Standardization of parvovirus B19 DNA extraction from serum and quantitative PCR.
Human parvovirus B19 (B19) is a newly emerging virus causing a wide spectrum of clinical conditions. The corner stone of diagnosis of acute B19 infection is demonstration of anti-B19 IgM antibodies and its DNA by PCR. Sera of patients infected with B19 acutely or persistently shows intense viraemia (up to 10(12) virus particles/ ml) but extraction of B19 DNA from serum is a problematic task. There is no single standardized, cost-effective in-house method, which can successfully extract DNA of B19 from serum samples and which has been subsequently tested repeatedly by many investigators over time. We describe here an efficient in-house method of extraction of B19 DNA from serum and quantitate extracted DNA by PCR. Firstly, a quantitative PCR was done using 3 microl of a cloned B19 DNA (33.3 pg/ml) mixed in 47 microl of sterile distilled water which were further diluted from 10(-1) up to 10(-7) to find the lower limit of DNA detection. To mimic human serum samples with known quantity of B19, 3 ml of cloned B19 DNA (33.3 pg/ml) were mixed with 47ml of fetal calf serum (FCS; free of B19) and were similarly log diluted from 10(-1) to 10(-7) in 50 ml volume. In-house method of DNA extraction from serum (FCS+B19 DNA) were then performed followed by quantitative PCR. In both the cases, we were able to detect B19 DNA up to 10(-4) dilution which contained 0.6 fg of B19 DNA corresponding to 12 B19 genome equivalents/PCR reaction or 2.4 x 10(2) genome quivalents/ml of serum. Further, the entire procedure was repeated two more times and identical results were obtained confirming its reproducibility. Using this in-house method we extracted and amplified B19 DNA successfully from sera of clinically suspected cases of B19 infection (data not shown). Compared to other studies, the sensitivity of our in-house method was found to be superior hence recommended for developing countries as commercial kits are too costly.
Kishore
Janak
J
Division of Virology, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow. janak@sgpgi.ac.in
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007194:India; D010322:Parvoviridae Infections / Q000175:diagnosis; D016732:Parvovirus B19, Human / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012015:Reference Standards
D004279:DNA, Viral