Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Mar 16;11(3):841.
doi: 10.3390/ani11030841.

Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV)

Carmen López-Vázquez  1 , Isabel Bandín  1 , Carlos P Dopazo  1
Affiliations

Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV)

Carmen López-Vázquez et al. Animals (Basel). .

Abstract

The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5-50 TCID50/mL), analytical specificity (intra and intergroup), efficiency (E = 100.0-101.1) and reliability (repeatability and reproducibility with CV < 5%, and standard curves with R2 < 0.95) with strains from any VHSV genotype have been widely demonstrated. The procedure is based on the 'binary multiplex RT-qPCR system (bmRT-qPCR)' previously reported by the same team, applied to arrays of 96-well PCR strip tubes plates, which can be stored at -25 °C for three months and up to one year before their use, without significant loss of efficiency.

Keywords: diagnosis; viral haemorrhagic septicaemia virus.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Standard curves with RT-qPCR. For each genogroup, the standard curves obtained with the macroarray applying RT-qPCR immediately after its preparation (0 h) and after the three storage times assessed (one day, one week, and three months) are shown. The equation of each curve (1), its reliability in terms of coefficient of determination (R2) (2), and the efficiency of the amplification (E) (3), calculated from the formula E = 10−1/S−1 (where S is the slope of the regression line), are indicated. For each genogroup, the average line (averaged from the 4 storage times) is also shown (4).
Figure 1
Figure 1
Standard curves with RT-qPCR. For each genogroup, the standard curves obtained with the macroarray applying RT-qPCR immediately after its preparation (0 h) and after the three storage times assessed (one day, one week, and three months) are shown. The equation of each curve (1), its reliability in terms of coefficient of determination (R2) (2), and the efficiency of the amplification (E) (3), calculated from the formula E = 10−1/S−1 (where S is the slope of the regression line), are indicated. For each genogroup, the average line (averaged from the 4 storage times) is also shown (4).
Figure 2
Figure 2
Standard curves with qPCR. For each genogroup, the standard curves obtained with the macroarray applying qPCR immediately after its preparation (0 h) and after the three storage times assessed (one day, one week, and three months) are shown. The equation of each curve (1), its reliability in terms of coefficient of determination (R2) (2), and the efficiency of the amplification (E) (3), calculated from the formula E = 10−1/S−1 (where S is the slope of the regression line) are indicated. For each genogroup, the average line (averaged from the 4 storage times) is also shown (4).
Figure 2
Figure 2
Standard curves with qPCR. For each genogroup, the standard curves obtained with the macroarray applying qPCR immediately after its preparation (0 h) and after the three storage times assessed (one day, one week, and three months) are shown. The equation of each curve (1), its reliability in terms of coefficient of determination (R2) (2), and the efficiency of the amplification (E) (3), calculated from the formula E = 10−1/S−1 (where S is the slope of the regression line) are indicated. For each genogroup, the average line (averaged from the 4 storage times) is also shown (4).
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Mortensen H.F., Heuer O.E., Lorenzen N., Otte L., Olesen N.J. Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea. Virus Res. 1999;63:95–106. doi: 10.1016/S0168-1702(99)00062-3. - DOI - PubMed
    1. Smail D.A. Viral haemorrhagic septicaemia. In: Woo P.T.K., Bruno D.W., editors. Fish Diseases and Disorders, Volume 3: Viral, Bacterial and Fungal Infections. CABI Publishing; New York, NY, USA: 1999. pp. 123–147.
    1. Ellis A.E. Innate host defense mechanism of fish against viruses and bacteria. Dev. Comp. Immunol. 2001;25:827–839. doi: 10.1016/S0145-305X(01)00038-6. - DOI - PubMed
    1. Einer-Jensen K., Ahrens P., Forsberg R., Lorenzen N. Evolution of the fish rhabdovirus viral haemorrhagic septicaemia virus. J. Gen. Virol. 2004;85:1167–1179. doi: 10.1099/vir.0.79820-0. - DOI - PubMed
    1. Snow M., Bain N., Black J., Taupin V., Cunningham C.O., King J.A., Skall H.F., Raynard R.S. Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV) Dis. Aquat. Organ. 2004;61:11–12. doi: 10.3354/dao061011. - DOI - PubMed

LinkOut - more resources