Sensitive Reverse Transcription Polymerase Chain Reaction Assay Research Articles

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used.In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes.The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy.

Analytic and Clinical Validation of a Prostate Cancer–Enhanced Messenger RNA Detection Assay in Whole Blood as a Prognostic Biomarker for Survival

BackgroundBiomarkers based on detecting prostate cancer (PCa)-specific transcripts in blood are associated with inferior outcomes, but their validation in a clinical context is lacking. ObjectiveTo determine whether detecting enhanced transcripts for PCa in whole blood using an analytically valid assay has prognostic significance relative to circulating tumor cell (CTC) enumeration. Design, setting, and participantsThe detection of KLK3, KLK2, HOXB13, GRHL2, and FOXA1 in whole blood by reverse transcription polymerase chain reaction (RT-PCR) was studied in 97 men with metastatic castration-resistant PCa (mCRPC) as a prognostic factor for overall survival. InterventionThe 2.5ml of blood was collected in PAXgene tubes for total RNA extraction and 7.5ml for CTC enumeration from patients with progressive mCRPC. Outcome measurements and statistical analysisPCa-enriched genes were detected using a sensitive RT-PCR assay in whole blood from patients with mCRPC. Analytical validity of the assay was established in a clinical laboratory environment. The frequency of detecting transcripts was compared to CTC enumeration using CellSearch in an independent data set and survival associations were explored by concordance probability estimate (CPE). Results and limitationsTwo or more genes were detected by PCR in 53% of patients (51 of 97; 95% confidence interval [CI], 43–63%), and unfavorable CTC counts (five of more cells) were seen in 46% (45 of 97; 95% CI, 36–56%). Importantly, transcripts were detectable in 11 of 52 patients with favorable CTC counts (21%; 95% CI, 8–35%). Transcript detection predicted overall survival in a proportional hazards model. Significantly, the predictive accuracy of RT-PCR detection in combination with CTC enumeration had a CPE of 0.752 (standard error: 0.038), although this was limited by the number of patients evaluated. ConclusionsThis validated RT-PCR assay detecting prostate-specific RNA in whole blood is prognostic for survival and may assess patient risk in tandem with CellSearch CTC enumeration. Its clinical utility is being prospectively explored.

Dermatofibrosarcoma protuberans COL1A1-PDGFB fusion is identified in virtually all dermatofibrosarcoma protuberans cases when investigated by newly developed multiplex reverse transcription polymerase chain reaction and fluorescence in situ hybridization assays

Dermatofibrosarcoma protuberans (DFSP) is a cutaneous, locally aggressive spindle cell tumor of intermediate malignancy. Tumor cells are reactive for CD34 and characterized by a t(17;22) translocation or a supernumerary ring chromosome that results in the fusion of exon 2 of PDGFB to various exons of the COL1A1 gene. We developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay to detect fusion transcripts for all possible COL1A1 breakpoints. Twenty-seven formalin-fixed, paraffin-embedded DFSP cases were analyzed using 18 COL1A1 forward primers and 1 exon 2 PDGFB reverse primer. Sequence analysis was performed to definitively characterize breakpoints. Results were correlated with histology, immunohistochemistry, PDGFB break-apart fluorescence in situ hybridization analysis, and cytogenetics when available. Fusion transcripts were detected by RT-PCR in all but one DFSP case. Sequencing revealed a PDGFB exon 2 breakpoint in all cases. COL1A1 breakpoints were in exons 7 (1 patient), 10 (1), 29 (2), 40 (1), 46 (3), and 49 (2), and intronic between exons 13:14 (1), 26:27 (2), 30:31 (1) 33:34 (1), 43:44 (7), 45:46 (1), and 46:47 (1). Three novel COL1A1 breakpoints were identified, intronic between exons 13:14 (1), 30:31 (1) and in exon 49 (2). There was no correlation found between breakpoints and age, sex, or histologic variants. Using this sensitive multiplex RT-PCR assay in combination with fluorescence in situ hybridization, we found COL1A1-PDGFB rearrangements appear more prevalent in DFSP than previously reported. Its detection may be particularly helpful in the differential diagnosis of atypical, fibrosarcomatous, and metastatic DFSP.

