Reverse Transcription-polymerase Chain Reaction Reaction Research Articles

Iris yellow spot virus: A New Onion Disease in Spain.

So far, only three viral diseases have been identified in onion crops grown in Spain. These are Tomato spotted wilt virus (TSWV), Onion yellow dwarf virus (OYDV), and Leek yellow stripe virus (LYSV). In September 2003, unusual virus-like symptoms including straw-colored, dry, tan, diamond-shaped lesions on the leaves and stalks, sometimes with necrotic lesions, curled leaves, and bulbs of reduced size, were observed on several onion plants (Allium cepa L.) in commercial fields in Albacete, Spain. Severely affected plants eventually died. To verify the identity of the disease found in the Spanish onions, double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was performed on leaf extracts of symptomatic onions using specific polyclonal antibodies against OYDV, LYSV, Cucumber mosaic virus (CMV) (Biorad Phyto-Diagnostics, Marnes-La Coquette, France), Iris yellow spot virus (IYSV), and TSWV (Loewe Biochemica, Sauerlach, Germany). All samples of infected onion tissue were positive for IYSV and negative for the other viruses tested. To confirm the ELISA results, viral RNA was extracted from five of the ELISA-positive onion samples, a healthy onion plant, and a positive control for IYSV (DSMZ, Braunschweig. Germany). The extracted RNA was used in a One-Step reverse transcription-polymerase chain reaction (RT-PCR) assay using SuperScript Platinum Taq (Invitrogen Life Technologies, Barcelona, Spain) in the presence of the IYSV1S and IYSV1A primers for the nucleocapsid gene of IYSV (1). The RT-PCR assay produced an amplicon of the expected size of 790 bp. No amplification products were observed when healthy plants or a water control were used as templates in the RT-PCR reaction. To establish the authenticity of the virus from onion, the PCR products were purified (High Pure PCR Product Purification Kit, Roche Diagnostics, Mannheim, Germany), sequenced, and the nucleotide sequences obtained were analyzed and compared with the published sequences in GenBank. The PCR product was 97% identical to the sequence of the IYSV nucleocapsid gene (Genbank Accession No. AB121026). IYSV, an emerging tospovirus that is potentially a devastating pathogen of onion, has been reported in many locations in Brazil, Japan, the Netherlands, Israel, Australia, the western United States, Slovenia, and Iran (2). IYSV is included in the European and Mediterranean Plant Protection Organization alert list of viruses (2), and to our knowledge, this is the first report of IYSV in Spain. This tospovirus is transmitted in a propagative manner by Thrips tabaci. Although the vector is present in large populations in the onion-growing areas in Spain, the efficiency of the Mediterranean ecotype in transmitting IYSV is not known.

RT–PCR‐Based Cloning of the Human BETA‐Amyloid Precursor Protein Gene and the Construction of its Expression Plasmids

Beta‐amyloid peptide (βA) is a major fibrillar component of neuritic plaques in Alzheimer's diseased brains and is related to the pathogenesis of the disease. βA generation depends on proteolytic cleavage of the amyloid precursor protein (APP). Obtaining full‐length APP is essential for the identification of biological activities that occur in the APP molecule; full‐length APP is also essential for the identification of proteases capable of creating βA as well as the factors that regulate the levels of βA. The present work offers a new approach that makes it easier for any laboratory interested in Alzheimer's disease research to obtain full‐length recombinant APP. Presented herein is the development of a new procedure for the cloning of human βA precursor protein gene (human APP gene) based on the reverse transcription (RT) and the polymerase chain reaction (PCR). This procedure for cloning human APP gene by means of RT–PCR reactions is cost‐effective, not time‐consuming, and is suited for any laboratory interested in research related to Alzheimer's disease. The present work also includes a new procedure for the construction of expression plasmids, (a) using the pFastBac™ HTb and the pBlueBacHis2 A transfer vectors for the purpose of obtaining human APP in insect cells; and (b) using the pET‐28a (+) transfer vector for the purpose of obtaining human APP in bacteria. The development of this new procedure is a significant contribution to the field in terms of augmenting the availability of full‐length APP critical for the exploration of therapeutic and preventative strategies toward the treatment of Alzheimer's disease.

