Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Abstract

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT-qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT-qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID mL , 50 pfu mL or 66 RNA copies mL , depending on the standard. All the standard curves showed high reliability (R > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.

Rapid detection and differentiation of carp oedema virus and cyprinid herpes virus‐3 in koi and common carp

“…All these reactions were performed according to the determined optimal conditions for each assay. Diagnostic sensitivity (probability of a positive test result in a CEV/KHV‐infected fish, using a gold standard test as reference) and diagnostic specificity (probability of a negative result in a non‐infected fish) and the positive predictive values (probability of a fish testing positive of actually being CEV/KHV‐infected) and negative predictive values (probability of an individual testing negative of not being infected) were calculated as previously described (Olveira, Soares, Engrola, Dopazo, & Bandın, ; Vazquez, Cutrin, Olveira, & Dopazo, ).…”

Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish

“…Sequence variations of Spring viremia of carp isolated from different geographic locations have been reported (Maj‐Paluch et al, ). D. Vazquez (Vazquez et al., ) developed an RT‐qPCR method that is specific for the detection of seven strains and 23 field viral isolates of infectious pancreatic necrosis virus. Currently, only the complete genome sequences of TiLV isolated from Israel and Thailand are available on the public database (Bacharach et al., ; Surachetpong et al., ).…”

“…Vazquez (Vazquez et al, 2017) developed an RT-qPCR method that is specific for the detection of seven strains and 23 field viral iso- ). In the present study, analysis of TiLV in six tissues: gills, liver, brain, heart, anterior kidney and spleen, using the developed RT-qPCR revealed that the virus distributed in different fish tissues.…”

“…A sensitive SYBR green‐based RT‐qPCR assay was developed to screen different strains of viral haemorrhagic septicaemia virus infection in Atlantic salmon ( Salmo salar ) and rainbow trout ( Oncorhynchus mykiss ) (Matejusova et al, ). Moreover, the specific primers and RT‐qPCR procedure have been established for the detection of infectious pancreatic necrosis virus in salmon (Vazquez et al, ). Comparison of the RT‐qPCR and conventional RT‐PCR methods revealed that the qPCR technique provides 100 times greater sensitivity for the detection of infectious salmon anaemia virus (Munir & Kibenge, ).…”

Quantitative Flow Cytometry to Measure Viral Production Using Infectious Pancreatic Necrosis Virus as a Model: A Preliminary Study

“…, Belgium), heated to 99 • C for 5 min and immediately cooled down to 4 • C. Then, 4 µL (5×) of First Strand Buffer, 1 µL dithiothreitol (DTT) (0.1 M), 1 µL of dNTPs (10 mM), 3.8 µL H 2 O and 0.2 µL Super Script III RT polymerase were added per reaction. The thermal profile continued with 10 min at 25 • C, 50 min at 50 • C and 5 min at 85 • C. Finally, the cDNA was maintained at 4 • C until use or stored at −20 • C.Real time PCR amplifications were performed in a Bio-Rad CFX96 (Bio-Rad Laboratories, Inc., Madrid, Spain) using 10 µL of 2x SYBR ® Green Supermix (iQ SYBR ® Green Supermix, Thermofisher, Bilbao, Spain), 500 nM of each primer (PP_WB 2370F, 5 -CAAGTTTGGCAGGCTCATCAG-3; PP_WB 2614R, 5 -CGTAGTCCTCGTACTCTTCTCC-3) and 2 µL of cDNA in a 20 µL final reaction, with the following thermal profile: 95 • C for 3 min, followed by 42 cycles of 95 • C for 15 s and 60 • C for 30 s, and a melting curve analysis of 55 • C to 95 • C with an increment of 0.2 • C for 10 s. Absolute quantification was performed using in vitro transcribed RNA standard from a cloned plasmid with a 364 bp fragment from RNA region 2317-2681 of reference strain WB[31].…”