Multiplex PCR using real time DNA amplification for the rapid detection and quantitation of HTLV I or II

Estes, Michael C.; Sevall, J. Sanders

doi:10.1016/s0890-8508(03)00002-1

Cited by 24 publications

(18 citation statements)

References 26 publications

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“…To evaluate intra-assay and interassay reproducibility, we calculated the coefficient of variation (CV) of the threshold cycle (C T ) values and the PVL results as follows: %CV ϭ (standard deviation/mean) ϫ 100. In the literature, CVs of C T values of Յ3% for intra-assay reproducibility and Յ10% for interassay reproducibility, based on standard dilutions, are acceptable (11,13). For comparison of the HTLV-1 PVL results measured with two different assays, we used GraphPad Prism 5 software to perform linear regression.…”

Section: Primers and Probesmentioning

confidence: 99%

“…Multiplex qPCR allows the simultaneous detection and amplification of two or more target DNA sequences in only one amplification reaction. To our knowledge, one specific and one generic biplex qPCR for HTLV-1 and -2 (13,26) and, just recently, one triplex qPCR for HTLV-1, -2, and -3 have been described (3). To address the current problems with HTLV diagnosis and quantitation, taking into account the diversity in HTLV genotypes and subtypes, we developed a novel triplex qPCR assay for simultaneous detection, genotyping, and quantification of PVL of HTLV-1, -2, and -3 infections.…”

mentioning

confidence: 99%

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Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

et al. 2009

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The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10 5 to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r 2 ‫؍‬ 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.Since the discovery of human T-lymphotropic virus type 1 (HTLV-1) in 1980 (16, 40), three other genotypes and 10 subtypes have been recognized. The precise geographical distribution and the clinical consequences of these infections are still a matter of debate. This can be attributed at least in part to the fact that there are insufficient accurate tools for HTLV diagnosis, genotyping, and measurement of viral burden.HTLV-1 is endemic in several geographical areas, including sub-Saharan Africa, South America, the Caribbean Islands, Japan, and Melanesia. It has been estimated that worldwide 10 to 25 million people are infected with this retrovirus (41, 53). Most HTLV-1-infected individuals remain asymptomatic throughout their lifetimes. However, 5 to 10% of infected people develop clinical complications, among which adult Tcell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are the most severe. Other manifestations of HTLV-1 infection include infective dermatitis (25), uveitis (34), arthritis (38), and Strongyloides stercoralis infection (53). Some of these manifestations could accelerate disease development and/or progression (12, 16). For HTLV-1, a distinction is made between seven subtypes: the worldwide, cosmopolitan subtype HTLV-1a; the Central African subtypes HTLV-1b, -d, -e, -f, and -g; and the Australo-Melanesic subtype 23,41,52).HTLV-2 was discovered in 1982. This retrovirus is endemic in Amerindian and pygmy populations and epidemic in intravenous drug users (16,49). In contrast to the case for HTLV-1, convincing epidemiological demonstra...

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Section: Primers and Probesmentioning

confidence: 99%

mentioning

confidence: 99%

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

et al. 2009

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“…In previous real-time PCR methods used for the detection and quantification of HTLVs, linear TaqMan probes were used (10,12). In our study, we used molecular beacon probes, which are dual-labeled oligonucleotide probes that fluoresce upon hybridization with a complementary target sequence (44).…”

Section: Discussionmentioning

confidence: 99%

“…A rapid, non-labor-intensive amplification technique for the accurate quantification of the viral target is real-time PCR (20), in which internal fluorescent probes provide specificity and allow the detection of amplification products that are directly related to the copy number of the target (36). Realtime PCR already has been used for detecting HTLV-1 and -2 proviral loads (10,12), including those of subtypes 1A, 1B, 1C, 2A, and 2B; however, the method did not detect HTLV-3 or various simian T-cell leukemia viruses (STLVs).…”

mentioning

confidence: 99%

One-Step, Multiplex, Real-Time PCR Assay with Molecular Beacon Probes for Simultaneous Detection, Differentiation, and Quantification of Human T-Cell Leukemia Virus Types 1, 2, and 3

