Single-tube nested quantitative PCR: a rational and sensitive technique for detection of retroviral DNA. Application to RERV-H/HRV-5 and confirmation of its rabbit origin
Introduction
Non-endogenous retroviral DNA related to genus Betaretrovirus has been found in a few humans (Griffiths et al., 1997, van Regenmortel et al., 2000, Weiss et al., 1999, Wilkinson et al., 1994). Originally referred to as human retrovirus 5 (HRV-5), it was reported recently to be a rabbit endogenous retrovirus (RERV-H) (Griffiths et al., 2002). HRV-5 was detected originally as retroviral particles in supernatants from a cell culture of human origin, fractionated in sucrose density gradients. The provirus lacks envelope gene and is related to intracisternal type A particles of rodents. Low concentrations of RERV-H/HRV-5 DNA has been detected in a few healthy individuals, and in patients suffering from autoimmunity and non-Hodgkin lymphomas (Brand et al., 1999, Griffiths et al., 1997, Kozireva et al., 2001, Murovska et al., 2000, Rigby et al., 1998, Rigby et al., 1997). A real-time PCR for detection and quantification of RERV-H/HRV-5 proviral DNA was developed. It is described here. The technique combines the specificity of hybridisation of a TaqMan® (Holland et al., 1991) probe with the specificity of two nested PCRs. The technique can be used to tailor a desired degree of specificity by use of conserved and unique sequences. The two nested primer pairs are isolated functionally from each other by Tm and concentration differences. The principle has not been used in the TaqMan® context before. It is valuable for detection of variable nucleic acids, like those of RNA viruses. Retroviruses, Orthomyxoviruses and Caliciviruses, since they are highly variable, constitute a special detection problem and should therefore be amenable to the technique. Despite the rather complicated amplification scheme, threshold cycles were linear versus the log of the amount of input viral nucleic acid over seven magnitudes. The closed-tube system also contains contamination protection due to uracil N-glycosylase (UNG). It was applied in healthy blood donors as well as in patients with autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis) and non-Hodgkin's lymphoma. The results confirm the previous finding of RERV-H/HRV-5 DNA in lymphoma patients (Kozireva et al., 2001), systemic lupus erythematosus and rheumatoid arthritis patients (Brand et al., 1999). RERV-H/HRV-5 DNA was also found in peripheral blood of one of 50 Swedish blood donors. The proviral copy numbers were low both in the patients and in the blood donor. Tests for laboratory contamination (water controls and wipe tests) were negative. Results were repeatable in three laboratories (Uppsala, Riga and London). Moreover, one non-Hodgkin's lymphoma patient was positive in four consecutive samples. The possible source of the rabbit DNA in a few human samples is discussed.
Section snippets
Patient material and cell lines
Two DNA samples, extracted previously from 0.5 ml whole blood (i) of a patient (117h; 971 206) diagnosed to have B-cell non-Hodgkin's lymphoma of low-grade malignancy (Murovska et al., 2000), and (ii) of a patient (385h) diagnosed to have a T-cell non-Hodgkin's lymphoma of high grade malignancy (Kozireva et al., 2001), and already known to be HRV-5 positive, were tested. Another whole blood sample from patient 117h (011101), previously not tested for HRV-5 DNA, was now included.
Nine lymphoma
Evaluation of the stnQPCR
The amplification curves of a run with final conditions as in Table 1, are shown in Fig. 2a. The sensitivity of the assay was around one copy per reaction, with two types of thermocyclers. This was determined in two ways. First, plasmid pHRV5.1 concentrations down to one copy per reaction (Fig. 2b) could be detected. Second, in a series of plasmid dilutions, signal was only obtained in a subset of wells at dilutions corresponding to 10 copies and lower. Such stochastic behaviour is
Discussion
Single-tube nested PCR was first described by Kemp et al. (1990). In spite of its potential advantages, single-tube nested real-time PCR has only been described by a few authors, among them Strauss et al. (2000) although this was in a SYBR Green format. The results emphasize that functional isolation of the amplification of two nested but simultaneously present primer pairs is a sensitive and rational technique. The strategy allows fine tuning of specificity by use of either short conserved or
Conclusions
- i
The stnQPCR developed to detect and quantify minute amounts of RERV-H/HRV-5 DNA is a rational and widely applicable technique. The principle allows optimal use of sequence conservation and uniqueness.
- ii
The data support the previous report that HRV-5 is a rabbit endogenous retrovirus.
- iii
stnQPCR results confirm the previous finding of RERV-H/HRV-5 DNA in lymphoma patients (Kozireva et al., 2001, Murovska et al., 2000, Rigby et al., 1998) and systemic lupus erythematosus patients (Brand et al., 1999).
Acknowledgements
David Griffiths kindly provided the plasmid pHRV5.1. Ola Söderberg and Kenneth Nilsson kindly provided lymphoid cell lines. The work was supported by grants from the Swedish Institute, contract #East 92 380/133 and 351, and the Swedish Society against Cancer, contract #01 6932.
References (24)
- et al.
Human retrovirus-5 in rheumatic disease
J. Autoimmun.
(1999) - et al.
Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients
Gene
(1990) - et al.
Advances in quantitative PCR technology: 5′ nuclease assays
Curr. Opin. Biotechnol.
(1998) - et al.
A new human retrovirus: a role in lymphoma?
Am. J. Med.
(1998) - et al.
Product differentiation by analysis of DNA melting curves during the polymerase chain reaction
Anal. Biochem.
(1997) - et al.
Interference of heparin with the polymerase chain reaction
Biotechniques
(1990) - et al.
A survey of synthetic HIV-1 peptides with natural and chimeric sequences for differential reactivity with Zimbabwean, Tanzanian and Swedish HIV-1-positive sera
AIDS
(1993) - et al.
Identification of regions of HIV-1 p24 reactive with sera which give ‘indeterminate’ results in electrophoretic immunoblots with the help of long synthetic peptides
AIDS Res. Hum. Retrov.
(1990) - et al.
A novel exogenous retrovirus sequence identified in humans
J. Virol.
(1997) - et al.
Novel endogenous retrovirus in rabbits previously reported as human retrovirus 5
J. Virol.
(2002)
Real time quantitative PCR
Genome Res.
Simultaneous amplification and detection of specific DNA sequences
Biotechnology (NY)
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