Development of a multiplex RT-PCR to detect transcription of varicella–zoster virus encoded genes

The reverse transcription polymerase chain reaction (RT-PCR) is used commonly to analyse transcription of genes. In the field of virology it is an extremely helpful method to analyse the transcriptional activity of both, DNA and RNA viruses. The standard RT-PCR allows an investigation of the activity of only one gene. Here, we describe the development of a sensitive multiplex RT-PCR assay for single tube amplification to analyse a set of different genes encoded by the varicella–zoster virus (VZV), a member of the human-pathogenous herpesviruses. This multiplex RT-PCR amplifies the genes ORF 4, ORF 21 and ORF 68; each of these three genes belongs to a distinct class of genes, which is expressed one after the other within the infectious cycle. This method provides the possibility for rapid but extensive examination of VZV transcriptional activity and could be used in both fields, fundamental research as well as clinical diagnostic work.

Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid leukemia may indicate risk for clinical relapse and may identify patients in a curable state

AbstractThe inv(16) cytogenetic subtype of acute myeloid leukemia (AML) has a relatively good prognosis. Many patients achieve complete remission (CR). The prognostic uncertainty of negative qualitative reverse transcription–polymerase chain reaction (RT-PCR) assays suggests the need to identify prognostically significant critical thresholds by real-time RT-PCR. A reliable and sensitive (10−5) real-time RT-PCR assay was set up for the evaluation of relevant CBFβ-MYH11/ABL transcript ratios and was applied to the 21 patients with inv(16) AML routinely referred for cytogenetic and molecular monitoring in Seràgnoli Institute (Bologna, Italy) since 1990. Among the 18 patients who underwent ablative chemotherapy, all achieved CR with a 3-year disease-free survival probability of 63% (95% CI, 40%-87%) and no recorded events after 26 months. Five patients had relapses; 2 died of disease and 3 entered second CR. Analysis of the 125 bone marrow (or peripheral blood) samples studied by real-time RT-PCR showed that transcript ratios of samples taken during CR at any time before a relapse were always greater than 0.12%, whereas those of samples taken during first or second CR from patients who did not subsequently have relapses were always less than 0.25%. This suggests that transcript ratios greater than 0.25% may correspond to high risk for relapse, whereas ratios below 0.12% might indicate the patient is in a curable state. If confirmed, such thresholds could open the way to a new phase in post-CR therapeutic decision making for patients with inv(16) AML.

Specific RT-PCR procedure for the detection of human parechovirus type 1 genome in clinical samples

Among the new genera of Parechoviruses, human parechovirus type 1, formerly ECHO virus 22, has recently been recognized on the basis of distinctive biological and molecular properties. This human pathogen generally causes mild gastro-enteritis, respiratory infections and is also responsible for central nervous system infections. To ensure reliable detection, these latter infections are diagnosed by using reverse transcription polymerase chain reaction (RT-PCR) procedures. Due to genetic differences, human parechovirus type 1 genome is not detected with an enterovirus specific RT-PCR procedure. Hence, a rapid, specific and sensitive RT-PCR assay was developed that can be used for the detection of human parechovirus type 1 in clinical samples.

Highly sensitive one-tube RT-PCR and microplate hybridisation assay for the detection and for the discrimination of classical swine fever virus from other pestiviruses

Rapid, sensitive and specific laboratory diagnostic methods are necessary to confirm outbreaks of classical swine fever. The detection of classical swine fever virus (CSFV) and its discrimination from other pestiviruses can be achieved by virus isolation on cell culture, antigen detection, or molecular methods. To reduce the time and the number of steps in the diagnostic procedure a sensitive and rapid detection method based on specific amplification of the pestiviral RNA by one-step reverse transcription-polymerase chain reaction (RT-PCR) followed by detection and differentiation of the amplification products by pestivirus-, bovine viral diarrhoea virus- (BVDV-) and CSFV-specific capture probe hybridisation and colorimetric assay in microwell plates (enzyme liked immunosorbent assay (ELISA)) was developed. Two different methods using two gene regions for pestivirus RT-PCR amplification were carried out. One pair of primers was selected from the 5′-UTR region and the second one from the gene region coding for N pro, C and E0 proteins. The designed oligonucleotide primers were used for several pestivirus reference strains as well as for some field isolates detection in cell culture supernatants and in clinical specimens. The specificity and sensitivity of both methods were compared using EZ r Tth RNA PCR kit and ACCESS RT-PCR system for combined RT-PCR assay. The use of one-step RT-PCR eliminates the additional manipulations that are generally required for a two reaction system and limits the risk of carry-over contamination. Labelling of PCR products with digoxigenin (DIG) during the amplification reaction enables colorimetric assessment of hybridisation reactions. For solution hybridisation pestivirus-, BVDV- and CSFV-specific biotin-labelled capture probes were used. By serial dilutions of DIG-labelled PCR products the RT-PCR-ELISA was found to be 100-times more sensitive than the conventional agarose gel electrophoresis. Higher sensitivity of RT-PCR-ELISA detection using specific biotin-labelled probes offers the opportunity to eliminate strain specific nested PCR and to overcome the problems with contamination and false positive results.