First Report of Cucurbit aphid-borne yellows virus in Spain.

In late spring 2003, field-grown melon plants (Cucumis melo L.) showing bright yellowing of older leaves were observed near Valladolises in Campo de Cartagena, Murcia, Spain. Symptoms resembled those caused by viruses of the genus Crinivirus (family Closteroviridae), but absence or very low populations of whiteflies were observed. However, diseased foci showed clear indications of heavy aphid infestations. Later, during the fall of 2003, squash plants (Cucurbita pepo L.) grown in open fields in the same area showed similar symptoms. Tissue print hybridizations to detect Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo yellows virus (BPYV) in symptomatic samples were negative. CYSDV and BPYV are two yellowing-inducing criniviruses previously described in Spain. In contrast, standard double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with antiserum against Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) that was kindly provided by H. Lecoq (INRA-Montfavet Cedex, France) were consistently positive. Definitive confirmation of CABYV associated with symptomatic samples was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for the CABYV coat protein gene. Total RNA extracts (TRI reagent; Sigma Chemical, St. Louis, MO) were obtained from symptomatic and asymptomatic leaf samples and RT-PCR reactions were carried out using the primers 5'-GAATACGGTCGCGGCTAGAAATC-3' (CE9) and 5'-CTATTTCGGGTTCTGGACCTGGC-3' (CE10) based on the CABYV sequence published by Guilley et al. (2). A single DNA product of approximately 600 bp was obtained only from symptomatic samples. Amplified DNA fragments from two independent samples (samples 36-2 and 37-5) were cloned in E. coli and sequenced (GenBank Accession Nos. AY529653 and AY529654). Sequence comparisons showed a 95% nucleotide sequence identity between the two sequences. A 97% and 94% nucleotide sequence identity was found among 36-2 and 37-5, respectively and the CABYV sequence published by Guilley et al. (2). CABYV seems to be widespread throughout the Mediterranean Basin (1,3) but to our knowledge, it has not previously been described in Spain. Additionally, our data suggest that significant genetic variability might be present in the Spanish CABYV populations.

Can the reverse transcriptase-polymerase chain reaction for prostate specific antigen and prostate specific membrane antigen improve staging and predict biochemical recurrence?

To evaluate the perioperative gene-specific primed nested reverse transcription-polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) for staging patients undergoing radical prostatectomy and predicting biochemical recurrence. In 80 consecutive patients undergoing radical prostatectomy for prostate cancer, blood samples were drawn before, during and 1 and 7 days after removing the prostate. After buffy coat and mRNA extraction, gene-specific primed nested RT-PCR was performed for PSA, PSMA and glyceraldehyde-3-phosphate dehydrogenase mRNA, and Southern blot analysis of the PCR reaction. The sensitivity of gene-specific RT-PCR to detect tumour cells was comparable with random primed RT-PCR. In the 80 patients the stage distribution was pT1 in two (2.5%), pT2 in 30 (37.5%) and > or = pT3 in 48 (60%); the nodal status was pN0 in 57 (71%), pN1 in 11 (14%) and pN2 in 12 (15%). The gene-specific RT-PCR reaction for PSA and PSMA was positive in no patients with pT1, 11 (37%) with pT2 and 23 (48%) with stage > or = pT3 disease. The result for PSA was positive in 12 (52%) and for PSMA in 11 (48%) of those with positive nodal status. Neither gene-specific RT-PCR for PSA or PSMA was able to predict organ-confined disease (P > 0.5). After a median (range) follow-up of 37 (11-67) months a biochemical recurrence was predicted in 65% of patients by preoperative RT-PCR for both PSA and PSMA, with a sensitivity, specificity, positive and negative predictive value of 58%, 80%, 87% and 47%, respectively; the assay after surgery predicted a recurrence in 73%, with respective values of 68%, 84%, 84% and 57%. Gene-specific primed nested RT-PCR for PSA and PSMA is a sensitive and simple assay; it might add substantial information for tumour staging in individual patients. RT-PCR before surgery allows the prediction of recurrence in 65% of cases and after surgery in 73%.

