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PubMed2002

Characteristics and applications of nucleic acid sequence-based amplification (NASBA).

Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA is also amplified though very inefficiently and only in the absence of the corresponding RNA target or in case of an excess (>1,000-fold) of target DNA over RNA. As NASBA is primer-dependent and amplicon detection is based on probe binding, primer and probe design rules are included. An overview of various target nucleic acids that have been amplified successfully using NASBA is presented. For the isolation of nucleic acids prior to NASBA, the "Boom" method, based on the denaturing properties of guanidine isothiocyanate and binding of nucleic acid to silica particles, is preferred. Currently, electro-chemiluminescence (ECL) is recommended for the detection of the amplicon at the end of amplification. In the near future, molecular beacons will be introduced enabling "real-time detection," i.e., amplicon detection during amplification. Quantitative HIV-1 NASBA and detection of up to 48 samples can then be performed in only 90 min.

DNA↗
PubMed2004

Expression level of MDR1 message in peripheral blood leukocytes from healthy adults: a competitive nucleic acid sequence-based amplification assay for its determination.

Accurate quantification of multidrug resistance-1 gene (MDR1) expression in target cells would be of important therapeutic value in predicting cellular response to anticancer drugs. Because certain normal cells in peripheral blood physiologically express MDR1, increasing the sensitivity of the detection methods might result in confounding low-degree expression in tumor cells with physiologic expression in normal cells. The purpose of this study was to determine MDR1 mRNA expression levels in peripheral blood leukocytes obtained from healthy adult volunteers using a competitive nucleic acid sequence-based amplification (NASBA) assay. We determined the reference intervals of MDR1 mRNA expression in peripheral blood obtained from 98 healthy adults by measuring its expression with the quantitative NASBA assay between 5.50 x 10(4) copies/microg RNA and 6.76 x 10(5) copies/microg RNA. The new reference intervals were evaluated using a number of sensitive or resistant cell lines as control; positive or negative MDR1 expression was clearly demonstrated. We also reevaluated MDR1 expression levels in leukemia cells obtained from patient peripheral blood; 18 of 31 samples (58%) exceeded the newly established upper reference limit. The cutoff value established could be used to distinguish significant MDR1 expression in tumor cells from physiologic expression in certain normal cells coexistent in peripheral blood.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

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.

Adult↗
PubMed2003

A NASBA method to detect high- and low-pathogenicity H5 avian influenza viruses.

Nucleic acid sequence-based amplification (NASBA) allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique was developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The described NASBA technique is a specific, rapid, and sensitive method of detection of influenza A subtype H5 viruses. More importantly, it can be used to distinguish high- and low-pathogenicity strains of the H5 subtype.

Allantois↗
PubMed2003

RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza.

A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected with low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard.

Allantois↗
PubMed2004

Multiparametric duplex real-time nucleic acid sequence-based amplification assay for mRNA profiling.

Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method. We have developed real-time NASBA assays to detect mRNA coding for the estrogen receptor alpha (ESR1) and the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB) as the normalizing gene. Both the ESR1/PPIB and PGR/PPIB duplex NASBA assays are highly sensitive, specific, and reproducible. Quantification is determined using external standard calibration curves and the ratio between the number of target and housekeeping gene mRNA copies. Amplification of the target gene in the duplex NASBA assay was disrupted when this latter was mixed with a large amount of the housekeeping PPIB gene, suggesting that it is preferable for the normalizing gene chosen to have an expression level comparable to the target gene. Sensitivity and robustness of the duplex NASBA assays were assessed in breast cancer cell lines. Such a rapid and easy-to-use multiparametric duplex real-time NASBA assay could also advantageously be set up for other mRNA profiling applications.

Biological Assay↗
PubMed2006

Effectiveness of dual and triple antiretroviral therapy in the treatment of HIV-infected children.

OBJECTIVE: The use of antiretroviral therapy in HIV-infected children has been a widely discussed issue. The aim of this study was to compare the effectiveness of dual nucleoside analogue reverse transcriptase inhibitor (NRTI) regimens and three-drug regimens [2NRTI+ non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI)] in a cohort of HIV-infected children. METHODS: The study was carried out in a referral center for the management of infected children, which is affiliated with the School of Medicine of Universidade Federal de Minas Gerais (UFMG). Those children whose antiretroviral therapy was implemented between January 1998 and December 2000 and who were followed until December 2001 were included in the study. Therapeutic failure or death was regarded as the endpoint in our analysis. RESULTS: A total of 101 patients were assessed, 58 (57.4%) on dual therapy and 43 (42.6%) on triple therapy. No statistically significant difference was observed between the groups in terms of gender, age, CD4+ count and baseline viral load. The average duration of dual therapy was 26.3 months (95%CI 21.3-31.3) and that of triple therapy was 34.3 months (95%CI 29.2-39.5%). There was therapeutic failure in 33 (56.9%) patients on dual therapy and in 11 (25.6%) patients on triple therapy (log rank = 5.03; p = 0.025). The relative risk of therapeutic failure of the dual therapy was 2.2 times higher (95%CI 1.3-3.9). The percentage of initial CD4+ T cells was a predictor of risk for therapeutic failure (p = 0.001). Patients on triple therapy showed a more remarkable reduction in their viral load (p = 0.001). CONCLUSION: Triple therapy was efficient for a longer time period and showed better virologic response than dual therapy in this cohort of HIV-infected children. Therefore, triple therapy should be the treatment of choice.