Quantification of porcine cytokine gene expression using RT-PCR, a homologous internal control and chemiluminescence for microplate detection

The polymerase chain reaction (PCR) has proved to be a sensitive and versatile method for the analysis of human and murine cytokine mRNA expression. This paper describes for the first time a reverse transcription-polymerase chain reaction (RT-PCR) at end-point for the quantification of five porcine cytokines: interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-10 and IL-18. The main features of the methodology are: (1) a unique RT for all quantifications, (2) the addition of homologous DNA internal controls (IC) of equal length to the corresponding cytokine and consequently co-amplification of the target cytokine and the IC with equivalent efficacy, (3) PCR and detection of amplicons for all cytokines simultaneously, (4) cytokine quantification in relation to a housekeeping gene control (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), (5) detection of the amplicons by enzyme linked immunosorbent assay (ELISA) using a chemiluminescent substrate with high sensitivity and wide dynamic range, (6) automation of the detection system for analysis of a large number of samples. This highly sensitive quantitative RT-PCR assay (able to detect 100–200 cytokines mRNA copies/75×10 3 cells) was validated on peripheral blood mononuclear cells (PBMC) from pigs infected or not with pseudorabies virus (PRV), re-stimulated in vitro by a mitogen or antigens.

A mutation (IVS8+0.6kbdelTC) creating a new donor splice site activates a cryptic exon in an Alu-element in intron 8 of the human beta-glucuronidase gene.

We have previously sequenced the complete coding region and the promoter region of the beta-glucuronidase gene of a patient with mild mucopolysaccharidosis type VII (MPS VII) and identified a nonsense mutation in the gene inherited from her mother. The mutation inherited from her father was not found. Here, we have extended the sequence analysis of the introns to cover all putative lariat branch points and putative intronic enhancers, although no nucleotide changes have been found in these regions. Careful analysis of mRNA structure by reverse transcription/polymerase chain reaction (RT-PCR) and direct sequencing has revealed the inclusion of a new exon derived from an antisense Alu-repeat in intron 8 and the skipping of exon 9 in a large proportion of the mRNA of our patient. A 2-bp deletion creating a strong 5'-splice site has subsequently been identified in the paternal gene of the patient (IVS8+0.6kbdelTC). With a sensitive RT-PCR assay, we demonstrate that both the inclusion of the Alu-cassette and the skipping of exon 9 are minor events in control samples and that mRNA with both alterations is only found in the IVS8+0.6kbdelTC carrier. The increased proportion of exon 9 skipping seems to be related to the premature termination of translation. This is the third report of a human disease mutation that creates a splice site and activates an antisense Alu-cassette; the question rises as to how these apparently strong cryptic exons are generally excluded from coding sequences.

Basal cell carcinoma development is associated with induction of the expression of the transcription factor Gli-1.

Recent work has established that activation of Hedgehog/patched signalling plays a key role in the development of basal cell carcinomas (BCCs). In Drosophila the effects of hedgehog signalling are mediated by the transcription factor Cubitus interruptus, which is homologous to the mammalian Gli family of transcription factors. In order to investigate the downstream consequences of patched gene inactivation in BCCs, we have investigated the expression of Gli-1 and Gli-3 in normal skin and BCCs by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Gli-3 was found to be expressed in both normal skin and BCCs by both RT-PCR and in situ hybridization using a Gli-3-specific probe. Using a sensitive RT-PCR assay we were unable to detect Gli-1 transcripts in normal skin. Gli-1 was expressed in 13 of 14 BCCs examined, and in situ hybridization confirmed that the transcripts were localized to the epithelial component of the tumours. Our results demonstrate that inactivation of the patched gene BCCs is associated with the accumulation of Gli-1 transcripts. These findings suggest that the Gli-1 transcription factor plays a key role in BCC development.

