Two-step Reverse Transcription-polymerase Chain Reaction Research Articles

Sensitivity of RNA viral nucleic acid-based detection of avian influenza virus, Newcastle disease virus, and African horse sickness virus on flinders technology associates card using conventional reverse-transcription polymerase chain reaction.

The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards. A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at -80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at -80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids. The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log10 EID50, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log10 ELD50. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log10 plaque-forming units, respectively. This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

First Report of Tomato Brown Rugose Fruit Virus on Greenhouse Tomato in Syria.

Tomato (Solanum lycopersicum L.) is an important vegetable crop worldwide. In spring and autumn 2017, virus-like symptoms were observed on greenhouse grown tomato plants in the east of Akkar plain (south of coastal region, Tartous governorate, Syria). These symptoms were: mild to severe mosaic on the apical leaves, brown necrosis on sepals, receptacle and flower's cluster carrier, and severe symptoms of brown rugose and discoloration on fruit. During next growing seasons, disease spread was observed in most of Syrian coastal region with disease incidence ranged from 40% to 70% by 2020. Tomato brown rugose fruit virus (ToBRFV) was suspected as a main causal agent of the disease, especially since its first report in Jordan, a neighboring country (Salem et al. 2016), Palestine (Alkowni et al. 2019), Turkey (Fidan et al. 2019), Germany (Menzel et al. 2019), Italy (Panno et al. 2019), America (Camacho-Beltrán et al. 2019), Egypt (Amer and Mahmoud, 2020), and recently in Spain (Alfaro-Fernandez et al. 2021). In November and December 2020, seventy-one leaf samples from symptomatic plants (59 from Tartous and 12 from Lattakia governorates) and seven from asymptomatic ones (5 from Tartous and 2 from Lattakia) were collected and tested for the presence of ToBRFV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using ToBRFV-commercial kit (LOEWE® Biochemia, Germany) following the manufacturer's instructions. Results showed, forty-three of symptomatic samples reacted positively (38 in Tartous and 5 in Lattakia) and none of asymptomatic ones. On the other hand, sap mechanical inoculation of 10 tomato cv. Mandaloun F1 (Enza Zaden, the Netherlands) plants using a positive tomato isolate gave systemic mosaic symptoms in all plants identical to those observed in the original plants in the field, after 13 days of inoculation, and necrotic local lesions on 10 plants of Nicotiana tabacum after 5 days, indicating the presence of a tobamovirus in general. ToBRFV infection was confirmed in all mechanically-inoculated plants by DAS-ELISA. Further tests were necessary to investigate ToBRFV presence, because of its serological relationships with another tobamoviruses. Six representative symptomatic samples (ELISA-positive) and two asymptomatic (ELISA-negative) samples were subjected to total RNA extraction using the SV-Total RNA Extraction kit (Promega, U.S.A.) following the manufacturer's instructions. The samples were tested by two-step reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers and protocols for most common tomato-infecting viruses, including: tomato chlorosis virus and tomato infectious chlorosis virus (Dovas et al. 2002), pepino mosaic virus (PepMV) and tomato torrado virus (Wieczorek et al. 2013), alfalfa mosaic virus (Parrella et al. 2000), tomato spotted wilt virus (Salem et al. 2012) and a pair of primers: ToBRFV-F2 (5'-CATATCTCTCGACACCAGTAAAAGGACCCG-3') and ToBRFV-R2 (5'-TCCGAGTATAGGAAGACTCTGGTTGGTC-3') targeting a region of the RNA dependent RNA polymerase (RdRp), of the ToBRFV genome (KT383474; Salem et al. 2016). First-strand cDNA synthesis was carried out using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Promega) and random primer according to the manufacturer's protocol, then followed by PCR with the seven species-specific primers. Only ToBRFV was detected among all tested viruses in symptomatic samples (ELISA-positive), and none of the tested viruses was detected in the asymptomatic plants. To confirm the presence of ToBRFV, two selected RdRp-specific PCR amplicons (872 bp) were purified and ligated into pGEM T-Easy Vector (Promega), and three clones were sequenced (GenBank accession nos. MZ447794 to 96). BLASTn analysis showed that the nucleotide sequences are 99.77-100% identical and shared around 99% identity to RdRp of ToBRFV isolate (MT118666) from Turkey available in the GenBank. Accordingly, the presence of ToBRFV was confirmed by bioassays on indicator plants, DAS-ELISA, RT-PCR, and further sequencing. To our knowledge, this is the first report of ToBRFV infecting tomato in Syria, and this requires special emphasis for further investigations because of the virus severity, easy transmission ability and absent of commercial resistance varieties till now.

