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- W2372865175 abstract "To establish the reverse transcription-nested polymerase chain reaction (RT-nested PCR) method for detecting Japanese encephalitis virus (JEV) in human biological products of porcine origin. Two pairs of primers were designed according to the complete genomic RNA sequence of JEV (GenBank accession number M55506). RNA was extracted from suckling mice brain infected with JEV and BHK-21 cell inoculated with JEV. The specific amplified fragments of E gene which encoding envelope glycoprotein of JEV were cloned into the pGEM-T easy vector and transformed into E.coli DH5. The recombinant plasmid were identified by electrophoresis, PCR, and restriction endonuclease analysis, and confirmed by sequencing. Porcine parvovirus (PPV), simian virus 40 (SV40), BHK-21 cell, PK15 cell, BSC-1cell, normal suckling mice brain (JEV free), and 210 clinical swine tissues specimens were submitted to detect JEV by PCR. All PCR products were detected by agarose gel electrophoresis. JEV RT-PCR amplification product of the external primers was 1 015 bp, and RT-nested PCR amplification product of the internal primers was 622 bp. Non-JEV specimens had not shown those specific amplification fragments. Using internal primers in a nested RT-PCR, the sensitivity was increased approximately 1000-fold compared to that of the RT-PCR. The procedure including RNA extraction, amplification, and detection could be finished within 8 h. The results of the study demonstrated that the RT-nested PCR established here was a rapid, specific, sensitive, and reliable method for the detection of JEV in human biological products of porcine origin." @default.
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- W2372865175 date "2005-01-01" @default.
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- W2372865175 title "Establishment and application of reverse transcription nested polymerase chain reaction assay for the detection of {sl Japanese encephalitis} virus" @default.
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