Gene expression of steroid hormone receptors in brain and peripheral tissues relative to reproduction.

In order to examine sex steroid hormone receptor expression in the central and peripheral tissues relative to reproduction, we have analysed estrogen (ER) and progestin receptors (PR) and their mRNAs in rat brain, and human uterine and other tissues, using immunocytochemical assay (ICA), reverse transcrip-tion-polymerase chain reaction (RT-PCR) and in situ hybridization technique (ISH) Summary and conclusion are as follows;1) ICA studies confirmed nuclear localization of ER or PR in human uterus and ovarian endometriosis. Good correlation exists between the ER-ICA and ligand binding assay (LH20 assay) or ER-enzyme immunoassay (EIA), indicating the usefulness of the ER-ICA for the semiquantitative measurement in humanuterine tissues. The ER score of ovarian endometriotic tissue was much less than that of the normal endometrium, suggesting much lower hormonal responsiveness in the pathologic tissue.2) ERmRNA analysis:(1) Northern blot analysis using rat ERcRNA probe demonstrated 6.6 kb ERmRNA in rat brain (the hypothalamus-preoptic area, HPOA), anterior hypophysis (AP), uterus, tube, ovary, and testis. Additional subspecies of 4.2, 2.6 and 2.0 kb were identified in the testis.(2) A very sensitive and specific RT-PCR assay for ERmRNA was developed to analyse the receptor ex-pression in more detail. The RT-PCR product 287 bp was generated from tissue RNA using the primer set derived from the rat ERcDNA sequence and its authenticity was confirmed by direct sequencing. Quan-tification of the RT-PCR assay was made possible by measurement of the hybridization signals in a Bio-Im-age Analysing System, BAS-2000. A standard curve was obtained from graded dilutions of the uterine RT-PCR products.The levels of ERmRNA were as follows; AP>HPOA, amygdala (AMY) >cerebral cortex (CC), cerebellum (Ce). The existence of a small amount of ERmRNA in the “non-target” CC and Ce implied adirect action of estrogen on these brain regions.3) PRmRNA analysis:In analogous experiments as ERmRNA, a very sensitive and specific RT-PCR assay for PRmRNA was also developed to detect and quantify PRcDNA in the tissues. Since rat PRcDNA has been not cloned and sequenced, rat PRmRNA was synthesized with the primer set derived from the human PRcDNA sequence. The RT-PCR product of 320 bp was generated from tissue RNA, and the authenticity of the rat uterine product was confirmed by direct sequencing.The levels of PRmRNA in adult rat brain were as follows; AP, HPOA, AMY, CC>Ce, with smaller differential distribution than that of ERmRNA, and roughly paralled the levels of PR with exception of the CC where the PR level is low. The expression mechanism of PR in the CC may be different from that in the other brain regions.The early postnatal development of PRmRNA was found. The increment of the cerebral PRmRNA might be associated with the early drastic increase of PR in the neonatal rat brain cortex.4) ER-and PRmRNAs analysis in human uterus:(1) Primary structure of the uterine ERmRNA, amplified by the RT-PCR and determined by direct sequencing of the product, showed that ERmRNA expressed in the normal human endometrium has Gly-400 (GGG) and Thr-594 (ACA).(2) Northern blot analysis of ER-and PRmRNAs using riboprobes demonstrated greater message levels in the endometrium than the myometrium, respectively. The results were in agreement with estrogendependent induction of PR.5) In situ hybridization studies.We have detected ER- and PRmRNAs and mapped out the distribution of the meassages in the intact female rat brain using rat ER- and PRcRNA synthetized. The distribution of both mRNAs was similar, with higher density in the arcuate nucleus and the ventrolateral part of the ventromedial nucleus of hypothalamus, indicating a possible coexpression in the same neurons.