FadP affects the virulence of Ralstonia pseudosolanacearum by altering nitrogen metabolism processes and reducing motility, cellulase, and extracellular polysaccharide production

Ralstonia pseudosolanacearum, the causative agent of bacterial wilt disease, is a significant pathogen of economically-important agricultural crops. FadP is a transcriptional regulator of fatty acid degradation; however, its function remains unknown in R. pseudosolanacearum. Herein, a R. pseudosolanacearum fadP deletion mutant (ΔfadP) was constructed to examine the role of FadP. Compared with wild-type strain Tm1401 and fadP complementation strain R-ΔfadP, ΔfadP showed reduced motility and cellulase and extracellular polysaccharide production, along with decreased resistance to tetracycline and virulence on tomato plants. Further, the mutant strain induced only a weak hypersensitive response in tobacco plants. To further understand the underlying virulence mechanisms, RNA-Seq technology was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 848 differentially-expressed genes, amongst which 505 were up-regulated and 343 were down-regulated in ΔfadP. Both flagella and chemotaxis genes were up-regulated, while genes coding for extracellular hydrolytic enzymes, cytochrome c oxidase, and carbon and nitrogen metabolism proteins were down-regulated. Two-step reverse-transcription polymerase chain reaction analysis targeting 13 genes confirmed that the expression of egl, rsl, pehC, pme, cspD3, narH, norB, ccoP, tek, and xpsR was significantly decreased. Our results suggest that FadP affects the virulence of R. pseudosolanacearum by altering nitrogen metabolism processes, reducing bacterial motility and extracellular polysaccharide and cellulase production, and affecting protein-carbohydrate interactions involved in host recognition, attachment, and invasion.

Global Regulator PhoP is Necessary for Motility, Biofilm Formation, Exoenzyme Production and Virulence of Xanthomonas citri Subsp. citri on Citrus Plants.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, ΔphoP. Compared with wild-type strain XHG3, ΔphoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of ΔphoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in ΔphoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.

Amplification of cDNA Generated by Reverse Transcription of mRNA: Two-Step Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

Reverse transcription-polymerase chain reaction (RT-PCR) is a powerful method to detect and synthesize cDNA copies of low-copy-number mRNAs. Two enzymes are used: reverse transcriptase to produce single-stranded cDNA copies, which are then used as templates in an amplification reaction catalyzed by a thermostable DNA polymerase. For this reason, the method is known as "two-step RT-PCR." This protocol describes the traditional method of RT-PCR in which the two synthetic reactions are performed separately and sequentially.

Integrated Graphene Oxide Purification-Lateral Flow Test Strips (iGOP-LFTS) for Direct Detection of PCR Products with Enhanced Sensitivity and Specificity.

