Single-step RT-PCR Research Articles

Differential seasonal prevalence of yellowing viruses infecting melon crops in southern California and Arizona determined by multiplex RT-PCR and RT-qPCR.

Viruses transmitted by the whitefly (Bemisia tabaci) are an increasing threat to cucurbit production in the southwestern United States as well as many other cucurbit production regions throughout the world. The crinivirus, cucurbit yellow stunting disorder virus (CYSDV), has severely impacted melon production in California and Arizona since its introduction to the region in 2006. Within the past few years, another crinivirus, cucurbit chlorotic yellows virus (CCYV), and the whitefly-transmitted ipomovirus, squash vein yellowing virus (SqVYV) were found infecting melon plants in California's Imperial Valley as well. Both CYSDV and CCYV, as well as an aphid transmitted polerovirus, cucurbit aphid-borne yellows virus (CABYV), occur together in the region and produce identical yellowing symptoms on cucurbit plants. Mixed infections of these four viruses both in the Sonoran Desert and other regions pose challenges for disease management and efforts to develop resistant varieties. A multiplex single-step RT-PCR method was developed that differentiates these viruses from one another, and this was used to determine the prevalence and distribution these viruses in melon samples collected from fields in the Sonoran Desert melon production region of California and Arizona, USA during spring and fall melon seasons from 2019 through 2021. TaqMan probes were also developed, optimized, and applied in a single-step multiplex RT-qPCR to quantify titers of these four viruses in plant samples, which frequently carry mixed infections. Results of the multiplex RT-PCR analysis demonstrated that CYSDV is the predominant virus during the fall season, whereas CCYV was by far the most prevalent virus during spring seasons each year. Multiplex RT-qPCR was used to evaluate differential accumulation and spatio-temporal distribution of viruses within plants and suggested differences in competitive accumulation of CCYV and CYSDV within melon. This study also provides the first official report of SqVYV in Arizona, USA, and offers an efficient method for virus detection and quantification for breeding and disease management in areas impacted by cucurbit yellowing viruses.

Simultaneous Detection of Influenza A/B, Respiratory Syncytial Virus, and SARS-CoV-2 in Nasopharyngeal Swabs by One-Tube Multiplex Reverse Transcription Polymerase Chain Reaction.

The treatment and outcome of respiratory virus infections differ. SARS-CoV-2, as well as other respiratory viruses such as influenza virus (A and B) and respiratory syncytial virus (RSV), require simultaneous, cost-effective, and rapid differential detection. We used a gold standard five-target single-step RT-PCR to detect influenza viruses, RSV, and SARS-CoV-2, and this method can be extended to detect influenza virus subtypes. As a result, this five-target single-step RT-PCR method is ideal for differentiating respiratory viruses. The 5' nuclease activity of Taq DNA polymerase is used in the real-time reverse transcription PCR assay. The Taq man fast viral 1-step enzyme is a 4× Master mix and five-target primer probe mix that detects influenza A, influenza B, SARS-CoV-2 ORF1ab, respiratory syncytial viruses A/B and actin. When compared with TaqMan TM and Invitrogen superscript TM III Platinum and the Meril Kit for SARS-CoV-2, the assay demonstrated 100% sensitivity, specificity, and amplification efficiency of 90.1% for target genes. In conclusion, our one-tube multiplex RT-PCR assay offers a rapid and reliable method for the simultaneous detection of influenza A/B, RSV, and SARS-CoV-2 from nasopharyngeal swabs. This assay has the potential to enhance diagnostic capabilities and improve public health responses during respiratory outbreaks, enabling timely interventions and informed decision making.

Detection of HEV RNA Using One-Step Real-Time RT-PCR in Farrow-to-Finish Pig Farms in Bulgaria.