Besson

Kazanji

2009

J Clin Microbiol

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A single-tube, multiplex, real-time PCR assay with molecular beacons was established in which various probes were used for the simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3 (HTLV-1, HTLV-2, and HTLV-3, respectively) and of simian T-cell leukemia virus types 1 and 3 (STLV-1 and STLV-3, respectively). The quantitative amplification of the standards with MT4 (HTLV-1) and C19 (HTLV-2) cell lines and a molecular clone of HTLV-3 was linear, with the simplex and multiplex methods having similar efficiencies. A maximum difference of 0.9 (mean, 0.4; range, 0.0 to 0.9) was found between threshold cycle values in single and multiplex reactions. The efficiency with each probe in the multiplex reaction was close to 100%, indicating strong linear amplification. The albumin gene was used to standardize the copy number. Comparable results for the detection and quantification of HTLV-1 were obtained with our new methods and with other real-time PCR methods described previously. With our new multiplex assay, however, we were able to detect and quantify HTLV-2 and -3 and STLV-1 and -3 in clinical specimens, with an excellent dynamic range of 10 6 to 10 0 copies per assay, which the other assays could not do. Thus, it will be possible to determine a wide range of HTLV types in both standard and clinical samples, with a detection of 1 to 10 HTLV copies in samples containing at least 100 cells. Furthermore, our system can provide evidence for multiple infections with the three HTLV types, with separate proviral load results. Our new method also could be used for epidemiological studies in Africa and in countries where HTLVs and STLVs are endemic.

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“…Ultimately, these individuals will be referred to an infectious diseases specialist. Confirmation of HTLV-1/2 infection has been recommended using Western Blot (Program CfDCME, 2001) and/or polymerase chain reaction (PCR) (Estes and Sevall, 2003), which are offered through many reference laboratories. Even so, many of these tests lack adequate specificity or sensitivity, and none are licensed for supplemental testing in the blood donor setting (Thorstensson et al, 2002).…”

Section: Diagnosis and Treatment Serologic And Nucleic Acid Testing Fmentioning

confidence: 99%

The human T‐cell leukemia virus type 1 (HTLV‐1): New insights into the clinical aspects and molecular pathogenesis of adult t‐cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV‐associated myelopathy (TSP/HAM)

Shuh

Beilke

2005

Microscopy Res & Technique

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Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus to be identified in the early 1980s. The isolation and identification of a related virus, HTLV-2, and the distantly related human immunodeficiency virus (HIV) immediately followed. Of the three retroviruses, two are associated definitively with specific diseases, HIV, with acquired immune deficiency syndrome (AIDS) and HTLV-1, with adult T-cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). While an estimated 10-20 million people worldwide are infected with HTLV-I, infection is endemic in the Caribbean, parts of Africa, southwestern Japan, and Italy. Approximately 4% of HTLV-I infected individuals develop ATLL, a disease with a poor prognosis. The clinical manifestations of infection and the current biology of HTLV viruses with emphasis on HTLV-1 are discussed in detail. The implications for improvements in diagnosis, treatment, intervention, and vaccination are included, as well as a discussion of the emergence of HTLV-1 and -2 as copathogens among HIV-1-infected individuals.

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Multiplex PCR using real time DNA amplification for the rapid detection and quantitation of HTLV I or II

Cited by 24 publications

References 26 publications

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

One-Step, Multiplex, Real-Time PCR Assay with Molecular Beacon Probes for Simultaneous Detection, Differentiation, and Quantification of Human T-Cell Leukemia Virus Types 1, 2, and 3

The human T‐cell leukemia virus type 1 (HTLV‐1): New insights into the clinical aspects and molecular pathogenesis of adult t‐cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV‐associated myelopathy (TSP/HAM)

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