An integrated graphene oxide purification-lateral flow test strip (iGOP-LFTS) was developed for on-strip purifying and visually detecting polymerase chain reaction (PCR) products with an improved sensitivity as well as a more stringent specificity. PCR products amplified with a pair of biotin- and digoxin-labeled primers were directly pipetted onto GO pads, on which graphene oxide selectively adsorbed residual primers and primer-dimers with the aid of a running buffer containing MgCl2 and Tween 20. By stacking up three GO pads to increase the surface area for adsorption, 83.4% of double-stranded DNA with a length of 30 bp and 98.6% of 20-nt primers could be removed from a 10-μL DNA mixture. Since no primers interfered with detection, the increase of the sample loading volume from 5 to 20 μL could improve the signal-to-noise ratio of the test line 1.6 fold using the iGOP-LFTS while no changes were observed using the conventional LFTS. The limit of detection of the iGOP-LFTS was determined to be 30 copies of bacteriophage λ-DNA with naked eyes and this limit could be further decreased to 3 copies by loading 20 μL of the sample, which corresponded to a 1000-fold improvement compared to that of the LFTS detected by naked eyes. When the ImageJ analysis was employed, a 100-fold decrease of the detection limit can be obtained. In addition, due to the removal of the primer-dimers, the dim test line observed in the negative control of the LFTS was eliminated using the iGOP-LFTS. A mock clinical specimen spiked with defective HIV-1 (human immunodeficiency virus) viruses was successfully analyzed using a two-step reverse transcription-PCR with 30 amplification cycles followed by the iGOP-LFTS detection. These significant improvements were achieved without introducing any additional hands-on operations and instrumentations.

Hepatitis E Virus in Wild Boar in Northwest Poland: Sensitivity of Methods of Detection.

In northwest Poland, 163 blood and 53 fecal samples of wild boars were collected in winter 2012/13 and 2013/14. All blood samples were tested for the presence of hepatitis E virus (HEV) ribonucleic acid (RNA) by two reverse transcription-polymerase chain reaction (RT-PCR) based methods and by anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). About 17.2% of blood samples were seropositive. One-step nested RT-PCR turned out to be too insensitive (11.6% were positive). Therefore a two-step nested RT-PCR was applied where 25.8% of the blood samples were tested positive for HEV RNA. About 50.0% of blood samples positive in ELISA were also positive in two-step nested RT-PCR. The prevalence of HEV RNA in feces was 9.4%. Based on the results of blood (ELISA, PCR) and fecal (PCR) tests, the overall prevalence of HEV in wild boars in northwest Poland was 36.8%. There was no correlation between the ELISA results and the presence of HEV RNA in plasma or in feces. According to the sequencing results of 348 bp PCR products of HEV, there were four different subtypes identified. Reports on the prevalence of HEV in wild boar populations are varying due to different sensitivities of the detection methods. However, this study reveals based on a highly sensitive method that HEV is widely spread in wild boar populations in the northwestern region of Poland and posing a potential risk to the consumer of game meat.

Validation of the sensitivities of one-step and two-step reverse-transcription PCR methods for detection of viral hemorrhagic septicemia virus (VHSV) IVa isolates from cultured olive flounder in Korea

Viral hemorrhagic septicemia (VHS) is one of the most serious viral diseases of cultured olive flounder in Korea. This study validated the sensitivities of one-step and two-step reverse-transcription PCR (RT-PCR) methods based on the OIE (World Organization for Animal Health) diagnostic manual for the detection of VHSV type IVa isolates from olive flounder in Korea. VHSV type I was used as a positive control for the VHSV VN primer set listed in the OIE manual; the PCR products amplified from VHSV I appeared as strong gel bands of the target size, but the PCR products amplified from the VHSV IVa Korean isolate appeared as faint bands. Sequence comparison revealed that the VHSV VN forward primer was mismatched at 4 out of 24 nucleotide positions within the corresponding VHSV type IVa sequence, but differed only by a single nucleotide in the case of VHSV I. Therefore, the VHSV VN IVa primer set was designed specifically to fit the VHSV IVa sequence, and this was used in RT-PCR. The PCR products amplified from VHSV IVa appeared as strong bands of the target size when the VHSV VN IVa primer set was used, but the PCR products of VHSV I appeared as faint bands. PCR titration results showed that the sensitivity of the VHSV VN IVa primer set for VHSV IVa was 100,000-fold higher than that of the VHSV VN primer set when one-step RT-PCR was used, and that the sensitivity was >10,000-fold higher than that of the VHSV VN primer set when the two-step RT-PCR method was used with oligo dT+random hexamer. The sensitivity of the VHSV VN IVa primer set for VHSV IVa was found to be 10,000-fold higher than that of the VHSV VN primer set when the two-step RT-PCR method was used with the target primer set. Therefore, the RT-PCR methods that are used in pathogen-diagnosis laboratories must be validated according to the specificity of the primer set and the one-step or two-step RT-PCR method employed, using either the target primer set or oligo dT+random hexamer.