Simultaneous detection of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) by multiplex reverse transcription-polymerase chain reaction (RT-PCR) in pacific white shrimp Penaeus vannamei.

An assay using a single-tube, 1-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the simultaneous detection of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV). Three primer sets, 9195 F/9992 R, 94 F2/R2, and ITS F/28S R, were mixed at a ratio of 3:1:1 to amplify specific fragments of the TSV, WSSV, and Penaeus vannamei genome, respectively, in the RT-PCR reaction. Shrimp samples were experimentally infected with WSSV and TSV. PCR-amplified products detected in the nucleic acid extraction of shrimp pleopods produced 4 kinds of results. With no virus infection, 1 fragment of 892 base pairs (bp) was amplified from a ribosomal RNA gene by primer set ITS F/28S R as an internal control. In samples only infected by WSSV or TSV, 2 fragments could be seen: either from WSSV (530 bp) plus the internal control or TSV (231 bp) plus the internal control, respectively. In cases of co-infection with both viruses, all 3 amplified products were detected simultaneously. This study is the first report of Penaeus vannamei specimens co-infected with WSSV and TSV being detected using a PCR method via experimental infection.

Detection of Encephalomyocarditis virus in clinical samples by immunomagnetic separation and one-step RT-PCR

A method of immunomagnetic separation and one-step reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Encephalomyocarditis virus (EMCV). EMCV was captured from sample on magnetic beads with homologous monoclonal antibody and then heat denatured. The heated beads were used directly in one-step RT-PCR reaction to amplify a 285-bp PCR fragment at the 3′ end of the genomic region that encodes the viral polymerase. This method detected as little as 3.5 TCID 50 of EMCV from infected cell culture. It was shown with this method that the sensitivity of RT-PCR increased when applied for the detection of EMCV added to fecal extract. Using this protocol EMCV was detected from heart homogenate samples containing less than 100 TCID 50/ml. The amplified product was sequenced to ensure specificity. The immunomagnetic-RT/PCR procedure described here should be useful for the rapid, specific and sensitive detection of EMCV in clinical samples. This technique is rapid, reliable and can be readily adapted to detect EMCV from other clinical samples.

Simultaneous Detection of the Three Ilarviruses Affecting Stone Fruit Trees by Nonisotopic Molecular Hybridization and Multiplex Reverse-Transcription Polymerase Chain Reaction

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Single-cell RT-PCR demonstrates expression of voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) in outer hair cells of rat cochlea

We investigated whether voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) are expressed in outer hair cells (OHCs) of rat cochlea using a single-cell reverse transcription–polymerase chain reaction (RT-PCR) technique. The OHCs were isolated from rat cochlea and the cytoplasm of each OHC was suctioned into a glass pipette containing RT-PCR reaction buffer with RNase inhibitor. RT-PCR revealed the presence of transcripts of ClC-1, ClC-2 and ClC-3, which were verified by DNA sequencing. The possible roles of these chloride channels in OHCs are discussed.

Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization

A rapid and sensitive assay for the specific detection of plant viroids using reverse transcription-polymerase chain reaction (RT-PCR) -probe capture hybridization (RT-PCR-enzyme-linked immunosorbent assay (ELISA)) was developed. The assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from viroid infected leaf tissue. Clarified sap extract from infected leaf tissue was treated first with GeneReleaser™ polymeric matrix to remove inhibitors of RT-PCR reactions. Viroid cDNA was then synthesized and amplified using viroid specific primers in RT-PCR assays and the amplified viroid cDNA (amplicon) was digoxigenin (DIG) -labelled during the amplification process. The amplicon was then detected in a colorimetric hybridization system in a microtiter plate using a biotinylated cDNA capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is at least 100-fold more sensitive than gel electrophoretic analysis in detecting the amplified product. Viroid cRNA may replace viroid cDNA as the capture probe. The cRNA probe was several fold more sensitive than the cDNA probe for viroid detection. Six to seven hours are needed to complete the RT-PCR-ELISA for viroid detection from infected leaf tissue.

