration of the plasmid was 5 x10^4 ag/µL or 10.5 x10^-2 molecules/mL. The findings of this study may be very useful for diagnostic, sequencing, cloning and expression purposes.
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Keywords
Chicken anaemia virus, Polymerase chain reaction sensitivity, Primers, Amplicon