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Detection of canine parvovirus and feline panleukopenia virus in fecal samples by strand exchange amplification.

ABSTRACT: Canine parvovirus 2 (CPV-2) and feline panleukopenia virus (FPLV) often cause acute enteric disease in their hosts. A simple, rapid, and effective method for the on-site detection of these viruses would be useful. We used a denaturation bubble-mediated strand exchange amplification (SEA) method to successfully detect CPV-2 and FPLV in fecal samples. SEA could detect as little as 3.6 pg/μL of CPV-2 and 6.6 pg/μL of FPLV genomic DNA following a 40-min incubation at an isothermal temperature of 61°C. Unlike PCR, SEA does not require complicated equipment, and positive samples produce a color change that can be visualized by the naked eye. Additionally, SEA is simpler than PCR because no extraction is needed, and heating of the fecal sample at 98°C can be performed with a heating block or water bath. This rapid and effective nucleic acid detection platform could be used as a point-of-care test for the detection of CPV-2 and FPLV.

SUBMITTER: Liu M

PROVIDER: S-EPMC7649555 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The infection in dogs due to canine parvovirus (CPV), is a highly contagious one with high mortality rate. The present study was undertaken for a detailed genetic analysis of partial VP2 gene i.e., 630 bp isolated from rectal swab samples of infected domestic and stray dogs from all areas of district Faisalabad. Monitoring of viruses is important, as continuous prevalence of viral infection might be associated with emergence of new virulent strains.In the present study, 40 rectal swab samples were collected from diarrheic dogs from different areas of district Faisalabad, Pakistan, in 2014-15 and screened for the presence of CPV by immunochromatography. Most of these dogs were stray dogs showing symptoms of diarrhea. Viral DNA was isolated and partial VP2 gene was amplified using gene specific primer pair Hfor/Hrev through PCR. Amplified fragments were cloned in pTZ57R/T (Fermentas) and completely sequenced. Sequences were analyzed and assembled by the Lasergene DNA analysis package (v8; DNAStar Inc., Madison, WI, USA).The results with immunochromatography showed that 33/40 (82%) of dogs were positive for CPV. We were able to amplify a fragment of 630 bp from 25 samples. In 25 samples the sequences of CPV-2a were detected showing the amino acid substitution Ser297Ala and presence of amino acid (426-Asn) in partial VP2 protein. Interestingly the BLAST analysis showed the of feline panleukopenia virus (FPV) sequences in 3 samples which were already positive for new CPV-2a, with 99% sequence homology to other FPV sequences present in GenBank.Phylogenetic analysis showed clustering of partial CPV-VP-2 gene with viruses from China, India, Japan and Uruguay identifying a new variant, whereas the 3 FPV sequences showed immediate ancestral relationship with viruses from Portugal, South Africa and USA. Interesting observation was that CPV are clustering away from the commercial vaccine strains. In this work we provide a better understanding of CPV prevailing in Pakistan at molecular level. The detection of FPV could be a case of real co-infection or a case of dual presence, due to ingestion of contaminated food.

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Detection of canine parvovirus and feline panleukopenia virus in fecal samples by strand exchange amplification.

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