Transcription-based Amplification System Research Articles

The new frontier of diagnostics: Molecular assays and their role in infection prevention and control.

Recent advances in technology over the last decade have propelled the microbiology laboratory into a pivotal role in infection prevention and control. The rapid adaptation of molecular technologies to the field of clinical microbiology now greatly influences infectious disease management and significantly impacts infection control practices. This review discusses recent developments in molecular techniques in the diagnosis of infectious diseases. It describes the basic concepts of molecular assays, discusses their advantages and limitations, and characterizes currently available commercial assays with respect to cost, interpretive requirements, and clinical utility.

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

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.

New nucleic acid amplification techniques in diagnostic pathology

AbstractIn 1985, Mullis discovered an exponentially increasing amplification system for nucleic acids, which became known as the polymerase chain reaction (PCR). This method permits the selective in vitro amplification of a particular DNA region by mimicking phenomena of in vivo DNA replication. Since then, many advances in PCR practice have been achieved, In this review, we will discuss the new techniques of in situ PCR, ligase chain reaction (LCR), transcription‐based amplification system (TAS) and the isothermal replication system, self‐sustained sequence replication (3SR). Many of the techniques described still remain largely research tools, however with further modifications they may become of use in routine diagnostic procedures in the next century.

RPCR: a powerful tool for random amplification of whole RNA sequences.

T h e polymerase chain reaction (PCR), at first used to amplify known DNA sequences present at very low levels in genomic DNA samples, ~1-3) was rapidly extended to amplif icat ion of RNA sequences by the use of cDNA. ~4's) The PCR method, which requires mult iple heat-denaturation steps and was originally established with the Klenow fragment of Escherichia coli DNA polymerase I, now employs thermostable forms of DNA polymerase and two gene-specific primers (GSP) that flank the DNA segment to be amplified. Although PCR is the most widely utilized amplif icat ion protocol, known RNA sequences can be amplified alternatively with a RNA transcription-based amplif icat ion system (TAS), ~6'7) changing the final product from double-stranded DNA to singlestranded RNA. The TAS reaction needs two GSP, with the downstream GSP linked to a RNA polymerase (T7, T3, or SP6) b inding site, and is carried out by using sequential cDNA synthesis and RNA transcription, which are displayed as seven steps, including three heat-denaturation steps. Since the original description of the TAS protocol, the reaction conditions have been modif ied to achieve more productive and quantitative amplification. Thus, the self-sustained sequence replication (3SR) ~8,9) reaction was modeled after the general scheme employed during retroviral replication and does not require heat-denaturation; 108 copies of each target molecule can be produced in less than 1 hr with all three enzyme activities from avian myeloblastosis virus reverse transcriptase, Escherichia coli RNase H, and T7 RNA polymerase operating in an efficient and concerted manner . Other protocols such as rapid amplification of cDNA ends (RACE) ~°-12) use only one GSP and permit amplif icat ion of a region between a single known short sequence in a cDNA molecule and its unknown 3' or 5' end. Also described as one-sided PCR ~13) or anchored PCR, ~4) RACE 3'-end and 5'-end procedures are performed by using a homopolymer ic nucleotide containing an adapter primer that anneals to the cDNA's 3' or 5' termin i and one GSP. RACE using a randomly primed first-strand cDNA has also been reported recently, ~1s,16) with the 3' extension technique assuming the amplification of DNA fragments localized both in large 3' unsequenced regions and downstream to a 5' known sequence. Last, an elegant method that employs head-to-tail ligation of specifically primed, single-stranded, firststrand cDNAs with RNA ligase followed by PCR amplification ~17) was developed to yield 5' double-stranded cDNA copies in sufficient abundance for cloning; although there is actually no available data about the efficiency of this technique, it has the potential advantage of c ircumventing the usual problems associated with the homopolymeric tailing.

Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR.

Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR.

Methodologies for in vitro nucleic acid amplification and their applications

The capability to detect the genetic elements (DNA or RNA) of a particular pathogen as a means of identifying the infectious agent has been the traditional function of nucleic acid hybridization assays. The low copy number of genetic material from several types of viral pathogens has fostered the development of in vitro nucleic acid amplification methods as a means to increase the copy number of the characteristic genetic elements of pathogenic agents. The polymerase chain reaction (PCR) and a transcription-based amplification system (TAS) are two amplification methods that have been developed to serve this function. Both methods have been employed to study both genetic and infectious disease problems. This review discusses the characteristics of these amplification methods and describes some of their applications, especially in the study of HIV-1.

Detection of Human Immunodeficiency Virus Type 1 in AIDS Patients Using Amplification-Mediated Hybridization Analyses: Reproducibility and Quantitative Limitations

Eighty-six peripheral blood mononuclear cell (PBMC) samples from 30 patients with AIDS were analyzed using a transcription-based amplification system (TAS) and the polymerase chain reaction (PCR). Human immunodeficiency virus type 1 (HIV-1) sequences were detected by amplification-mediated hybridization in 98% of the samples, 52% of which were positive for p24 antigen by ELISA. Neither TAS (93%) nor PCR (95%) detected HIV-1 sequences in all 86 samples. The hybridization-detection methods (slot blot, bead-based sandwich, and solution) used to detect the HIV-1-specific TAS products had a clear influence on the efficiency of detecting and quantitating the levels of HIV-1 present in these samples. The reproducibility of amplification of constant amounts of HIV-1 RNA and beta-globin DNA by TAS and PCR was studied over 3 months. The results indicated that variations of 10- and 5-fold in the HIV-1 sequence levels could be detected between samples by TAS and PCR, respectively. Within the range of sensitivities for each assay used, the administration of zidovudine did not appear to reduce the amount of HIV-1 nucleic acid sequences as observed in PBMC obtained serially from six AIDS patients.

Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format.

The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 10(6)-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 10(6) uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.