| 摘要 |
PURPOSE: Simultaneous detection method of Porcine epidermic diarrhea (PEDV) and Transmissible gastroenteritis (TGEV) using multiplex reverse transcription polymerase chain reaction (RT-PCR) and primers for RT-PCR are provided. CONSTITUTION: Simultaneous detection method comprises the following steps of: a) design of primers, b) PCR reaction with primer, reaction mixture and RNA purified from feces or small intestine of infected porcine, and c) agarose gel electrophoresis of PCR products. Forward primer for PEDV is oligonucleotide containing the complement sequence to nucleotide 927-946 of M gene of PEDV and reverse primer is oligonucleotide containing the complement sequence to nucleotide 1319-1338. The size of PCR product is 412 base pairs and very well conserved region of PEDV. Forward primer for TGEV is oligonucleotide containing the complement sequence to nucleotide 1226-1245 of N-gene of TGEV and reverse primer is oligonucleotide containing the complement sequence to nucleotide 1818-1837. The size of PCR product is 612 base pairs and very well conserved region of TGEV. The four primers, AMV reverse transcriptase, Tfl DNA polymerase, reaction buffer, MgSO4, dNTP and RNA sample purified from infected porcine are combined to perform RT-PCR. The reaction buffer can be used for reverse transcriptase and Tfl DNA polymerase.
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