First report of Dahlia latent viroid and Potato spindle tuber viroid mixed‐infection in commercial ornamental dahlia in Japan

Dahlia (Asteraceae) has been cultivated since 1841 in Japan. Two viroids belonging to the family Pospiviroidae, Potato spindle tuber viroid (PSTVd) and Dahlia latent viroid (DLVd), were identified separately from dahlia plants in Japan and in the Netherlands, respectively (Tsushima et al., 2011; Verhoeven et al., 3). Because DLVd was characterised as a novel species, the findings drew attention towards the study of DLVd infection in dahlia and its potential risk in domestic dahlia cultivation. A total of four dahlia samples infected with PSTVd-dahlia (Tsushima et al., 2) were collected, and total RNA was extracted from approximately 200 mg of leaf tissue using TRIzol® reagent (Life Technologies, Carlsbad, USA) according to manufacturer's instructions. RNA was separated in 2-dimensional polyacrylamide gel (2D-PAGE) (Schumacher et al., 1) and two specific bands appeared, one corresponding to PSTVd infection and the other being unknown (Fig. 1). The unknown circular RNA samples were gel-excised and first tested with PSTVd-specific primers using a two-step reverse-transcription-polymerase chain reaction (RT-PCR) (Tsushima et al., 2), with, however, negative results. Therefore, a DLVd-specific primer set, DLVd-P1 (5'-GGGGCTCCTCAGAGAGTCTC-3') and DLVd-P2 (5'-GGGGCAACTCCGAGAGTGCTG-3') (Verhoeven et al., 3), was then used in two-step RT-PCR for the unknown viroid-like circular RNAs. The amplified DNA bands of the expected size (~340 bp) from these positive samples were purified, ligated to pGEM®-T easy vector (Promega, Madison, USA) and sequenced. Identical 342 bp sequences were obtained from all four dahlia isolates (GenBank Accession No. LC036322), corresponding with DLVd reported from the Netherlands (JX263426). Furthermore, a total of 78 dahlia leaf samples were collected from fields around Japan, irrespective of disease symptoms, and verified by two-step RT-PCR using the same DLVd-specific and pospiviroid-specific primers (Tsushima et al., 2). Of these samples, DLVd infection was detected in 38, suggesting that DLVd is widespread in Japan. New PSTVd infection was not found. To evaluate the pathogenicity of DLVd and PSTVd, these viroids were co-inoculated into wild hop (Humulus lupulus var. cordifolius) and dahlia seedlings. DLVd alone was inoculated into members of the Asteraceae family, Chrysanthemum pacificum, Tithonia rotundifolia, Helianthus annuus and Glebionis coronaria. Inoculated dahlia seedlings were positive for both DLVd and PSTVd, but wild hop and the remaining members of Asteraceae were negative. Dahlia seedlings that were positive for DLVd and PSTVd showed mild leaf curling in the early stage of growth (Fig. 2). To the best of our knowledge, this is the first report of DLVd infecting commercial dahlia in Japan and also of the mixed-infection of DLVd and PSTVd in commercial dahlia plants. A possible negative synergistic interaction of DLVd and PSTVd during dahlia cultivation must be considered. This work was supported in part by Japan Society for the Promotion of Science KAKENHI grant no. 24380026. TT received Research Fellowship of the Japan Society for the Promotion of Science.

Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection

Being beneficial from dramatic process in bio-chemical and micro-electro-mechanical technologies, this study presents an integrated microfluidic system capable of performing a continuous-flow reverse transcription-polymerase chain reaction (RT-PCR) combined with fluorescence microscopy for rapid diagnosis of RNA-based viruses. The device consists of two heated cylinders for heating the different reaction zones of the reverse transcription and the amplification. In the amplification cylinder, bath refrigerating is used for thermal protection of the annealing region, which is heated possibly due to the thermal effects of radiation, conduction, and/or convection from denaturation and extension regions, thus resulting in a space-saving design of the whole device. Detection of the amplified products is performed on-line by a fluorescence microscopy with SybrGreen I, a widely used intercalating dye. In this article, the proposed miniature RT-PCR system is used to amplify and detect two RNA-based viruses (Noroviruses (NVs) and Rotaviruses (RVs)), which are now recognized as the most common etiological agents of acute viral gastroenteritis causing numerous outbreaks worldwide. The experimental data have demonstrated the ability of the presented system to perform a two-step or one-step RT-PCR process. On this device, the NVs and RVs RNA samples were successfully reverse transcribed and amplified within 1 h, and the limit of detection of the RNA concentration was 6.4 × 104 copies μl−1 using one-step RT-PCR process. Consequently, the developed microfluidic system can provide a promising platform for fast diagnosis of RNA-based viruses.

No borna disease virus-specific RNA detected in blood of race horses and jockeys.

Borna disease virus (BDV) predominantly infects horses and sheep, causing a broad range of behavioural disorders. It is controversial whether BDV infects humans and causes psychiatric disorders. We searched for BDV-derived nucleic acids in blood of race horses and jockeys riding the horses. We assayed for the BDV genome in RNA extracted from peripheral blood mononuclear cells (PBMC) of 39 race horses and 48 jockeys. Two polymerase chain reaction protocols [one-tube reverse transcription-polymerase chain reaction (RT-PCR) and two-step RT-PCR] were used to assay BDV p24 and p40 transcripts. The p24 and p40 viral nucleic acid sequences were not detected in the PBMC RNAs from any of the race horses or jockeys. These data do not support an epidemiological association between BDV infection, race horses and humans.

Reverse Transcription Loop-Mediated Isothermal Amplification of DNA for Detection of Potato virus Y.

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

Methylenetetrahydrofolate Reductase Genotype Affects Risk of Relapse after Hematopoietic Cell Transplantation for Chronic Myelogenous Leukemia

Methylenetetrahydrofolate reductase (MTHFR) directs intracellular folate toward homocysteine metabolism and away from nucleotide synthesis. Two common MTHFR polymorphisms, C677T and A1298C, are associated with reduced enzyme activity. We evaluated the association of these polymorphisms with risk of relapse and bcr-abl mRNA transcript detection among 336 Caucasian patients who underwent allogeneic hematopoietic cell transplantation for chronic myelogenous leukemia. Data on the transplant course and folate-related exposures were abstracted from medical records. MTHFR C677T and A1298C genotypes were determined using polymerase chain reaction/restriction fragment length polymorphism and TaqMan assays. Qualitative bcr-abl mRNA testing was conducted using a two-step reverse transcription-polymerase chain reaction assay. Cox regression analysis was used to assess the association between MTHFR genotypes and time to relapse and bcr-abl mRNA detection. A statistically significant decreased risk of relapse was observed in patients with the variant A1298C genotype [1298AC, hazard ratio (HR)=0.48 and 95% confidence interval (CI)=0.26-0.88; 1298CC, HR=0.28 and 95% CI=0.09-0.84; P-trend <0.01). For the joint C677T/A1298C genotype, variant genotypes were associated with a decreased risk of relapse when compared with the wild-type 677CC/1298AA genotype. This risk was lowest for the 677CC/1298CC genotype (HR, 0.23; 95% CI, 0.08-0.72). MTHFR genotypes were not associated with bcr-abl transcript detection. These findings suggest that individuals with the 677CC/1298AA genotype are at higher risk of relapse after hematopoietic cell transplantation and that the balance of intracellular folate metabolites available for nucleotide synthesis (regulated by the relative activity of the MTHFR enzyme) may affect the progression from bcr-abl positivity to clinical relapse.

TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus

One step TaqMan reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probe was developed for detection of Japanese encephalitis virus (JEV). Real-time RT-PCR was optimized to quantify JEV using the detection system (Rotor Gene 2000 detector) and dual-labeled fluorogenic probes. The gene specific labeled fluorogenic probe for the 3' non-translated region (3' NTR) was used to detect JEV. When the specificity of the assay using specific JEV primers was evaluated by testing three different JEV strains, other swine viruses and bovine viral diarrhea virus, no cross-reactions were detected with non-JE reference viruses. A single tube TaqMan assay was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method. Detection limits of two step and real-time RT-PCR for JEV were 112 TCID(50) /ml and 11.2 TCID(50) /ml, respectively. Quantification of JEV was accomplished by a standard curve plotting cycle threshold values (C(t)) versus infectivity titer. Real-time RT-PCR assay using single tube method could be used as a sensitive diagnostic test, and supplied the results in real time for detection and quantification of JEV. We could detect JEV RNA genome in plasma samples of pigs inoculated with KV1899 strain at 2 days post inoculation, but couldn't in 41 fetus samples. This assay was sensitive, specific, rapid and quantitative for the detection of JEV from laboratory and field samples.

Establishment of a rapid procedure for one-step real-time reverse transcription-polymerase chain reaction in combination with automated mRNA extraction: a clinical application to intra-operative molecular diagnosis of cancer cells

Reverse transcription-polymerase chain reaction (RT-PCR) technique is extremely useful for sensitive detection of cancer cells, but it requires at least 8 h. If the precise information on the cancer spread based on RT-PCR is available during surgery, we can give the optimal therapy to each patient during operation. We aimed to establish the ultra-rapid procedure of RT-PCR for application to the intra-operative diagnosis. We have established a one-step real-time RT-PCR using LightCycler with hybridization probes, in combination with automated mRNA extraction on MagNA Pure LC. The method can be completed in only 70 min after sampling and has a comparable sensitivity to conventional RT-PCR and/or two-step real-time RT-PCR with a wide (10–10 6) dynamic range. When this method was applied to the intra-operative detection of free cancer cells in peritoneal lavage, the results were available during operation in all cases. We could detect cancer cells more effectively compared to conventional cytology and RT-PCR. This protocol is easily accomplished, delivers rapid, sensitive and reproducible results, and is therefore applicable to intra-operative diagnosis.

Molecular Monitoring of Acute Promyelocytic Leukemia by DzyNA Reverse Transcription-PCR

Acute promyelocytic leukemia (APL) is caused by inhibition of apoptosis and a block in myeloid differentiation resulting from a translocation invariably involving the retinoic acid α (RARα) gene (1)(2). Ninety percent of breakpoints lie within intron 3 or 6 of the PML gene, producing fusion transcripts of the S-type (exon 3 PML /exon 3 RARα ) or L-type (exon 6 PML /exon 3 RARα ) isoform. The remaining patients have V-type transcripts, which vary in length depending on where the breakpoint lies within exon 6 of the PML gene. Although a combination of all- trans -retinoic acid (ATRA) differentiation therapy and chemotherapy successfully induces remission in the majority of patients harboring the PML/RARα transcript, approximately one-third of these patients relapse (3). Patient survival rates are improved by initiating salvage therapy at molecular relapse rather than waiting for overt hematologic relapse (4)(5). Recent preliminary results suggest that the predictive power of molecular monitoring may be improved by the application of new quantitative methods to patient management (6)(7). This report provides the most recent patient results obtained with the single-tube DzyNA reverse transcription-PCR (RT-PCR) assay recently developed in our laboratory for diagnosing and monitoring APL (8). DzyNA RT-PCR assays for quantification of PML/RARα transcripts and endogenous BCR control transcripts were used to analyze total RNA from patients with APL. Specimens were analyzed with the investigator blinded to the patients’ clinical histories. Primers, substrate, and reaction conditions for DzyNA RT-PCR and confirmatory two-step nested qualitative RT-PCR were as described previously (8). The PML/RAR α primers amplify all L-type, and most V-type, transcripts. PML/RAR α concentrations were normalized by use of BCR concentrations, thus controlling for variations in RNA integrity from clinical specimens. Quantitative data were expressed as ng-equivalents of the total NB4 RNA. Relative transcript amounts were expressed …

