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

Forsman, Anna; Užameckis, Dmitrijs; Rönnblom, Lars; Baecklund, Eva; Åleskog, Anna; Bindra, Amarinder; Pipkorn, Rüdiger; Lejniece, Sandra; Kozireva, Svetlana; Murovska, Modra; Blomberg, Jonas

doi:10.1016/s0166-0934(03)00127-7

Cited by 20 publications

(17 citation statements)

References 18 publications

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“…The strategy of using a low concentration of the specific primers contributes to the specificity, since the production of primer dimers from the long and numerous specific primers is suppressed (see Table S1). The temperature switch allows all primers to be added as one mix in the tube, which minimizes the risk of contamination (see Table S1) (2). As presented here, the concentration of each specific primer was 5 nM in the 22-plex sepsis VOCMA experiment and 50 nM in the 7-plex gastro VOCMA experiment.…”

Section: Discussionmentioning

confidence: 99%

Variation-Tolerant Capture and Multiplex Detection of Nucleic Acids: Application to Detection of Microbes

et al. 2012

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In contrast to ordinary PCRs, which have a limited multiplex capacity and often return false-negative results due to target variation or inhibition, our new detection strategy, VOCMA (variation-tolerant capture multiplex assay), allows variation-tolerant, target-specific capture and detection of many nucleic acids in one test. Here we demonstrate the use of a single-tube, dual-step amplification strategy that overcomes the usual limitations of PCR multiplexing, allowing at least a 22-plex format with retained sensitivity. Variation tolerance was achieved using long primers and probes designed to withstand variation at known sites and a judicious mix of degeneration and universal bases. We tested VOCMA in situations where enrichment from a large sample volume with high sensitivity and multiplexity is important (sepsis; streptococci, enterococci, and staphylococci, several enterobacteria, candida, and the most important antibiotic resistance genes) and where variation tolerance and high multiplexity is important (gastroenteritis; astrovirus, adenovirus, rotavirus, norovirus genogroups I and II, and sapovirus, as well as enteroviruses, which are not associated with gastroenteritis). Detection sensitivities of 10 to 1,000 copies per reaction were achieved for many targets. VOCMA is a highly multiplex, variation-tolerant, general purpose nucleic acid detection concept. It is a specific and sensitive method for simultaneous detection of nucleic acids from viruses, bacteria, fungi, and protozoa, as well as host nucleic acid, in the same test. It can be run on an ordinary PCR and a Luminex machine and is suitable for both clinical diagnoses and microbial surveillance.

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Section: Discussionmentioning

confidence: 99%

Variation-Tolerant Capture and Multiplex Detection of Nucleic Acids: Application to Detection of Microbes

et al. 2012

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“…Single-tube nested quantitative PCR (stnQPCR) for RERV-H StnQPCR was performed as previously described (Forsman et al 2003). Briefly, an outer pair of primers, 330f (5¢-GGACCTAGCC CAGCCCAAAG-3¢) and 609r (5¢-CAGCCCGTAAATCATGCA ACAA-3¢), and an inner pair of primers, 466f (5¢-AAAAGTTGGC TGCGGTA-3¢) and 540r (5¢-ATGGGGAGGTTGATGGT-3¢) were used, together with the TaqMan probe 5¢-TTACAACAATT AGAGGCAGGCCATTT-3¢.…”

Section: Collection Of Blood Samplesmentioning

confidence: 99%

“…Immunological detection of RERV-H peptides was performed as previously described (Forsman et al 2003). The synthetic, biotinylated peptides P1982 (HRV5-RT1, (bio)PHSHAPRVLYTQGI AVADLILTAIKK) and P1983 (HRV5-RT2, (bio)RKQAEGIIR SCEKFVVAIVPKIPA) derived from the polymerase gene sequence of RERV-H, and P1985 (HRV5-CA-CTERM1, (bio)ANKTC REALRPWQHKDIPTFLKI) and P1986 (HRV5-CA-CTERM2, (bio)LRPWQHKDIPTFLKICRDVMDDV) from the C-terminal region of the capsid protein, were synthesised using the Fmocstrategy on an ABI 433 automated synthesiser (Applied Biosystems) and purified by preparative HPLC.…”

