Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA)

Deiman, Birgit; Aarle, Pierre van; Sillekens, Peter

doi:10.1385/mb:20:2:163

Cited by 240 publications

(184 citation statements)

References 67 publications

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“…37 NASBA reagents were obtained from BioMerieux (NucliSens basic kit, BioMerieux, Boxtel, The Netherlands). Oligonucleotide primers for LMP2 and (nonspliced) BARF1 mRNA were described before.…”

Section: Nucleic Acid Sequence Based Amplification For Ebv Rna Detectionmentioning

confidence: 99%

Noninvasive diagnosis of nasopharyngeal carcinoma: Nasopharyngeal brushings reveal high Epstein‐Barr virus DNA load and carcinoma‐specific viral BARF1 mRNA

Stevens

Verkuijlen

Hariwiyanto

et al. 2006

Intl Journal of Cancer

106

130

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Nasopharyngeal carcinoma (NPC) is the most prevalent ENTtumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N 5 106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p < 0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at 280°C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool. ' 2006 Wiley-Liss, Inc.Key words: EBV; NPC; diagnosis; viral load; oncogene; carcinoma Undifferentiated nasopharyngeal carcinoma (NPC WHO type III) is virtually 100% associated with Epstein-Barr virus (EBV) and has a reported high incidence in most of South-East Asia and intermediate incidence in North-African populations and in Inuit. [1][2][3] In Indonesia, with an ethnically diverse population of 225 million people, NPC is the most common ENT tumour with high prevalence among native populations and a yearly overall incidence estimated at 6.2/100,000. 4 Extremely high incidence was recently documented in native populations living on the island of Sulawesi. 5 In Yogyakarta, Central Java, NPC is the most prevalent tumour among man and 4th most prevalent among females, with a male/female ratio of 2.4, constituting respectively 22 and 8% of all diagnosed malignancies. 4 The strong etiological link between EBV and NPC has been known for over 3 decades [1][2][3]6 and is reflected by abnormal anti-EBV antibody profiles, increased circulating EBV DNA levels and by distinct EBV gene expression in the tumour cells. 7-11 Classically, NPC is considered to have a latency type 2 EBV transcription, with expression of EBV-encoded small RNAs 1 and 2 (EBER1/2), BamHI A rightward transcript...

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Section: Nucleic Acid Sequence Based Amplification For Ebv Rna Detectionmentioning

confidence: 99%

Noninvasive diagnosis of nasopharyngeal carcinoma: Nasopharyngeal brushings reveal high Epstein‐Barr virus DNA load and carcinoma‐specific viral BARF1 mRNA

Stevens

Verkuijlen

Hariwiyanto

et al. 2006

Intl Journal of Cancer

106

130

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“…Human papillomavirus RNA was detected by real-time NASBA (Compton, 1991;Chan and Fox, 1999;Deiman et al, 2002). Reagents were obtained from NorChip (Klokkarstua, Norway), as they are presented in the commercially available PreTect HPVProofer kit.…”

Section: Nasba Protocol: Amplification and Detection Of Hpv Rnamentioning

confidence: 99%

Human papillomavirus oncogenic expression in the dysplastic portio; an investigation of biopsies from 190 cervical cones

