Characterization of the quantitative HCV NASBA assay

Damen, Marjolein; Sillekens, Peter; Cuypers, H. T. M.; Frantzen, Inge; Melsert, Roel

doi:10.1016/s0166-0934(99)00079-8

Cited by 37 publications

(19 citation statements)

References 17 publications

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“…Both LCR and isothermal amplification were first described in 1989 [89,90]. Although these techniques are well known and used for viral load detection in infection with, for example, HIV and hepatitis C virus, they are not used for the detection or identification of bacteria in blood cultures [91][92][93][94]. LCR has only been used for the detection of Mycobacterium tuberculosis in respiratory specimens, and the isothermal transcription-mediated technique has only been used for the rapid identification of Candida spp.…”

Section: Multiplex Assaysmentioning

confidence: 99%

Conventional and molecular techniques for the early diagnosis of bacteraemia

Paolucci

Landini

Sambri

2010

International Journal of Antimicrobial Agents

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Bloodstream infections account for 30-40% of all cases of severe sepsis and septic shock, and are major causes of morbidity and mortality. Diagnosis of bloodstream infections must be performed promptly so that adequate antimicrobial therapy can be started and patient outcome improved. An ideal diagnostic technology would identify the infecting organism(s) and their determinants of antibiotic resistance, in a timely manner, so that appropriate pathogen-driven therapy could begin promptly. Unfortunately, despite the essential information it provides, blood culture, the gold standard, largely fails in this purpose because time is lost waiting for bacterial or fungal growth. Several efforts have been made to optimize the performance of blood culture, such as the development of technologies to obtain rapid detection of microorganism(s) directly in blood samples or in a positive blood culture. The ideal molecular method would analyze a patient's blood sample and provide all the information needed to immediately direct optimal antimicrobial therapy for bacterial or fungal infections. Furthermore, it would provide data to assess the effectiveness of the therapy by measuring the clearance of microbial nucleic acids from the blood over time. None of the currently available molecular methods is sufficiently rapid, accurate or informative to achieve this. This review examines the principal advantages and limitations of some traditional and molecular methods commercially available to help the microbiologist and the clinician in the management of bloodstream infections.

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Section: Multiplex Assaysmentioning

confidence: 99%

Conventional and molecular techniques for the early diagnosis of bacteraemia

Paolucci

Landini

Sambri

2010

International Journal of Antimicrobial Agents

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“…Diagnostic procedures based on NASBA methodology have been described for several viruses including Human Immunodeficiency Virus Type 1 (de Baar et al 1999), cytomegalovirus (Witt et al 2000), enterovirus (Heim & Schumann 2002), West-Nile and St Louis Encephalitis viruses (Lanciotti & Kerst 2001), parainfluenza virus (Hibbitts et al 2003) and hepatitis C virus (Damen et al 1999). In the NASBA procedure, target-specific amplification is achieved through oligonucleotide primers and the co-ordinated activity of 3 enzymes: reverse transcriptase, RNase H, and T7 RNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA)

Starkey

Millar²,

Jenkins³

et al. 2004

Dis. Aquat. Org.

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Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41°C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses. KEY WORDS: Nodavirus · NASBA · RT-PCR · Diagnostics · Nucleic acid amplification Resale or republication not permitted without written consent of the publisherDis Aquat Org 59: [93][94][95][96][97][98][99][100] 2004 ular diagnostic methods based on RT-PCR (Nishizawa et al. 1994, Grotmol et al. 2000 or nested RT-PCR (Dalla Valle et al. 2000) have been developed for betanodaviruses, and have contributed significantly to the diagnosis and control of VNN. However, these assays are relatively time-consuming, may be compromised by limited sensitivity, and are susceptible to false positive reactions arising from amplicon contamination.Nucleic acid sequence based amplification (NASBA) (Compton 1991) is an isothermal method for nucleic acid amplification that is particularly suited to RNA targets (Kievits et al. 1991). Diagnostic procedures based on NASBA methodology have been described for several viruses including Human Immunodeficiency Virus Type 1 (de Baar et al. 1999), cytomegalovirus (Witt et al. 2000, enterovirus (Heim & Schumann 2002), West-Nile and St Louis Encephalitis viruses (Lanciotti & Kerst 2001), parainfluenza virus (Hibbitts et al. 2003) and hepatitis C virus (Damen et al. 1999). In the NASBA procedure, target-specific amplification is achieved through oligonucleotide primers and the co-ordinated activity of 3 enzymes: reverse transcriptase, RNase H, and T7 RNA polymerase. The final amplification product is a singlestranded RNA, the polarity of which is opposite to that of the target. Real-time detection in NASBA can be performed using...

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“…Among the methodologies that may have more widespread practical application is transcription-mediated amplification (TMA). As it is similar to some aspects of NASBA ® technology, the amplification portion of this new assay has been shown to be useful in the field of HCV research (7,(9)(10)(11). In the current study, we report on the performance characteristics of a newly developed TMA-based assay that has been validated prior to commercialization.…”

Section: Introductionmentioning

confidence: 99%

Performance characteristics of a transcription‐mediated nucleic acid amplification assay for qualitative detection of hepatitis C virus RNA

Roß¹,

Viazov²,

Hoffmann³

et al. 2001

Clinical Laboratory Analysis

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The detection of hepatitis C virus (HCV) RNA by nucleic acid amplification techniques is the method of choice to differentiate between ongoing and past infection, and can be used to monitor the course of HCV infection. In this study, we evaluated the performance characteristics of a newly developed transcription-mediated amplification (TMA)-based assay, the VERSANT HCV RNA qualitative assay, which was designed to qualitatively detect HCV RNA. Samples tested by the TMA assay included 100 HCV antibody negative sera; serial dilutions of an HCV genotype 1a panel; the WHO HCV RNA standard 76/790; an HCV genotyping panel; and 150 clinical specimens, including sera from patients who had received alpha interferon (IFN) treatment or liver transplants. TMA test results were compared with the Cobas Amplicor HCV polymerase chain reaction (PCR) assay. The analytical specificity of the HCV TMA assay was > 98%. No carry-over contaminations were observed. The assay demonstrated an analytical sensitivity of 100% at 41 HCV RNA copies/mL (genotype 1a panel) and 5 IU/mL (WHO standard), respectively. HCV genotypes and subtypes did not affect the results. Qualitative RNA detection by diagnostic Amplicor PCR and TMA was in agreement in > 97% of all 150 clinical samples tested. In our study, the TMA-based assay proved to be a specific and sensitive method for qualitative HCV RNA detection. The test may turn out to be an attractive alternative to already established techniques for HCV RNA amplification in routine clinical laboratories.

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Characterization of the quantitative HCV NASBA assay

Cited by 37 publications

References 17 publications

Conventional and molecular techniques for the early diagnosis of bacteraemia

Conventional and molecular techniques for the early diagnosis of bacteraemia

Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA)

Performance characteristics of a transcription‐mediated nucleic acid amplification assay for qualitative detection of hepatitis C virus RNA

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