(1) Background: HEV is a zoonotic, foodborne pathogen. It is spread worldwide and represents a public health risk. The aim of this study was to evaluate the presence of HEV RNA in farrow-to-finish pig farms in different regions of Bulgaria; (2) Methods: Isolation of HEV RNA from pooled samples of feces was performed using a QIAamp® Viral RNA Mini Kit followed by HEV RNA detection using a single-step real-time RT-PCR with primers and probes targeting the ORF 3 HEV genome; (3) Results: HEV RNA was detected in 12 out of 32 tested farms in Bulgaria (37.5%). The overall percentage of HEV-positive pooled fecal samples was 10.8% (68 of 630 samples). HEV was detected mostly in pooled fecal samples from finisher pigs (66/320, 20.6%) and sporadically from dry sows (1/62, 1.6%) and gilts (1/248, 0.4%); (4) Conclusions: Our results confirm that HEV circulates in farrow-to-finish pig farms in Bulgaria. In our study, we found HEV RNA in pooled fecal samples from fattening pigs (4-6-months age), shortly before their transport to the slaughterhouse indicating a potential risk to public health. The possible circulation of HEV throughout pork production requires monitoring and containment measures.

Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains.

Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010-2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact.

Abstract 5405: Multiplex profiling of miRNA biomarkers for discovery and verification using the FirePlex® platform

Abstract To address the needs for microRNA (miRNA) biomarker discovery and verification, we developed the FirePlex® miRNA assay. This assay enables the detection of 5-400 miRNA targets per sample in 96-well format, with readout on standard flow cytometers and analysis with an included bioinformatics software package. The FirePlex miRNA assay combines particle-based multiplexing, using patented FirePlex® hydrogel particles, with single step RT-PCR signal amplification using universal primers. Thus, the FirePlex miRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The miRNA assay can reliably detect as few as 1000 miRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification, making the assay ideally suited for profiling in serum, plasma, exosomes, urine, and directly from FFPE tissues. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the FirePlex miRNA assay are displayed and interpreted using the integrated FirePlex Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid biomarker discovery studies, we offer pre-designed panels of 400 miRNAs based on a research-curated list of miRNAs published to be found in circulating biofluids. For studies focused on specific research areas of interest, we offer pre-designed focus panels for Oncology, Cardiology, Neurology, Immunology, and Organ Toxicity. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating miRNA profiles, as well as miRNA profiles obtained directly from FFPE tissues, using the FirePlex miRNA Assay Panels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and verification of miRNA biomarker signatures from biofluid samples. Citation Format: Elnaz Atabakhsh, Ivona Kelley, Michael Tackett, Izzuddin Diwan, Gensis Tejada, Conor Rafferty, Daniel Pregibon. Multiplex profiling of miRNA biomarkers for discovery and verification using the FirePlex® platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5405.

Development of single step RT-PCR for detection of Kyasanur forest disease virus from clinical samples

BackgroundKyasanur Forest Disease (KFD), a tick borne flavivirus, which was earlier endemic to Karnataka state, India, has been confirmed and detected from neighboring states of Tamil Nadu, Maharashtra, Goa and Kerala states in India. Increased human and vector surveillance therefore becomes essential for the identification of KFD affected regions and control of further spread of the disease. Currently, available KFD detection assays include realtime RT-PCR and nested RT-PCR assays. Here we describe the development of a sensitive single step RT-PCR assay for the detection of KFD viral RNA. This can be easily used in any BSL-2 laboratory for screening of KFD suspected cases or for differential diagnosis of viral hemorrhagic fever panel. MethodThree primer sets were designed and checked for sensitivity using known dilutions of KFD viral RNA (Ranging from 106 copies to 10 copies). The primer set (2) was found to be most sensitive was selected and tested for specificity for Kyasanur forest disease virus (KFDV) by testing against zika, dengue, chikungunya, crimean congo hemorrhagic fever (CCHF), yellow fever, japanese encephalitis (JE) and west nile viruses. A total of 104 samples (human, monkey and tick positive and negative samples) were tested using this assay. ResultNo false positive or false negative results were seen for human, monkey or tick samples. The assay was specific for KFD and could detect upto 100 copies of KFD viral RNA. Discussion and conclusionThe previously published sensitive real time RT-PCR assay requires higher cost in terms of reagents and machine setup and technical expertise has been the primary reason for development of this assay. A single step RT-PCR is relatively easy to perform and more cost effective than real time RT-PCR in smaller setups in the absence of Biosafety Level-3 facility. This study reports the development and optimization of single step RT-PCR assay which is more sensitive and less time-consuming than nested RT-PCR and cost effective for rapid diagnosis of KFD viral RNA.

