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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.19.0525    [Accepted] Published online December 24, 2019.
An improvement of real-time PCR system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam
Ha Thi Thanh Tran1  , Anh Kieu Dang1, Duc Viet Ly1, Hao Thi Vu1, Tuan Van Hoang1, Chinh Thi Nguyen1, Nhu Thi Chu1, Vinh The Nguye1, Huyen Thi Nguyen1, Anh Duc Truong1  , Ngoc Thi Pham1  , Hoang Vu Dang1,* 
Department of Biochemistry and Immunology, National Institute of Veterinary Research (NIVR), 86 Truong Chinh, Dong Da, Hanoi 100000, Vietnam
Correspondence:  Hoang Vu Dang, Tel: +84-02438695140, Fax: +84-24-3869-4082, Email: dangnivr@yahoo.com
Received: 21 June 2019   • Revised: 23 September 2019   • Accepted: 12 November 2019
Abstract
Objective
The rapid and reliable detection of the African Swine Fever Virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including real-time PCR. However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on OIE protocol for accurate detection of ASFV in field samples in Vietnam.
Methods
Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE
Results
Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of Cq values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78).
Conclusion
We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.
Keywords: African Swine Fever; Real-time PCR; Conventional PCR; PAMs Cell, Virus Isolation; Molecular Diagnosis
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