Acquired Immunodeficiency Syndrome↗
PubMed2006

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.

Animals↗
PubMed2002

A nucleic acid sequence-based amplification method to detect Salmonella enterica serotype enteritidis strain PT4 in liquid whole egg.

Nucleic acid sequence-based amplification (NASBA) was applied to the detection of Salmonella enterica cells in liquid whole egg. Samples (25 g) of liquid whole egg inoculated with Salmonella cells were enriched for 16 h in buffered peptone water, and the NASBA procedure was effective in detecting the presence of Salmonella in samples inoculated with 10 to 100 CFU prior to enrichment.

Animals↗
PubMed2002

Significant differences between plasma HIV-1 RNA assays in HIV-1 subtype E infected patients treated with antiretroviral therapy.

UNLABELLED: A total of 72 HIV-1 infected Thai patients treated with didanosine (ddI) or stavudine (d4T) plus ddI at the time of interim analysis were analyzed. Sixty patients (83%) carried subtype E documented by HIV-1 V3 serotyping. HIV-1 RNA levels were measured using three commercial viral load assays. At baseline (n = 57), Quantiplex 2.0 and NucliSens 2.0 showed mean log10 HIV-1 RNA of 0.7 log10 or 5 fold lower than Amplicor 1.5 (mean 4.29 versus 5.0 log10, respectively, p < 0.001). At week 20 of treatment (n = 29), HIV-1 RNA levels were detected in 55.2%, 31%, and 33.5% of subjects tested by Amplicor 1.5, Quantiplex 2.0, and NucliSens 2.0, respectively. IN CONCLUSION: plasma HIV-1 RNA analyses showed comparable values with Quantiplex 2.0 and NucliSens 2.0 assays. In contrast, Amplicor 1.5 resulted in approximately 5 folds higher HIV-1 RNA levels and a 25% higher rate of detection of plasma HIV-1 RNA as compared to the other two assays. As the current goal of therapy is to suppress plasma viral load below the detection limit of the assays, the significant differences between the assays may influence antiretroviral efficacy evaluation and management.

Adult↗
PubMed2004

Once-daily saquinavir and ritonavir in treatment-experienced HIV-1-infected individuals.

To assess the efficacy of 48 weeks' treatment with saquinavir 1600 mg and ritonavir 100 mg, both given once daily (SQVOD), in drug-experienced HIV-infected patients, a SQVOD-based therapy was offered to 100 treatment-experienced patients via their own physicians. The patients starting this regimen were followed up for 48 weeks. HIV-RNA was assessed by means of NASBA (limit of quantification = 80 copies/mL). Fifteen patients received the SQVOD-based therapy. Six discontinued before week 48 because of failure, toxicity or intolerance due to the high pill burden and gastrointestinal side effects. The median baseline CD4+ cell counts and plasma HIV-RNA levels were 317 cells/microL (range 44-698) and 4.18 log copies/mL (range 2.65-6.18). At week 4, there was a mean decrease of 1.96 log copies/mL (P < 0.0001) in HIV-RNA, with 75% of the patients having fewer than 400 copies/mL; seven of the nine patients treated for 48 weeks reached fewer than 400 copies/mL. No substantial change in cholesterol or triglyceride values was observed over 48 weeks. As this SQVOD-based regimen had considerable short-term virologic activity in treatment-experienced HIV-infected patients, it may be a reasonable option when non-nucleoside reverse transcriptase inhibitors cannot be administered and once-daily dosing is preferred by the patient. However, the high pill burden and frequent gastrointestinal side effects of the soft gel capsule formulation of saquinavir may limit its long-term efficacy.

Adult↗
PubMed2003

Molecular techniques to detect and identify protozoan parasites in the environment.

