Establishment of a random PCR method for detecting unknown virus

SHI Bi-sheng; ZHANG Xiao-nan; GU Shi-ming; TIAN Di; YUAN Zheng-hong; HU Yun-wen

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Journal of Microbes and Infections ›› 2008, Vol. 3 ›› Issue (3) : 134-137.

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Establishment of a random PCR method for detecting unknown virus

SHI Bi-sheng; ZHANG Xiao-nan; GU Shi-ming; TIAN Di; YUAN Zheng-hong; HU Yun-wen

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Abstract

Objective To establish a molecular biology method for detecting sequences of unknown viruses. Methods The hepatitis B and hepatitis C viruses were used as hypothetically unrecognized DNA, RNA viruses respectively to test the efficacy of this random PCR-based method. Virus-containing serum was first filtered through a 0.22-μm disk filter followed by DNase I treatment to eliminate the host genomic DNA. After the 26mer 5’ end anchored random primer was annealed to the extracted nucleic acids either by Klenow polymerase extension (DNA as template) or by reverse transcription (RNA as template), the virus genomes were randomly amplified with the 20mer anchor primer. Purified PCR products were cloned, sequenced and subjected to BLAST search for further analysis. Results HBV and HCV genomes were amplified by random PCR and their fragments could be detected in the picked clones. The detective rate was about 15% at 1106 copies/ml viral load. Positive clones could still be obtained with as few as 1104 copies/ml viral load. Conclusion This culture-independent random PCR-based method can be used to investigate unknown viruses and will be useful for the early diagnosis of emerging infectious disease.

Key words

Unknown virus / Molecular diagnosis / Random polymerase chain reaction

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SHI Bi-sheng; ZHANG Xiao-nan; GU Shi-ming; TIAN Di; YUAN Zheng-hong; HU Yun-wen. Establishment of a random PCR method for detecting unknown virus[J]. Journal of Microbes and Infections. 2008, 3(3): 134-137