Real-time Nucleic Acid Sequence-based Amplification Research Articles

WITHDRAWN: The use of dry respiratory swabs for the detection of respiratory viruses in older children and adults using real-time nasba with molecular beacon technology

WITHDRAWN: The use of dry respiratory swabs for the detection of respiratory viruses in older children and adults using real-time nasba with molecular beacon technology

WITHDRAWN: Development and evaluation of a real-time NASBA for the detection of noroviruses

WITHDRAWN: Development and evaluation of a real-time NASBA for the detection of noroviruses

Development of conventional and real-time NASBA® for the detection of Legionella species in respiratory specimens

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1–1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1–10 000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1–10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.

Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of Chlamydophila pneumoniae in Respiratory Specimens

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 mul sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.

Presence of E6 and E7 mRNA from Human Papillomavirus Types 16, 18, 31, 33, and 45 in the Majority of Cervical Carcinomas

The oncogenic potential of the human papillomavirus (HPV) early genes E6 and E7 is well established and a source of interest with regard to HPV testing for cervical carcinoma. Here we present a study performed with 204 histologically confirmed invasive cervical squamous cell carcinomas (SCCs) in which we evaluated the HPV E6 and E7 mRNA detection assay PreTect HPV-Proofer for detection of high-risk HPV types 16, 18, 31, 33, and 45. For further evaluation, detection of E6 and E7 mRNA from HPV types 35, 52, and 58 by real-time multiplex nucleic acid sequence-based amplification was also included. For comparison and to assess the overall prevalence of various HPV types, samples were also tested for HPV DNA by both consensus and type-specific PCR, reverse line blotting, sequencing, and in situ hybridization. The overall prevalence of HPV was 97%. HPV E6 and E7 transcripts were detected in 188 of 204 (92%) biopsy specimens, of which 181 contained one of the following HPV types: 16, 18, 31, 33, or 45. Consensus PCR and type-specific PCR detected HPV in 187 of 204 and 188 of 204 (92%) specimens, respectively. In conclusion, this study verifies the presence of HPV E6 and E7 mRNA in SCCs and demonstrates that HPV infections among Norwegian women with SCCs are limited mainly to the five high-risk types, 16, 18, 31, 33, and 45. This, together with the fact that PreTect HPV-Proofer detects the HPV oncogenic transcripts, suggests that the assay is a valuable approach in the field of HPV detection in cervical carcinoma.

Enhanced clinical utility of the NucliSens EasyQ RSV A+B Assay for rapid detection of respiratory syncytial virus in clinical samples

The aim of the present study was to compare traditional methods for the detection of respiratory syncytial virus with a newly developed commercial assay based on real-time nucleic acid sequence based amplification. Respiratory syncytial virus is a major cause of severe respiratory infection in infants and in certain groups of older children and adults. Treatment options are limited, but a rapid diagnosis improves patient management and infection control. The rapid diagnosis of respiratory syncytial virus currently relies on antigen detection assays. These tests are limited to use in certain good-quality types of samples, which are rarely obtained from adult patients. Molecular-based assays for the detection of respiratory syncytial virus are shown to be highly sensitive, specific, and more rapid than cell culture techniques. This retrospective study compared traditional laboratory techniques for the detection of respiratory syncytial virus in 508 respiratory samples collected during the winter months of 2003-2004 against the recently developed, commercially available NucliSens EasyQ Respiratory Syncytial Virus A+B assay (bioMérieux, Marcy l'Etoile, France), which is based on real-time nucleic acid sequence based amplification using molecular beacons and an internal control. Using traditional techniques, the prevalence of respiratory syncytial virus in the samples tested was found to be 21%. Using the real-time nucleic acid sequence-based amplification assay, an additional 41 samples from patients with a clinically diagnosed respiratory illness were found to be positive for respiratory syncytial virus. The NucliSens EasyQ assay was shown to be sensitive and specific for the detection of respiratory syncytial virus A+B in different types of respiratory samples. Moreover, the time required to complete the assay was <4 h, so results could be obtained on the same day as sample receipt in the laboratory.

Development of a real-time NASBA assay for the detection of Campylobacter jejuni cells

The objectives of this study were the development of a real-time NASBA assay for the detection of Campylobacter jejuni mRNA and the evaluation of its potential to determine the viability of the detected C. jejuni cells. A set of specific primers and probes was chosen to amplify the mRNA of the tuf-gene and the GTPase-gene. Only the tuf-assay was able to detect as low as 10 2 cells per NASBA reaction and was specific for Campylobacter. However, as the assay was able to detect dead cells, it cannot be used to demonstrate the viability of C. jejuni cells . The tuf-gene mRNA is no good viability indicator due to its stability.

(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate

The effects of drugs on Plasmodium falciparum transmission stages may reduce the spread of parasites in the population and contribute to malaria control. Detailed quantitative studies on (sub)microscopic gametocytaemia have become feasible with the availability of real-time Pfs25 quantitative Nucleic Acid Sequence-based Amplification (QT-NASBA), which can be used to detect gametocyte densities above 20 gametocytes per millilitre from in vitro cultures. Gametocyte dynamics were investigated in children with uncomplicated P. falciparum malaria after treatment with sulphadoxine-pyrimethamine (SP) or a combination of SP and artesunate (SP+AS), in a 28-days drug efficacy study. This study demonstrated that gametocyte prevalence in 873 samples from symptomatic Kenyan children was 2.8 times higher by QT-NASBA compared with microscopy. Microscopy-positive cases showed a significant correlation with QT-NASBA for gametocyte density. At enrolment, gametocyte prevalence was 86% by QT-NASBA compared with 22% by microscopy. Gametocytes were detected in 97% of children in at least one blood sample and in 38% of children in all samples obtained during the 28-days follow-up. Both the risk of gametocyte carriage and gametocyte density were considerably higher after treatment with SP compared with SP+AS. Gametocyte prevalence and density decreased with time in the SP+AS group, but not in the SP-treated children. Our data suggest that the potential of malaria transmission remains high even after treatment with artemisinin combination therapy, although prevalence and density of gametocytes is lower after SP+AS.