Detection of European Isolates of Oat Mosaic Virus

Oat mosaic virus (OMV) is a fungally-transmitted virus which causes yield losses in winter oats in five European countries. Detection of the virus has depended upon the recognition of transient symptoms or electron microscopy. Recent research has confirmed that the virus is a Bymovirus, yet OMV could not be reliably detected by the enzyme-linked immunosorbent assay (ELISA) using a range of antisera raised against other members of the genus. Therefore, a reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed to specifically detect the virus. Using total RNA isolated from 16 field OMV isolates collected from throughout Europe, these primers were shown to reliably detect the virus in either one-step or two-step RT-PCR. The primers were specific and no PCR product was obtained with either Oat golden stripe virus (OGSV), which is frequently associated with OMV, or with other members of the Bymovirus genus. The two-step protocol was able to detect as little as 5 × 10−3μl (10ng) of total RNA isolated from an infected plant. Both protocols were as reliable as electron microscopy, but were more sensitive and were able to detect infection earlier than in mechanically-inoculated plants. However, this protocol did not detect three American isolates of the virus nor was amplification achieved using alternative primers raising the possibility that these isolates may represent a separate strain or virus. This protocol enables sensitive, rapid and reliable detection of OMV and will therefore assist management of the disease.

Detection and Discrimination of Wheat Spindle Streak Mosaic Virus and Wheat Yellow Mosaic Virus Using Multiplex RT-PCR

Wheat spindle streak mosaic virus (WSSMV) and wheat yellow mosaic virus (WYMV) are two closely related bymoviruses which cause significant yield losses in wheat. There is no molecular diagnostic protocol available for either virus nor are serological methods able to discriminate them. A multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for their detection and discrimination. Twenty-three isolates of WSSMV and WYMV were collected from a range of countries and the sequence of 834–837 nucleotides of the coat protein gene of three representative WSSMV isolates and one WYMV isolate was determined. This sequence data provided further evidence that the two viruses are distinct species and was used together with previously published sequence data to design specific oligonucleotide primers to discriminate the two viruses. Using total RNA isolated from eighteen WSSMV and five WYMV isolates in either one-step or two-step RT-PCR, these primers were shown to detect and discriminate the two viruses reliably. The primers were specific to either WSSMV or WYMV and no PCR product was obtained with either soil-borne wheat mosaic virus, which is frequently associated with both diseases, or with the closely related viruses, barley yellow mosaic virus, barley mild mosaic virus or oat mosaic virus. This new diagnostic protocol will enable more effective management of the diseases caused by these viruses by enabling correct identification of the causal pathogen and earlier detection than is possible by serological methods.

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

Partial nucleotide sequences of amplification products obtained from four European apple stem grooving virus (ASGV) isolates using degenerate primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Based on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were designed to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV isolates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to constitute good templates for reliable amplification of ASGV sequences from leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermore, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark tissues of apple trees during both active growth and the dormant season.

Long-term persistence of activated cytotoxic T lymphocytes after viral infection of the central nervous system.

Mice intranasally inoculated with influenza A/X-31 are protected against a subsequent intracerebral challenge with the neurovirulent influenza A/WSN and this heterotypic protection is mediated by CD8(+) cytotoxic T lymphocytes. We have studied the kinetics of this secondary immune response and found that despite the elimination of replication-competent virus by day 10, we were able to recover activated influenza-specific cytotoxic T lymphocytes (CTLs) that killed freshly ex vivo from the brains of mice for at least 320 d after the intracerebral inoculation. The activated antiviral CTLs expressed high levels of the early activation marker CD69, suggesting continuing TCR signaling despite a lack of viral protein and major histocompatibility complex staining by immunohistochemistry in the brain parenchyma and barely detectable levels of viral nucleic acid by single and two-step reverse transcription PCR. Local persistence of activated lymphocytes may be important for efficient long-term responses to viruses prone to recrudesce in sites of relative immune privilege.