title: Comparison of four molecular assays for the detection of Tembusu virus.
abstract: Tembusu virus (TMUV) belongs to the genus Flavivirus that may cause severe egg drop in ducks. In order to evaluate the most efficient TMUV detection method, the performances of a conventional RT-PCR (C-RT-PCR), a semi-nested PCR (SN-RT-PCR), a reverse-transcriptase real-time quantitative PCR (Q-RT-PCR), and a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) targeting the TMUV virus-specific NS5 gene were examined. In order to compare the sensitivity of these four techniques, two templates were used: (1) plasmid DNA that contained a partial region of the NS5 gene and (2) genomic RNA from TMUV-positive cell culture supernatants. The sensitivities using plasmid DNA detection by C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 2 × 10(4) copies/μL, 20 copies/μL, 2 copies/μL, and 20 copies/μL, respectively. The sensitivities using genomic RNA for the C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 100 pg/tube, 100, 10, and 100 fg/tube, respectively. All evaluated assays were specific for TMUV detection. The TMUV-specific RNA was detected in cloacal swabs from experimentally infected ducks using these four methods with different rates (52-92%), but not in the control (non-inoculated) samples. The sensitivities of RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs collected from suspected TMUV infected ducks within 2 weeks of severe egg-drop were 38/69 (55.1%), 52/69 (75.4%), 57/69 (82.6%), and 55/69 (79.7%), respectively. In conclusion, both RT-LAMP and Q-RT-PCR can provide a rapid diagnosis of TMUV infection, but RT-LAMP is more useful in TMUV field situations or poorly equipped laboratories.
authors.affiliation: a College of Veterinary Medicine , Shandong Agricultural University , Tai'an , Shandong 271018 , People's Republic of China.|b Department of Veterinary and Biomedical Sciences , The Pennsylvania State University , University Park , PA 16802 , USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.affiliation: c Department of Biomedical Engineering , The Pennsylvania State University , University Park , PA 16802 , USA.
authors.forename: Yin-Ting
authors.initials: YT
authors.lastname: Yeh
authors.affiliation: a College of Veterinary Medicine , Shandong Agricultural University , Tai'an , Shandong 271018 , People's Republic of China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: a College of Veterinary Medicine , Shandong Agricultural University , Tai'an , Shandong 271018 , People's Republic of China.
authors.forename: Chunmei
authors.initials: C
authors.lastname: Yu
authors.affiliation: a College of Veterinary Medicine , Shandong Agricultural University , Tai'an , Shandong 271018 , People's Republic of China.
authors.forename: Xuhui
authors.initials: X
authors.lastname: Gao
authors.affiliation: a College of Veterinary Medicine , Shandong Agricultural University , Tai'an , Shandong 271018 , People's Republic of China.
authors.forename: Youxiang
authors.initials: Y
authors.lastname: Diao
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004372:Ducks / Q000821:virology*; D005416:Flavivirus / Q000235:genetics / Q000302:isolation & purification*; D018177:Flavivirus Infections / Q000662:veterinary* / Q000821:virology; D010063:Ovum / Q000821:virology; D011201:Poultry Diseases / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; C096041:NS5 protein, flavivirus; D017361:Viral Nonstructural Proteins