Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates

Sauvaigo, Sylvie; Barlet, Valérie; Guettari, N.; Innocenti, P; Parmentier, Françoise; Bastard, Christian; Seigneurin, Jean‐Marie; Chermann, J.-C.; Téoule, R.; Marchand, Joseph

doi:10.1128/jcm.31.5.1066-1074.1993

Cited by 17 publications

(7 citation statements)

References 33 publications

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“…Proteinase K digestion with 500 l of whole blood or with samples containing blood was done after lysis of the erythrocytes in TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA) as described by Sauvaigo et al (31).…”

Section: Methodsmentioning

confidence: 99%

PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs

1995

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A PCR assay for the diagnosis of leishmaniosis was developed by using primers that were selected from the sequence of the small-subunit rRNA gene. The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (lysis of erythrocytes in Tris-EDTA buffer, digestion with proteinase K directly in PCR buffer, and no further purification steps). Furthermore, an internal control is included in every specimen in order to detect the presence of PCR inhibitors. The PCR was compared with diagnostic in vitro cultivation of promastigote stages for the detection of Leishmania spp. in clinical specimens from humans and dogs with a tentative diagnosis of leishmaniosis. PCR and cultivation gave identical results with all but 1 of the 95 specimens from humans. The PCR result in this case was false negative, possibly because of unequal apportionment of this sample. With 10 skin biopsies from six patients with cutaneous leishmaniosis, the sensitivity was 60%. For six human immunodeficiency virus-positive patients with visceral leishmaniosis, all bone marrow biopsies and 7 of 11 whole blood samples (after isolation of leukocytes by Ficoll-Paque) were positive in both tests. PCR detected one more case with the use of 500 l of whole blood with direct lysis of the erythrocytes in Tris-EDTA buffer. With dog lymph node aspirates, the sensitivity was 100% (16 of 16 samples) for both methods; furthermore, PCR was positive for 5 of 13 whole blood samples from dogs with leishmaniosis. The specificity of the PCR was 100% (70 specimens from patients without leishmaniosis). This PCR assay proved to be feasible as a routine diagnostic test, being reliable and faster than in vitro cultivation.

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Section: Methodsmentioning

confidence: 99%

PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs

1995

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“…The number of input target molecules for LD-PCR in DNA extracted from PBMCs of infected individuals was estimated by semiquantitative PCR of the HIV-1 gag gene ( Table 1). Decadic dilutions of DNA were assayed in a classical PCR reaction with Taq polymerase and gag A1 primer, 5Ј-GATTTAAACACC ATGCTAAACACAGTGG-3Ј (nt 882 to 909, sense), and gag A2 primer, 5Ј-TT TGGTCCCTGTCTTATGTCCAAAATGC-3Ј (nt 1177 to 1204, antisense), for the presence of a 322-nt amplification product (29). Decadic dilutions of the plasmid pNL4-3 standard DNA were amplified in the parallel.…”

Section: Subjectsmentioning

confidence: 99%

“…Analysis of PCR products. Southern blots were hybridized with the 32 P-labeled oligonucleotide probes 5ЈNCS, 5Ј-TTGCTGAAGCGCGCACGGCAA-3Ј (nt 249 to 269, sense, NDK1) (10); gag, 5Ј-TTTGTTCCTGAAGGGTACTAGTAG TTCC-3Ј (nt 1047 to 1074, antisense, gagDTRA) (29); pol, 5Ј-TGCCCACACTA ATGATGTAAAACAATTA-3Ј, (nt 3208 to 3235, sense, polA1) (29); tat, 5Ј-TT GGGTGTCGACATAGCAGAATAGGCGT-3Ј (nt 5361 to 5388, sense, tatA2) (29); env, 5Ј-ATCCTCAGGAGGGGACCCAGAAATT-3Ј (nt 6906 to 6930, sense); nef, 5Ј-TTTCCAGGTCTCGAGATACTGCTCC-3Ј (nt 8480 to 8504, antisense); and LTR(U3), 5Ј-CTACAAGGGACTTTCCGCTGG-3Ј (nt 9022 to 9042, sense).…”

Section: Subjectsmentioning

confidence: 99%

Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals

Sanchez

Chermann

et al. 1997

J Virol

Self Cite

145

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Human immunodeficiency virus type 1 (HIV-1) genomes present in peripheral blood mononuclear cells (PBMCs) of infected persons or in lymphocytes infected in vitro were studied by long-distance PCR (LD-PCR) using primers localized in the HIV-1 long terminal repeats. The full-length 9-kb DNA was the only LD-PCR product obtained in peripheral and cord blood lymphocytes from seronegative donors infected in vitro. However, a high proportion (27% to 66%) of distinct populations of extensively deleted HIV-1 genomes of variable size was detected in PBMCs of 15 of 16 HIV-1-infected persons. Physical mapping of defective genomes showed that the frequency of deletions is proportional to their proximity to the central part of HIV-1 genome, which is consistent with a deletion mechanism involving a single polymerase jump during reverse transcription. Sequencing of deletion junctions revealed the presence of short direct repeats of three or four nucleotides. The number of defective HIV-1 genomes decreased after in vitro activation of PBMCs. Persistence of full-length and deleted genomes in in vitro activated PBMCs correlated with isolation of an infectious virus. Our results represent the first quantitative assessment of intragenomic rearrangements in HIV-1 genomes in PBMCs of infected persons and demonstrate that, in contrast to in vitro infection, defective genomes accumulate in PBMCs of infected persons.

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“…High extraction efficiency (75%) in small elution volumes and increased blood capacity (up to 1.5 μL) were observed due to low protein binding to the chitosan phase. While the volume capacity of 1.5 μL of blood represents a 7-fold improvement, larger capacity still is required for many clinical analyses. , …”

mentioning

confidence: 99%

Microfluidic-Based DNA Purification in a Two-Stage, Dual-Phase Microchip Containing a Reversed-Phase and a Photopolymerized Monolith

et al. 2007

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In this report, we show that a novel capillary-based photopolymerized monolith offering unprecedented efficiency (approximately 80%) for DNA extraction from submicroliter volumes of whole blood (Wen, J.; Guillo, C.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78, 1673-1681) can be translated to microfluidic devices. However, owing to the large mass of protein present in blood, both DNA binding capacity and extraction efficiency were significantly decreased when extraction of DNA was carried out directly from whole blood (38+/-1%). To circumvent this, a novel two-stage microdevice was developed, consisting in a C18 reversed-phase column for protein capture (stage 1) in series with a monolithic column for DNA extraction (stage 2). The two-stage, dual-phase design improves the capability of the monolith for whole blood DNA extraction by approximately 100-fold. From a 10-microL load of whole blood containing 350 ng of DNA, 99% (340+/-10 ng) traverses the C18 phase while approximately 70% (1020+/-45 ug) of protein is retained. A total of 240+/-2 ng of DNA was eluted from the second-stage monolith, resulting in an overall extraction efficiency of 69+/-1%. This provided not only an improvement in extraction efficiency over other chip-based DNA extraction solid phases but also the highest extraction efficiency reported to-date for such sample volumes in a microfluidic device. As an added bonus, the two-stage, dual-phase microdevice allowed the 2-propanol wash step, typically required to remove proteins from the DNA extraction phase for successful PCR, to be completely eliminated, thus streamlining the process without affecting the PCR amplifiability of the extracted DNA.

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Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates

Cited by 17 publications

References 33 publications

PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs

PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs

Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals

Microfluidic-Based DNA Purification in a Two-Stage, Dual-Phase Microchip Containing a Reversed-Phase and a Photopolymerized Monolith

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