et al. 2004

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In this study, we investigated the presence of E6/E7 transcripts of seven common high-risk human papillomavirus (HPV) types in 190 cervical biopsies. The RNA-based real-time nucleic acid sequence-based amplification assay (NASBA) and type-specific PCR, both detecting HPV 16, 18, 31, 33, 45, 52, and 58, as well as consensus PCR, were performed on all 190 biopsies. High accordance between type-specific and consensus PCR confirms that the HPV types included in this study are the most common types present in cervical dysplasia. Furthermore, we see a clear increase in the incidence of HPV, both DNA and RNA, along with the histological severity of dysplasia. HPV RNA was detected in all but two PCR-positive cases, confirming that the virus exerts E6/E7 mRNA expression in cases of high-grade dysplasia. Out of 19 women given a normal or borderline diagnosis at conisation, only four were found HPV positive, which may suggest that unnecessary conisations can possibly be reduced by introducing HPV testing into the preoperative routine assessment. Human papillomavirus (HPV) infections are associated with the development of cervical neoplasia and are a necessary factor in the aetiology of cervical carcinoma (Walboomers et al, 1999;Bosch et al, 2002;zur Hausen, 2002). The most frequent HPV types found in high-grade cervical intraepithelial dysplasia (CIN II/III) and in cervical carcinomas are HPV 16, 18, 31, 33, and 45, which are often referred to as high-risk HPV or cancer-associated HPV types. However, the association between a positive HPV DNA test and the risk of subsequent development of dysplasia is partly unknown; the presence of HPV DNA does not necessarily indicate a risk for developing a more severe lesion. In fact, HPV is a very common virus among sexually active women and most infections are transient and asymptomatic (zur Hausen, 1991;Ho et al, 1998;Moscicki et al, 1998). Only very few infections cause morphologic changes in the epithelium and only a small percent of these will eventually develop cervical cancer (Bosch et al, 1995). On the other hand, several studies have shown that in cervical carcinogenesis, the expression of HPV E6 and E7 ORF is required for cell transformation and immortalisation (zur Hausen, 1988;Munger et al, 1989;Stoler et al, 1992). Consequently, persistent expression of these oncogenes may serve as an indicator of progression to CIN and invasive cancer (Sotlar et al, 1998), and detection of E6 and E7 mRNA transcripts may therefore be of higher prognostic value than the detection of HPV DNA. Moreover, new methods for preservation of RNA in cells have made gene expression analysis more feasible.In this study, we investigated the presence of high-risk HPV E6/E7 transcripts in cervical biopsies from patients undergoing conisation. The analysis was performed on a biopsy taken from the ectocervix/transformation zone at the time of conisation, and the pathological diagnosis of this particular biopsy was not always representative for the existing lesion that was later revealed in the cone. This s...

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“…It performed well in multicenter evaluation compared with other commercially available assays (Ginocchio et al, 2003;Murphy et al, 2000). The NucliSens HIV-1 QT is a NASBA with end-point electro-chemiluminesence (ECL) detection (Deiman et al, 2002). Further refinement has led to the development of the CE marked NucliSens EasyQ HIV-1-a combination of NASBA amplification and real-time detection using molecular beacons utilizing the NucliSens EasyQ analyzer (van Beuningen et al, 2001;Leone et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…It is specific for RNA-based virus in the presence of DNA background. It makes use of the simultaneous enzymatic activities of avian reverse transcriptase, ribonuclease H and bacteriophage T7 RNA polymerase (Compton, 1991;Deiman et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Quantitative detection of HIV-1 RNA using NucliSens EasyQ HIV-1 assay

Yao

Liu²,

et al. 2005

Journal of Virological Methods

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HIV-1 RNA viral load has become the major biological marker for disease prognosis and outcome of antiretroviral therapy in the treatment of HIV-infected individuals. The aim of this study was to compare the performance of the new CE marked NucliSens EasyQ HIV-1 assay with NucliSens HIV-1 QT assay (reference method). NucliSens EasyQ HIV-1 (EasyQ) couples nucleic acid sequence-based amplification (NASBA) with real-time detection using molecular beacons utilizing the NucliSens EasyQ analyzer. NASBA is a sensitive, isothermal, transcription-based amplification system designed specifically for the detection of RNA targets. There was significant correlation (r = 0.878, P < 0.0001) between the two different assays in the analysis of clinical samples and the frequency of concordant results (log difference <0.5) was 74%. The two assays detected HIV-1 RNA in 81 specimens, and neither detected (below the lower detection limit, 400 copies/ml for NucliSens HIV-1 QT and 500 IU/ml for EasyQ) HIV-1 RNA in 12 specimens. Three clinical specimens had detectable HIV-1 RNA using the EasyQ only, and two specimens had detectable HIV-1 RNA using the NucliSens HIV-1 QT only. The EasyQ procedure can analyze 48 clinical samples within 6 h. The coefficient of variation of EasyQ ranged from 3.0 to 9.5% (3% at 4.9 log; 5.7% at 3.7 log; 9.5% at 2.7 log). The new assay is shown to be a rapid, convenient, and reliable procedure for HIV-1 RNA viral load monitoring.

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Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA)

Cited by 240 publications

References 67 publications

Noninvasive diagnosis of nasopharyngeal carcinoma: Nasopharyngeal brushings reveal high Epstein‐Barr virus DNA load and carcinoma‐specific viral BARF1 mRNA

Noninvasive diagnosis of nasopharyngeal carcinoma: Nasopharyngeal brushings reveal high Epstein‐Barr virus DNA load and carcinoma‐specific viral BARF1 mRNA

Human papillomavirus oncogenic expression in the dysplastic portio; an investigation of biopsies from 190 cervical cones

Quantitative detection of HIV-1 RNA using NucliSens EasyQ HIV-1 assay

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