Section: Antibody Binding To Synthetic Rerv-h Peptidesmentioning

confidence: 99%

Uptake of amplifiable fragments of retrotransposon DNA from the human alimentary tract

et al. 2003

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Few attempts have been made to study the transfer of DNA from ingested food across the intestinal barrier. A low uptake of ingested DNA has been observed in mice, cattle and poultry. There have been no reports on humans so far. Maintenance of species barriers, protection against retrotransposons, optimisation of oral DNA vaccines and the fate of genetically modified foodstuffs are issues where this topic is of importance. We therefore used the high-copy-number rabbit retrotransposon RERV-H, and rabbit mitochondrial DNA, to study the transfer of DNA from ingested rabbit meat into the bloodstream of two human volunteers. A quantitative PCR was used to measure RERV-H levels in food and in the blood. Amplification with the primers selected results in the generation of a 250-bp fragment of RERV-H. Transfer across the intestinal epithelium could be demonstrated in both subjects. Levels of the fragment in the bloodstream peaked at 1-3 h after ingestion of the experimental meal. One hour after a meal of rabbit meat containing 10(14) copies of RERV-H DNA, a maximum concentration of 200 copies of RERV-H DNA per ml of peripheral blood was observed, which corresponds to the uptake of approximately 10(6) RERV-H DNA copies in 1 h. RERV-H DNA was detected in both cellular and plasma compartments. Both rabbit retrotransposon and mitochondrial DNA was taken up from the human alimentary tract. The size of the fragments detected is similar to that of SINE retrotransposons (approximately 300 bp). The fate and functionality of alimentary DNA in humans will require further study.

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“…Three integration sites were identified (from three different individuals), but none of the flanking sequences was present in human genome sequence databases, and they could not be detected experimentally in human DNA by Southern blot or PCR. A survey of other mammalian species demonstrated that HRV-5 is in fact an ERV of the European rabbit (Oryctolagus cuniculus) and that the rabbit genome harbors around 1,000 to 5,000 copies of this element, now designated rabbit ERV-H (148,190).…”

Section: Human Retrovirusmentioning

confidence: 99%

“…The possibility that HRV-5 is a replication-competent rabbit virus that has crossed species and infected humans is very remote, since flanking rabbit genomic sequences and mitochondrial DNA were also detected in some human samples (148,190), so this appears to be a simple case of PCR contamination. However, contamination cannot easily explain the initial discovery of HRV-5 as an RNA element or its disproportionate detection in specific diseases.…”

Section: Human Retrovirusmentioning

confidence: 99%

Human RNA “Rumor” Viruses: the Search for Novel Human Retroviruses in Chronic Disease

Voisset

Weiss

Griffiths

2008

Microbiol Mol Biol Rev

143

156

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SUMMARY Retroviruses are an important group of pathogens that cause a variety of diseases in humans and animals. Four human retroviruses are currently known, including human immunodeficiency virus type 1, which causes AIDS, and human T-lymphotropic virus type 1, which causes cancer and inflammatory disease. For many years, there have been sporadic reports of additional human retroviral infections, particularly in cancer and other chronic diseases. Unfortunately, many of these putative viruses remain unproven and controversial, and some retrovirologists have dismissed them as merely “human rumor viruses.” Work in this field was last reviewed in depth in 1984, and since then, the molecular techniques available for identifying and characterizing retroviruses have improved enormously in sensitivity. The advent of PCR in particular has dramatically enhanced our ability to detect novel viral sequences in human tissues. However, DNA amplification techniques have also increased the potential for false-positive detection due to contamination. In addition, the presence of many families of human endogenous retroviruses (HERVs) within our DNA can obstruct attempts to identify and validate novel human retroviruses. Here, we aim to bring together the data on “novel” retroviral infections in humans by critically examining the evidence for those putative viruses that have been linked with disease and the likelihood that they represent genuine human infections. We provide a background to the field and a discussion of potential confounding factors along with some technical guidelines. In addition, some of the difficulties associated with obtaining formal proof of causation for common or ubiquitous agents such as HERVs are discussed.

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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

Cited by 20 publications

References 18 publications

Variation-Tolerant Capture and Multiplex Detection of Nucleic Acids: Application to Detection of Microbes

Variation-Tolerant Capture and Multiplex Detection of Nucleic Acids: Application to Detection of Microbes

Uptake of amplifiable fragments of retrotransposon DNA from the human alimentary tract

Human RNA “Rumor” Viruses: the Search for Novel Human Retroviruses in Chronic Disease

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