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Published on: November 1, 2018
Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline
Siti Tasnim Makhtar1, Sheau Wei Tan2, Nur Amalina Nasruddin1
1Faculty of Veterinary Medicine, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia.
Background:
Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.
Results:
A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34-0.53% and 1.38-2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.
Conclusions:
The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.
Insights
A new TaqMan real-time RT-PCR assay effectively detects Feline morbillivirus (FeMV) in cats. This sensitive method improves early FeMV infection diagnosis compared to conventional RT-PCR.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Feline Infectious Diseases
Background:
- Morbilliviruses, including Feline morbillivirus (FeMV), are associated with significant animal diseases and economic losses.
- FeMV is suspected to cause chronic kidney disease in cats, but its role remains unclear.
- FeMV has a global distribution, detected across multiple continents.
Purpose of the Study:
- To develop a sensitive TaqMan real-time RT-PCR (qRT-PCR) assay for detecting FeMV.
- To target the N gene of FeMV for early infection detection in clinical samples.
Main Methods:
- Development of a specific TaqMan real-time RT-PCR assay targeting the FeMV N gene.
- Validation of the assay's specificity against related viruses (CDV, NDV, MeV) and other feline pathogens (FCoV, FeLV).
- Determination of the assay's limit of detection and reproducibility (intra- and inter-assay variation).
Main Results:
- The developed qRT-PCR assay demonstrated high specificity, with no cross-reactivity to other morbilliviruses or feline viruses.
- The assay achieved a lower detection limit of 1.74 × 10^4 copies/μL.
- Clinical sample testing revealed a significantly higher detection rate (35.2%) compared to conventional RT-PCR (15.5%), indicating enhanced sensitivity.
Conclusions:
- The TaqMan-based real-time RT-PCR assay is a sensitive, specific, rapid, and reproducible tool for FeMV detection.
- This assay facilitates the early diagnosis of FeMV infection in cats.
- The improved diagnostic capability aids in understanding and managing FeMV-related health issues in feline populations.