The environmental route of transmission is important for many protozoan and helminth parasites, with water, soil and food being particularly significant. Both the potential for producing large numbers of transmissive stages and their environmental robustness pose persistent threats to public and veterinary health. The introduction of molecular techniques, in particular those based on the amplification of nucleic acids, has provided researchers with highly sensitive and specific assays for the detection and identification of these pathogens. The application of these techniques to clinical, environmental, and food samples is instrumental for a thorough understanding of the epidemiology of the infection and for the implementation of control measures. Here, the advantages and drawbacks of some molecular techniques (Polymerase Chain Reaction--PCR; Reverse-Transcriptase PCR--RT-PCR; Real-time PCR--qPCR; Nucleic Acid Sequence-Based Amplification--NASBA) will be briefly reviewed. Some application of these techniques will be illustrated with reference to two important and widespread human parasites, the apicomplexan Cryptosporidium and the flagellate Giardia.

Animals↗
PubMed2006

[Study on Rotavirus detection by nucleic acid sequence based amplification].

Rotavirus is one of the major cause of the viral gastroenteritis throughout the world. A nucleic acid sequence-based amplification(NASBA) technique for the detection of rotavirus in faecal specimens was developed and compared to the RT-PCR technique. Primers were designed according to the high conserved region of VP7 gene. Amplicons were detected by denaturating agarose gel, and then demonstrated by Northern hybridization with digoxigenin-labeled oligonucleotide probe. The anticipative specific amplification product is 392bp,and no nonspecific products appear even the concentration of nontarget nucleic acid as high as 1 microg/microL. A detection limit of 50 pg target RNA/mL is obtained when the optimal amplification time of 3h used.The NASBA assay will be a favourable alternative to RT-PCR for the investigation of rotavirus outbreaks as a routine diagnostic test in the near future because it is shown to be highly sensitive, specific and do not require specialized equipment.

DNA Primers↗
PubMed2006

Detection of animal viruses using nucleic acid sequence-based amplification (NASBA).

As seen in recent avian influenza outbreaks in Asia, prevention is the key to fighting infectious disease successfully. Efficient disease surveillance systems on the basis of molecular diagnostics will help monitor the emergence of viruses in the early stage and thus prompt containment measures can be in place to minimize disease spread. Here we describe and review molecular diagnostics focusing on nucleic acid sequence-based amplification (NASBA) technology in detecting viruses causing animal diseases, such as avian influenza, foot-and-mouth disease, and Newcastle disease. NASBA offers high sensitivity, specificity, accuracy, and speed of availability of results, and NASBA would be the most applicable molecular diagnostics for disease surveillance and control.

Animal Diseases↗
PubMed2006

Practical experiences of moving molecular diagnostics into routine use at the Veterinary Laboratories Agency.

The practical bench application of molecular tests (here defined as nucleic acid-based tests) in a routine testing situation is not without its challenges. This paper outlines the approaches we take at the Veterinary Laboratories Agency (VLA) and highlights some of the practical issues which we have found to be important for the successful introduction and use of molecular tests in a routine testing environment. The potential advantages of molecular tests, and factors which dictate which tests are adopted for routine testing, are discussed before giving some examples of molecular tests in routine use at the VLA. The instrumentation, reagents and assays we use are outlined, followed by sections of how we approach validation and how we manage and resource the transfer of molecular tests into routine use.

Laboratories↗
PubMed2006

[Application of nucleic acid sequence-based amplification in detection of cytomegalovirus mRNA of recipients after allogeneic peripheral blood stem cell transplantation].

The study was purposed to investigate diagnostic value of late-mRNA detection by nucleic acid sequence-based amplification (NASBA) technique for human cytomegalovirus (HCMV) infection of the recipients after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and to evaluate the clinical significance for guiding antiviral therapy. 352 samples were collected from 128 transplant patients after allo-PBSCT. A molecular biological diagnostic technique--NASBA was used to detect human cytomegalovirus (HCMV) late mRNA encoding the viral structural protein PP67 (UL65) expression in peripheral blood of recipients after allo-PBSCT, and the detected results were compared with HCMV DNA detection by PCR. The sensitivity, specificity and early diagnostic value of HCMV mRNA detection for HCMV disease were evaluated. The results showed that out of 352 detected blood specimens from 84 patients 183 specimens (51.99%) were positive of HCMV DNA by PCR, 105 specimens (29.83%) were positive of HCMV mRNA by NASBA. 45 patients were infected by HCMV. The sensitivity and specificity of HCMV DNA and HCMV mRNA for detecting HCMV disease were 95.56% (43/45), 93.33% (42/45) and 60.24% (50/83), 97.59% (81/83). The results of specificity showed significant difference between two groups of HCMV mRNA and HCMV DNA (P < 0.05). It is concluded that the detection of late-mRNA of HCMV by NASBA technique is rapid, sensitive and specific detection for HCMV active infection. The detected result correlates with clinical symptoms. It can monitor HCMV infection of allo-PBSCT transplanted recipients and provide indication to antiviral therapy.

Adolescent↗