title: Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus.
abstract: Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China; Institute of Military Veterinary Medicine, Academy of Military Medical Sciences of PLA, Jilin, 130117, People's Republic of China.
authors.forename: Peng
authors.initials: P
authors.lastname: Jia
authors.affiliation: US Geological Survey, Western Fisheries Research Center, 6505 Northeast 65th Street, Seattle, WA 98115, USA.
authors.forename: Maureen K
authors.initials: MK
authors.lastname: Purcell
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Guang
authors.initials: G
authors.lastname: Pan
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Jinjin
authors.initials: J
authors.lastname: Wang
authors.affiliation: Shenzhen Supervision and Testing Center for Quality and Safety of Agri-products, Shenzhen, 518005, People's Republic of China.
authors.forename: Shifu
authors.initials: S
authors.lastname: Kan
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Xiaocong
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Xiujie
authors.initials: X
authors.lastname: Shi
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Junqiang
authors.initials: J
authors.lastname: He
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Yu
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Qunyi
authors.initials: Q
authors.lastname: Hua
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Tikang
authors.initials: T
authors.lastname: Lu
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Lan
authors.affiliation: US Geological Survey, Western Fisheries Research Center, 6505 Northeast 65th Street, Seattle, WA 98115, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Winton
authors.affiliation: Institute of Military Veterinary Medicine, Academy of Military Medical Sciences of PLA, Jilin, 130117, People's Republic of China.
authors.forename: Ningyi
authors.initials: N
authors.lastname: Jin
authors.affiliation: Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China. Electronic address: liuhong@szciq.gov.cn.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
mesh_terms: D000818:Animals; D017931:DNA Primers; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology; D029142:Infectious hematopoietic necrosis virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics; D019562:Viral Load
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D012194:RNA-Directed DNA Polymerase
keywords: Analytical sensitivity; Analytical specificity; IHNV; RT-ddPCR; RT-qPCR