Multiplex nested RT-PCR for the detection of porcine enteric viruses

Abstract

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (PRV-A) are major viruses causing enteric diseases of piglets. A multiplex nested reverse transcription polymerase chain reaction (multiplex nested RT-PCR) was developed for the detection of these viruses in field samples from piglets with diarrhea. A mixture of (1) three external pairs of primers, yielding in the amplification step two different amplicons with sizes of 950 bp and 317 bp and (2) three pairs of internal primers in a second round of PCR (nested PCR), yielding two different amplicons with sizes of 792 bp and 208 bp for TGEV and porcine PRV-A, respectively. The genome of PEDV was not detected after the amplification step but it was detected in the second round of PCR, yielding amplicon with size of 291 bp. Multiplex nested RT-PCR can detect TGEV, PRV-A, and PEDV up to concentration 10(2) TCID(50)/mL, 10(1) TCID(50)/mL, and 27.2 microg/microl of RNA, respectively. A total of 175 field samples were collected from swine with diarrhea from January 2005 until July 2007. The samples were tested for the presence of three viruses by a multiplex nested RT-PCR. Dual infections with PEDV and PRV-A were identified in seven specimens (4%) (n = 6). Twenty-one (25%) infections were caused by PEDV and thirty-four infections (41%) were caused by PRV-A. The genome of TGEV was not detected in any of these field samples, however TGEV was detected in piglets infected experimentally. The multiplex nested RT-PCR is rapid, sensitive, and a cost-effective detection method for the detection of porcine enteric viruses.

Fig. 1

Electrophoretic profile of multiplex nested RT-PCR products. M, DNA Ladder φX174/HaeIII. Lane 1, TGEV, TMK22 strain; PEDV isolate; and PRV-A, KR-47 strain. Lane 2, negative control (MilliQ water). Lane 3, PEDV isolate. Lane 4, PRV-A, KR-47 strain. Lane 5, TGEV, TMK22 strain.

Fig. 2

Electrophoretic profile of the multiplex nested RT-PCR product for PEDV Moscow/07/05 isolated from porcine stool sample and inoculated into Vero cell culture. M, DNA marker φX174/HaeIII. Lane 1, 11th passage, lane 2, 10th passage; lane 3, 9th passage; lane 4, 8th passage; lane 5, 7th passage; lane 6, 6th passage; and lane 7, 5th passage.

Fig. 3

Comparison of the nucleotide sequences of the amplified PEDV M gene region. The positions of specific primers (M3: forward primer, M4: reverse primer) are underlined. Russian isolates are in bold.

Fig. 3

Comparison of the nucleotide sequences of the amplified PEDV M gene region. The positions of specific primers (M3: forward primer, M4: reverse primer) are underlined. Russian isolates are in bold.

Fig. 3

Comparison of the nucleotide sequences of the amplified PEDV M gene region. The positions of specific primers (M3: forward primer, M4: reverse primer) are underlined. Russian isolates are in bold.

Fig. 3

Comparison of the nucleotide sequences of the amplified PEDV M gene region. The positions of specific primers (M3: forward primer, M4: reverse primer) are underlined. Russian isolates are in bold.

Fig. 3

Comparison of the nucleotide sequences of the amplified PEDV M gene region. The positions of specific primers (M3: forward primer, M4: reverse primer) are underlined. Russian isolates are in bold.

Fig. 4

Evolutionary relationships of PEDV Russian isolates and published strains based on the expected fragment comparison. (A) Cluster A and (B) cluster B. NCBI accession numbers, strain and isolate designations are given. Russian isolates are in bold.