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

doi:10.1016/S0166-0934(03)00127-7

Journal of Virological Methods

Volume 111, Issue 1, July 2003, Pages 1-11

https://doi.org/10.1016/S0166-0934(03)00127-7 Get rights and content

Abstract

It was reported earlier that a few patients suffering from non-Hodgkin's lymphoma had low amounts of DNA from the so-called fifth human exogenous retrovirus, HRV-5. A sensitive and rational method for large-scale screening for HRV-5 DNA was therefore developed. It is a single-tube nested quantitative PCR (stnQPCR), which uses two functionally isolated primer pairs and one probe target distinct from related endogenous retroviral sequences, yet encompassing known HRV-5 variation, allowing optimal use of sequence conservation. DNA from lymphoma, myeloma, and follicular dendritic cell lines was tested for HRV-5 positivity, as was DNA from whole blood of blood donors, non-Hodgkin's lymphoma and systemic lupus erythematosus patients, as well as DNA from lymph node biopsies of rheumatoid arthritis patients with lymphoma. One blood donor, one systemic lupus erythematosus patient, two previously known positive non-Hodgkin's lymphoma patients, and one rheumatoid arthritis lymphoma patient, came out positive. They had 24, 2, 148, 480 and 30 proviral copies per μg of DNA from PBMC or lymphoma tissue, respectively. During the completion of this work it was reported that HRV-5 is a rabbit endogenous retrovirus (RERV-H), and that HRV-5 positivity was due to presence of rabbit DNA. DNA from six RERV-H/HRV-5 positive samples was therefore retested. Three also contained rabbit mitochondrial DNA. A search for HRV-5 antibodies using synthetic peptides was negative in sera from three RERV-H/HRV-5 positive individuals, as well as in 144 other sera, according with a noninfectious origin of the RERV-H/HRV-5 DNA in human samples. A search for possible sources of rabbit DNA contamination was negative. Methods for prevention of PCR contamination were strictly adhered to. Three samples from RERV-H/HRV-5 positive individuals positive at the Uppsala laboratory were retested at one or two other laboratories, and all three were positive. Two other samples, which were positive in the Riga laboratory, were tested also in London and also found positive. One non-Hodgkin's lymphoma patient was RERV-H/HRV-5 positive in four consecutive samples, showing that positivity was a property of that patient. It is concluded that the stnQPCR developed to detect and quantify minute amounts of RERV-H/HRV-5 DNA is a principle which can be applied widely and HRV-5 is a RERV-H. Its presence in a few human blood samples could not be explained.

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 T_m 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)

A. Brand et al.
Human retrovirus-5 in rheumatic disease
J. Autoimmun.
(1999)
D.J. Kemp et al.
Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients
Gene
(1990)
Y.S. Lie et al.
Advances in quantitative PCR technology: 5′ nuclease assays
Curr. Opin. Biotechnol.
(1998)
S.P. Rigby et al.
A new human retrovirus: a role in lymphoma?
Am. J. Med.
(1998)
K.M. Ririe et al.
Product differentiation by analysis of DNA melting curves during the polymerase chain reaction
Anal. Biochem.
(1997)
E. Beutler et al.
Interference of heparin with the polymerase chain reaction
Biotechniques
(1990)
J. Blomberg 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)
J. Blomberg 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)
D.J. Griffiths et al.
A novel exogenous retrovirus sequence identified in humans
J. Virol.
(1997)
D.J. Griffiths et al.
Novel endogenous retrovirus in rabbits previously reported as human retrovirus 5
J. Virol.
(2002)

C.A. Heid et al.

Real time quantitative PCR

Genome Res.

(1996)

R. Higuchi et al.

Simultaneous amplification and detection of specific DNA sequences

Biotechnology (NY)

(1992)

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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

Abstract

Introduction

Section snippets

Patient material and cell lines

Evaluation of the stnQPCR

Discussion

Conclusions

Acknowledgements

J. Autoimmun.

Gene

Curr. Opin. Biotechnol.

Am. J. Med.

Anal. Biochem.

Interference of heparin with the polymerase chain reaction

Biotechniques

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

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.

A novel exogenous retrovirus sequence identified in humans

J. Virol.

Novel endogenous retrovirus in rabbits previously reported as human retrovirus 5

J. Virol.

Real time quantitative PCR

Genome Res.

Simultaneous amplification and detection of specific DNA sequences

Biotechnology (NY)