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

Comparative performance of the Amplicor HIV-1 monitor assay versus NucliSens EasyQ in HIV subtype C-infected patients

In facing global programs for treating HIV-infected patients in the developing countries, there is a real need for viral load assays that are accurate for the local subtypes. The present study was designed to evaluate viral load measurements using the newer version of the NASBA assay in subtype C-infected patients. The performances of this new version, a real-time nucleic acid sequence-based amplification HIV-1 assay (NucliSens EasyQ), were compared to Amplicor HIV-1 Monitor Assay version 1.5 in 79 samples of subtype C-infected patients originating from Ethiopia. Twenty HIV-1 subtype B-infected patients served as a control group. Blood samples from patients in both groups were tested by both assays. The results were compared by a paired, two-tailed Student's t-test. The disparity between the results of the two viral load assays was highly significant in subtype C samples (P = 0.005), such that in the vast majority, higher values of viral load were obtained by the Amplicor assay. However, no differences between the two assays were found in subtype B samples (P = 0.77). CD4 measurements were available for 78 samples of subtype C-infected patients. Of these, a CD4-to-viral load discrepancy (CD4 <or= 200, viral load <or=5,000 IU/ml) was found in 11.5% of the samples when using the EasyQ assay, compared with 5.1% of the samples using the Amplicor assay. In conclusion, the performance of the NucliSens EasyQ assay was inferior to that of the Amplicor assay in assessing viral load levels in subtype C-infected patients. This difference may have a significant bearing on patient care.

HSV1 and HSV2 detection using a real-time DNA NASBA assay

HSV1 and HSV2 detection using a real-time DNA NASBA assay

Development and evaluation of a real time NASBA for the detection of noroviruses

Development and evaluation of a real time NASBA for the detection of noroviruses

Nucleic acid extraction and real-time NASBA to diagnose atypical pneumonia

Nucleic acid extraction and real-time NASBA to diagnose atypical pneumonia

The use of dry respiratory swabs for the detection of respiratory viruses in older children and adults using real-time NASBA technology

The use of dry respiratory swabs for the detection of respiratory viruses in older children and adults using real-time NASBA technology

Real-time NASBA assay for the detection of influenza A and B

Real-time NASBA assay for the detection of influenza A and B

Development of a real-time NASBA for respiratory syncytial virus

Development of a real-time NASBA for respiratory syncytial virus

Mumps meningitis in south-east Scotland—a retrospective study

Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies.

NASBA: a novel approach to assess hormonal receptors and ERBB2 status in breast cancer

In human breast cancer, estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and human epidermal growth factor receptor (ERBB2) status are currently determined using different techniques. We propose to assess the mRNA expression of these three clinically relevant markers using a unique technique, real-time nucleic acid sequence-based amplification (NASBA). Gene expression of hormone receptors was analyzed and compared to the cytosolic functional protein content as determined with a ligand binding assay (LBA), while ERBB2 mRNA expression was compared to quantitative PCR and ELISA. We observed that the three markers are significantly overexpressed at the mRNA level in positive tumors, as measured by DNA- or protein-based techniques. Biostatistical analysis of the receiver operating characteristic (ROC) curve demonstrated high concordance between NASBA and LBA [area under the curve (AUC) for ROC of 0.899] and showed that ERalpha status could be predicted using the molecular assay with a sensitivity of 72.7% and a specificity of 93.5%. Similar results were obtained for PR (AUC ROC 0.938, sensitivity 75.3%, specificity 100%). Moreover, excellent concordance was observed between NASBA, quantitative PCR and ELISA with respect to ERBB2 (AUC ROC 0.92, sensitivity 90%, specificity 89.7%; and AUC ROC 0.98, sensitivity 100%, specificity 91.5%, respectively). These results suggest that NASBA is well suited for assessing ER, PR and ERBB2 status in breast tumor samples. This approach is rapid, highly sensitive and a standardized method that could be complementary to the existing techniques, especially for small tumors.

Evaluation of a real-time nucleic acid sequencebased amplification (nasba) assay for diagnosis of urogenital chlamydial infection

This study was aimed to evaluate a new realtime nucleic acid sequence-based amplification (NASBA-Real- Time) assay for diagnosis of urogenital Chlamydia trachomatis infection. A total of 193 patients aged 16 to 42 (mean age, 22,8 years) were examined, with most of them having symptoms of urogenital infection. Cervical and urethral swabs from women and men, respectively, were investigated with the use of cell culture method, polymerase chain reaction (PCR) and NASBA- Real-Time. C. trachomatis infection was diagnosed in 29 patients (15 %): in 21 patients - by all the three methods, whereas 8 samples were culture negative. Sensitivity of PCR and NASBA- Real-Time methods were 100 %, cell culture method - 78,4 %. Negative predictive value of PCR and NASBA-Real-Time was found to be 100 %, and that of cell culture - 95,3 %. Specificity as well as positive predictive values of all the three methods equaled 100%. Thus, the new real-time NASBA-Real-Time assay was shown to be a very sensitive and specific test, which can be recommended as a confirmatory method for diagnosis of urogenital chlamydial infection.

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.