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Comparative Study
. 2005 Nov;43(11):5574-80.
doi: 10.1128/JCM.43.11.5574-5580.2005.

TaqMan real-time reverse transcription-PCR and JDVp26 antigen capture enzyme-linked immunosorbent assay to quantify Jembrana disease virus load during the acute phase of in vivo infection

Affiliations
Comparative Study

TaqMan real-time reverse transcription-PCR and JDVp26 antigen capture enzyme-linked immunosorbent assay to quantify Jembrana disease virus load during the acute phase of in vivo infection

Meredith Stewart et al. J Clin Microbiol. 2005 Nov.

Erratum in

  • J Clin Microbiol. 2007 Jun;45(6):2100

Abstract

Jembrana disease virus (JDV) is an acutely pathogenic lentivirus that affects Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute phase of the disease process. We report for the first time two techniques that enable quantification of the virus and the use of these techniques to quantify the virus load during the acute phase of the disease process. A one-step JDV gag [corrected] TaqMan real-time reverse transcription-PCR (RT-PCR) assay was developed for the detection and quantification of JDV RNA in plasma. The limit of detection was 9.8 x 10(2) JDV viral RNA copies over 35 cycles, equivalent to 4.2 x 10(4) JDV genome copies/ml, and a peak virus load of 1.6 x 10(12) during the acute febrile period. An antigen capture enzyme-linked immunosorbent assay (ELISA) was also developed to quantify the levels of JDV capsid (JDVp26) over a linear range of 10 to 200 ng/ml. Viral RNA and JDVp26 levels were correlated in 48 plasma samples obtained from experimentally infected cattle. A significant positive correlation (R = 0.860 and r(2) = 0.740) was observed between the two techniques within the range of their detection limits. The relatively insensitive capture ELISA provides an economical and feasible method for monitoring of virus in the absence of more sensitive techniques.

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Figures

FIG. 1.
FIG. 1.
Standard curve generated to indirectly quantify the amount of virus/reaction mixture based on serial dilutions of JDV plasmid 139. The equation generated to predict the amount of virus/reaction was log10 JDV viral RNA copies = 12.896 − 0.283 × CT (Spearman's correlation = 0.986; r2 = 0.973; P < 0.0001). The different amount of JDV genome copies/reaction mixture used and the average CT for each amount are indicated.
FIG. 2.
FIG. 2.
TaqMan real-time RT-PCR using serial dilutions of viral RNA as a template. (A) Gel electrophoresis of TaqMan RT-PCR results. Lane 1, 1-kb-plus ladder (Invitrogen, Australia); lane 2, undiluted RNA sample; lane 3, 10−1 dilution; lane 4, 10−2 dilution; lane 5, 10−3 dilution; lane 6, 10−4 dilution; lane 7, 10−5 dilution; lane 8, 10−6 dilution; lane 9, ultrapure water. (B) Amplification plot generated during a standard TaqMan RT-PCR using the same RNA samples (linear representation). The RNA dilutions and baseline threshold are indicated. ΔRn, recorded fluorescence emission adjusted for the baseline fluorescence.
FIG. 3.
FIG. 3.
Febrile response and viral dynamics of circulating JDV RNA in the plasma during the acute phase of infection for animal CB61, animal CB62, and animal CB63. The animals were challenged with approximately 1,000 ID50 of Tabanan/87 strain of JDV on day 0. The daily rectal temperatures (°C) of each animal (bars) and the level of JDV genome copies/ml (dots) are shown. The results for samples with amounts below the limit of detection are not depicted.
FIG. 4.
FIG. 4.
Reproducibility of standard curve of recombinant JDVp26 protein detected by antigen capture ELISA. The concentrations of JDVp26 protein were measured in duplicate for each capture ELISA plate, and the mean and standard deviations were plotted against the OD450.
FIG. 5.
FIG. 5.
Correlation of the levels of JDVp26 antigen (ng/ml) and JDV RNA (number of copies/ml) from 48 plasma samples. The correlation coefficient was 0.860, and the equation used to determine the log10 amount of viral RNA/ml was (log10 JDVp26 (ng/ml) × 0.7) + 9.188 (n = 48; r2 = 0.744; P < 0.0001).
FIG. 6.
FIG. 6.
Comparison during the acute phase of JDV infection of the estimated titer of virus generated from three sets of data: the previously reported animal bioassay (ID50/ml) data (33), the predicted number of RNA genome copies/ml determined by the JDV pol TaqMan real-time RT-PCR, and the capsid levels were determined by the JDVp26 antigen capture ELISA. The data were normalized to the onset of fever (rectal temperature, ≥39.5°C) after JDV infection. Bioassay ID50/ml data (34) are indicated by the black columns, the numbers of RNA genome copies/ml generated by TaqMan real-time RT-PCR are indicated by the white columns, and the capsid levels were determined by JDVp26 capture ELISA are indicated by the striped columns. Bars indicate standard deviations when sample numbers were >1.

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References

    1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. - PMC - PubMed
    1. Bobrow, M. N., T. D. Harris, K. J. Shaughnessy, and G. J. Litt. 1989. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods 125:279-285. - PubMed
    1. Bustin, S. A. 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29:23-39. - PubMed
    1. Chadwick, B. J., R. J. Coelen, G. E. Wilcox, L. M. Sammels, and G. Kertayadnya. 1995. Nucleotide sequence analysis of Jembrana disease virus: a bovine lentivirus associated with an acute disease syndrome. J. Gen. Virol. 76:1637-1650. - PubMed
    1. Che, X. Y., L. W. Qiu, Y. X. Pan, K. Wen, W. Hao, L. Y. Zhang, Y. D. Wang, Z. Y. Liao, X. Hua, V. C. Cheng, and K. Y. Yuen. 2004. Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome. J. Clin. Microbiol. 42:2629-2635. - PMC - PubMed

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