Abstract 2756: Multiplexing done differently: flexible, sensitive, and accurate detection of miRNA and protein analytes using Firefly particle technology

Abstract Detection of molecular biomarkers in biological fluids is used as an indicator of normal biological processes, pathogenic processes, and response to therapeutic intervention1,2. Using a combined signature of multiple biomarkers can better account for patient and epidemiological heterogeneity, and provide a more accurate indication of patient health2. This has necessitated the development of technologies that can be used for multiplex biomarker profiling directly from biofluids such as plasma, serum, and urine. Conventional assays consume excessive quantities of precious clinical samples, time and budget when used for discovery and validation of multi-analyte biomarker signatures. To address this need, we developed the Firefly Technology platform, which allows for sensitive and accurate detection of up to 75 protein analytes or 68 miRNAs directly from a biological sample. The Firefly platform uses patented Firefly® hydrogel particles and a three-region encoding design that allows for true, in-well multiplexing, providing flexibility over which analytes can be quantified in the same well and allowing for custom panel design. For the detection of protein analytes, the Firefly immunoassays use high-performance matched antibody pairs that reduce cross reactivity between individual analytes, provide up to 5 logs dynamic range, and typically demonstrate single-digit pg/ml sensitivity, while requiring only 12.5 µl biofluid input. Similarly, the Firefly microRNA assay can reliably detect as few as 1000 microRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification. This assay utilizes single step RT-PCR signal amplification using universal primers, thus leveraging PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The Firefly assays have been validated in a wide range of biological samples including plasma, serum, urine and cell culture supernatant, providing experimental flexibility. The 96well plate assay format enables high-throughput screening of samples, with readout conducted on standard flow cytometers, thereby omitting the need for complex and expensive dedicated instrumentation. Finally, the integrated Firefly Analysis Workbench software enables easy and rapid data analysis, visualization, and export in under ten minutes, and includes key features such as standard curve analysis and publication-quality heatmaps and graphs. Here we present data from several studies investigating cytokine profiling in human and rodent samples, and circulating and tumor microRNA profiles, using the Firefly Platform. Together, this novel combination of bioinformatics tools and multiplex, high-sensitivity assays enables rapid discovery and validation of biomarker signatures from fluid samples. Citation Format: Elnaz Atabakhsh, Graeme Doran, Conor Rafferty, Jennifer Heath, Sarah Albertson, Peter Gillis, Jonathan Pinney, Russell Neuner, Michael Chipchase, James Murray, Daniel Pregibon. Multiplexing done differently: flexible, sensitive, and accurate detection of miRNA and protein analytes using Firefly particle technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2756. doi:10.1158/1538-7445.AM2017-2756

Abstract 1082: High-throughput,purification-free, multiplexed profiling of circulating miRNA for discovery,validation, and diagnostics

Abstract To address the needs for circulating miRNA biomarker validation, we developed the Multiplexed Circulating microRNA assay. This assay enables the detection of up to 68 microRNA targets per sample in 96-well format with readout on standard flow cytometers and analysis with an included bioinformatics software package. The Circulating microRNA assay combines particle-based multiplexing, using patented Firefly hydrogel particles, with single-step RT-PCR signal amplification using universal primers. Thus, the Circulating microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the Circulating microRNA assay are displayed and interpreted using our included Firefly Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid discovery and validation of biomarkers, we have generated fixed panels for Oncology, Cardiology, Neurology, Immunology, and Liver Toxicology. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating and tumor microRNA profiles using the Firefly Circulating microRNA Assay Fixed Panels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and validation of microRNA biomarker signatures from fluid specimens. Citation Format: Jessica Tytell, Issac Stoner, Michael Tackett, Graeme Doran, Conor Rafferty, Andreas Windemuth, Daniel Pregibon. High-throughput,purification-free, multiplexed profiling of circulating miRNA for discovery,validation, and diagnostics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1082.