Analysis of mRNA from Microdissected Frozen Tissue Sections without RNA Isolation

Molecular study of gene expression in solid tumors is based largely on mRNA extracted from crushed frozen tumor samples. As most tumors are heterogeneous in composition, molecular alterations acquired by neoplastic cells may be masked by normal epithelial, stromal, and inflammatory cells, which may make up a significant volume of many tumors. We have developed a technique whereby reverse transcription polymerase chain reaction (RT-PCR) can be performed on lesions microdissected directly from frozen tumor sections. This allows for molecular analysis of mRNA from histologically homogeneous cell populations. Cryostat sections are placed onto a thin layer of 2% agarose on a glass slide and stained briefly. Microdissected tissue is immersed in a freezing solution to lyse the cells; aliquots are used directly in RT-PCR reactions without further purification. We successfully amplified cDNA fragments of the beta2-microglobulin, p21Waf1, and BRCA1 genes from small microdissected lesions. Also, we examined the effect of varying thickness of cryostat sections (20 versus 40 microm) and several tissue staining dyes. We estimate that a small microdissected region, containing no more than 200 cells, can provide enough mRNA to make cDNA for 80 to 100 PCR reactions. We believe that this technique will be a useful tool to study gene expression in histologically defined tissues.

Competitive reverse transcription polymerase chain reaction for quantifying pre-MRNA and mRNA of major acute phase proteins

Competitive reverse transcription polymerase chain reaction (RT-PCR) is an increasingly used method for quantifying RNA. The technique involves co-amplification from test RNA with an internal standard using common primers in a single reaction. The standard competes for primers and enzyme and it is therefore referred to as a competitor. A RT-PCR polycompetitor for use in quantifying acute phase serum amyloid A, constitutive serum amyloid A, serum amyloid P component, C-reactive protein, apolipoprotein A1, apolipoprotein A2, glyceraldehyde-3-phosphate dehydrogenase and β-actin mRNAs has been generated and used in quantitative PCR. The polycompetitor was synthesised from ten oligonucleotides of 77–90 bases using primer extension and contains sequences which permit amplification using priming sites that are present in both hnRNA (pre-mRNA) and mRNA. The polycompetitor was cloned into the expression vector pSP64(polyA) and a polycompetitor transcript with a poly(A)-tail sequence at the 3′-end was generated by in vitro transcription. The poly(A)-tail sequence allows the option of performing reverse transcription using oligo(dT) in addition to directed reverse transcription using the specific 3′-reverse PCR primers. cDNA products generated from amplification of the internal polycompetitor standard and endogenous RNA species were separated by capillary electrophoresis and quantified by UV absorbance at 254 nm. Reproducibility was determined to be very high when starting ratios of internal standard and target mRNA are at an approximate equivalence point. Relative standard deviations were less than 5% between independent RT-PCR reactions with the same sample mix of internal standard and total RNA. Applying the method to total RNA samples harvested at various timepoints following cytokine induction of acute phase mRNAs in endothelial cells demonstrated that quantitative readout from the RT-PCR method correlates well with that obtained from Northern-blotting and is at least 100-fold more sensitive. This method will be useful for studying regulation of acute phase proteins in in vitro tissue culture experiments and may also be applied to clinical tissue samples from patients with inflammatory diseases.

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Molecular cloning and sequencing of the cDNA encoding mouse glutamate-cysteine ligase regulatory subunit

Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify and clone the regulatory subunit of mouse glutamate-cysteine ligase (Glclr) using primers adapted from the published rat Glclr cDNA sequence, and from mouse genomic DNA. Amplified cDNA was cloned into a plasmid vector, and additional RT-PCR reactions coupled with 3′ RACE were used to amplify and sequence 3′ regions covered by the rat primer. Comparison of the mouse Glclr cDNA sequence and predicted protein sequence with that of rat Glclr and human GLCLR revealed extensive homology in cDNA and amino acid sequences among these species.

