Comparison and verification of quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) and real time RT-PCR for avian leukosis virus subgroup J

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Abstract

Avian leukosis virus subgroup J (ALV-J) infections cause significant economic losses because of increased mortality, tumor production, decreased production, and cost for eradication. Current quantification methods for ALV-J expressed by TCID50 are difficult to determine because of the lack of cytopathic effect in cell cultures and non-specificity of currently available antigen-capture ELISA tests. In this study, a one-tube fluorescent probe based real time RT-PCR method was developed for quantification of ALV-J and compared with available quantification methods. Cell lysates with different TCID50s determined by cell culture and antigen capture ELISA (ag-ELISA) were used for one-tube real time RT-PCR using fluorogenic probe and quantitative competitive RT-PCR (QC-RT-PCR). The results of QC-RT-PCR and real time RT-PCR were highly correlated to the TCID50s determined by conventional culture methods. They were also very specific, sensitive, easy to perform, reproducible, and rapid compared with conventional methods. These RT-PCR based quantification methods of ALV-J viral RNA will be useful for virological and pathogenesis studies.

Introduction

Avian leukosis viruses (ALVs) of chickens are classified into six subgroups, A through E, and newly identified J (Payne and Fadly, 1997). Subgroup classification is based on differences in viral envelope glycoprotein antigens, which determine virus-serum neutralization properties, viral interference patterns, and host range of in vivo and in vitro infectivity (Coffin, 1996). The prototype strain of avian leukosis virus subgroup J (ALV-J), HPRS-103, was isolated from commercial meat-type chickens associated with myelocytomatosis in England (Payne et al., 1991). The strain was sequenced completely and designated a novel subgroup. It was hypothesized to be a recombinant between exogenous ALVs and the EAV family of endogenous avian retroviruses (Bai et al., 1995). The virus has a wide host range and is capable of infecting jungle fowl, turkeys, and all of 11 genetic lines of chickens studied (Payne et al., 1992). Susceptibility to tumors varies markedly between different genetic lines of chickens, but meat-type chickens are particularly prone to tumors.

ALV-J infection occurs worldwide and is ubiquitous in broiler breeders and commercial broilers. Infection causes significant economic losses in the broiler industry caused by increased mortality, tumor production, decreased weight gain, and cost for eradication (Stedman and Brown, 1999). In the field, presence of infection is associated with variable tumor types including myelocytoma, renal tumors and others (Venugopal, 1999).

Because no observable cytopathic effect is produced in vitro, TCID50 of ALV-J is determined by serially diluting a sample, inoculating it into cells resistant to growth of endogenous viruses, incubating it for 7–9 days, and detecting group specific antigen p27 using an antigen capture ELISA (Fadly and Witter, 1998). This technique is laborious, expensive, requires cells resistant to endogenous viruses, and takes 8–10 days to complete.

Polymerase chain reaction (PCR)-based techniques have been used to detect the presence of cellular and viral genes and also have been used to quantify these genes (Foley et al., 1993, Jung et al., 2000, Killeen, 1997). Recently, real time monitoring systems of PCR amplification have been developed and are used widely for detection and quantification of genes (Orlando et al., 1998). For diagnosis of ALV-J infection, several PCR systems have been developed and are widely used for both laboratory and field samples (Smith et al., 1998). This manuscript describes development of a new one-tube real time reverse transcription (RT)-PCR system using fluorogenic probe for ALV-J RNA quantification. This study was also performed to verify and compare this new real time RT-PCR with a previously developed quantitative competitive (QC)-RT-PCR (Kim et al., 2000) and the conventional quantitative method using cell culture systems and ag-ELISA.

Section snippets

Production of viral samples

ALV-J (strain ADOL-7501, Avian Disease Diagnostic Lab, East Lansing, MI) was propagated in primary or secondary C/E chicken embryo fibroblasts (C/E CEF, Kestrel Inc., Waukee, IA, USA) grown at 37 °C. After propagation, samples were collected and TCID50s determined as follows. The stock virus was diluted serially ten-fold to 10−8 with cell culture media and each dilution of the virus was cultivated at 37 °C and 5% CO2 for 8 days in monolayer of C/E CEF. After freezing and thawing (3×), 100 μl of

Production of control RNA

By insertion into the plasmid, transformation, and the in vitro transcription reaction, a 636-bp RNA was produced successfully, and the quantitiy was measured by spectrophotometer (data not shown). In this experiment, the slopes of the standard curve of control RNA and viral RNA were −3.632 (R2=0.985) and −3.55 (R2=0.998), respectively, showing the control RNA transcripts were reliable for ALV-J quantification.

Real time RT-PCR

Amplification plots and standard curves were generated after completion of the PCR

Discussion

Both QC-RT-PCR and real time RT-PCR used in this study highly correlated with TCID50 determined by conventional cell culture and ag-ELISA methodologies (Fig. 3). Both methods detected the amount of RNA in samples with different TCID50s of 106.5, 105.5, and 103.5. These results were reproducible in separate trials using both methods.

Real time RT-PCR is a newly introduced technique but has been widely used for detection and quantification of genes. It has a higher sensitivity than conventional

Acknowledgements

Support for this work was provided by the United States Poultry and Egg Association. We thank Drs C. Baile and S. Gullicksen, Department of Animal Science in University of Georgia, for technical support with the ABI Prism 7700.

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