Establishment of internally controlled Real-time PCR for the detection of human parvovirus B19 DNA in serum

Abstract

To establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum. Two DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA. The standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively. The Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.