A method to remove environmental inhibitors prior to the detection of waterborne enteric viruses by reverse transcription-polymerase chain reaction

A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the reverse transcription-polymerase chain reaction (RT-PCR). Environmental inhibitors, concentrated along with viruses during water sample processing, are removed by the method through a series of steps that includes dialysis, solvent extraction, ultrafiltration and glass purification. The method was tested by spiking sodium phosphate with poliovirus type 1 with or without humic or fulvic acids and then measuring virus recovery by plaque assay and RT-PCR. Results of the study indicated that (i) 90% of the spiked virus could be recovered from samples at the end of the ultrafiltration step, (ii) virus was detected in the final eluate of samples containing as much as 0.5 mg of humic acid or 5.0 mg of fulvic acid, and (iii) as little as 0.06 plaque forming units (PFU) was detectable per RT-PCR reaction. These results indicate that the described purification method along with RT-PCR is a feasible approach for detecting waterborne human enteric viruses in the presence of interfering substances.

Coexpression of ϵ, Gγ and Aγ Globin mRNA in Embryonic Red Blood Cells from a Single Copy β-YAC Transgenic Mouse

We utilized reverse transcription polymerase chain reaction (RT-PCR) to amplify ϵ, Gγ and Aγ globin cDNAs from single red blood cells isolated from a day-10 transgenic fetus harboring a single copy of the human β-YAC. A detailed structural analysis of the β-YAC showed a single copy of each β-like globin gene is present and linked to the locus control region (LCR). RNAse protection analysis of RNA isolated from all erythroid tissues from day-8 to day-16 of development and the adult stage showed proper developmental switching of the β-like globin gene expression. Using ϵ/γ and Gγ/ Aγ primer sets in separate RT-PCR reactions on RNA from single day-10 red blood cells we observed an intercellular variation in the ϵ and γ RT-PCR products that may be indicative of a change in the LCR preference from the ϵ gene promoter to the γ gene promoter during switching. We also found that the majority of the red blood cells examined contain all three globin mRNA species. These observations suggest that either the LCR is capable of interacting simultaneously with more than one globin gene promoter or alternatively, the LCR may interact with only one promoter at any given time, but its interaction oscillates between promoters (flip-flop mechanism) resulting in similar expression of more than one gene from a single β-globin locus.

Diagnosis of the African horse sickness virus serotype 4 by a one-tube, one manipulation RT-PCR reaction from infected organs

A single tube reverse transcription — polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV) in splenic tissues from infected horses is described. Double stranded RNA was extracted from infected organs of horses and used to produce complementary DNA (cDNA) with the two primers selected for the PCR. The 1179 bp amplified product (the segment 7 which encodes for VP 7), detected by electrophoresis on agarose gel and ethidium bromide staining, was hydrolysed with eight restriction endonucleases for characterization of the AHSV. The sensitivity of this method is discussed. Application of the RT-PCR method should improve detection and shorten the time required to confirm a clinical diagnosis of AHSV infection.

A one-tube, one manipulation RT-PCR reaction for detection of Ross River virus

A sensitive, single tube reverse transcription-polymerase chain reaction (RT-PCR) protocol for the detection of Ross River virus (RRV) is described. All components necessary for both reverse transcription and PCR were combined in a single tube, and reverse transcription and PCR carried out sequentially in a single, non-interrupted thermal cycling program. The antisense oligonucleotide from the two primers selected for use in the PCR also served to prime specifically for the reverse transcription. The 549 bp product was detected by electrophoresis and ethidium bromide staining. The detection limit using this system was 18 fg of purified viral RNA or 1.3 pfu of whole virus. Greater sensitivity cannot reasonably be expected unless a more sensitive method than electrophoresis and ethidium bromide staining is used for PCR product detection, such as nested PCR or hybridisation with labelled probe. This PCR detection system will be adapted for detection of RRV in mosquito populations for virus surveillance programs.