Development of a Sensitive Real-Time Fast-qPCR Based on SYBR (R) Green for Detection and Quantification of Chicken Parvovirus (ChPV)

Author(s)

Nunez, Luis F. ; Santander-Parra, Silvana H. ; Chaible, Lucas ; De la Torre, David, I ; Buim, Marcos R. ; Murakami, Alexandre ; Zaidan Dagli, Maria Lucia ; Astolfi-Ferreira, Claudete S. ; Piantino Ferreira, Antonio J.

• Document type: Journal article
• Source: VETERINARY SCIENCES; v. 5, n. 3, p. 13-pg., 2018-09-01.

Abstract

Many viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs of enteric diseases worldwide. Therefore, the present study aims to develop a sensitive real-time fast-qPCR assay based on SYBR (R) Green for detection and quantification of ChPV. A 561-bp non-structural (NS) gene was amplified and cloned, and a pair of primers was designed based on conserved nucleotide sequences on the NS gene of ChPV, the intercalating DNA reagent SYBR (R) Green was employed, and the Fast mode of a thermocycler was used. The assay detects 10(9) to 10(1) copies of the genome (CG). The limit of detection (LoD) was estimated to five CG, and the limit of quantification (LoQ) was estimated at ten CG. The standard curve efficiency was 101.94%, and the melting curve showed a unique clean peak and a melting temperature of 79.3 degrees C. The assay was specific to amplify the ChPV NS gene, and no amplification was shown from other viral genomes or in the negative controls. A total of 141 samples were tested using the assay, of which 139 samples were found positive. The highest CG value of ChPV was 5.7 x 10(6) CG/uL of DNA without apparent clinical signs of enteric disturbance, and 4.6 x 10(6) CG/uL DNA were detected in chickens with RSS. (AU)

• FAPESP's process: 15/09348-5 - Molecular detection, quantitation, tissue distribution and determination of pathological changes of enteric viruses related to enteric disease in chickens.
• Grantee: Luis Fabian Nuñez Naranjo
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 13/08560-5 - Pathogenicity study of chicken enteric viruses (Astrovirus, avian nephritis virus and Parvovirus) in experimentally infected chickens
• Grantee: Antonio José Piantino Ferreira
• Support Opportunities: Regular Research Grants