A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector

Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host–virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.

Molecular and seroprevalence of imported dengue virus infection in Al-Madinah, Saudi Arabia

Recently, urban outbreaks of dengue virus (DENV) have occurred in western areas of Saudi Arabia; Jeddah and Makkah, neighbouring cities to Al-Madinah, where there is growing population traffic. DENV activity has not previously been reported in Al-Madinah. Molecular detection of DENV RNA using type-specific single-step real-time RT-PCR and seroprevalence of anti-DENV antibodies using ELISA was reported among Al-Madinah population for the first time through a cross-sectional study from May 2008 to July 2009. A total of 351 febrile, clinically suspected patients were identified, and acute dengue infection was identified in 71 of them during the first week of onset of fever; 5 (1.4 %) by real-time reverse transcription PCR alone, 45 (12.8 %) cases by IgM-ELISA alone and 13 (3.7 %) by both, while 8 (2.3 %) cases were identified during the second week of fever by the presence of IgM-ELISA only. Anti-DENV IgG antibodies were not detected in any of the tested samples. Of the 71 cases, 55 were resident in Al-Madinah (37 Saudi and 18 non-Saudi); however, all of them were imported cases. DENV-1 and DENV-2 were identified in 7 and 11 cases, respectively, while DENV-3 and DENV-4 were not detected in any cases. It was observed that the middle-aged group was the most infected group. DENV anti-DENV IgM antibodies showed a positive correlation of high significance with the number of days with fever. Nationality and gender were found to be significant independent predictors.

Isolation-Amplification of ENV-TM Subunit Genes of Jembrana Disease Virus by a Single Step RT-PCR and Its Direct Cloning in PCR2.1-TOPO Plasmid

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Iris yellow spot virus on onion crops in Calabria, Southern Italy.

The red onion (Allium cepa) cv. Cipolla rossa di Tropea, is a crop of utmost relevance in Calabria (southern Italy) as it consti- tutes the fourth most important agricultural product of Italy en- dowed with a certified protected geographical indication. In May 2012, symptoms resembling those of Iris yellow spot virus (IYSV), consisting of diamond-shaped lesions on the scapes ac- companied by chlorotic or necrotic spots on the leaves, were ob- served in several commercial plots. Symptomatic plants were col- lected from bulb and seed crops and tested for the presence of IYSV, a virus recorded from northern Italy since 2008 (Tomassoli et al., 2009). Total RNA was extracted from scapes and leaves and tested by single-step RT-PCR using primers specific to the vi- ral nucleocapsid (N) gene (Tomassoli et al., 2009). Five of 14 test- ed samples were IYSV-positive, two from fresh market bulb and three from seed crops. Amplicons (ca. 600 bp) were purified, se- quenced and three of the sequences were deposited in GenBank (accession Nos. JX310661, JX310662, JX310663). IYSV isolates from Calabria showed 98.5 to 100% identity at the amino acid level with the N gene sequences of the isolates previously identi- fied in Italy (FJ185142), Serbia and Brazil (EU750697 and AF067070). Real time RT-PCR assays disclosed a higher number (10) of infected samples, confirming the highly sensitive and rap- id detection of IYSV in onion afforded by this technique (Tiberi- ni et al., 2011). To our knowledge, this is the first report of IYSV in Calabria. It confirms spreading of the virus within Italy and calls for surveillance for reducing the impact of IYSV infections on onion crops and other allium species.

Clearance of Macrobrachium rosenbergii nodavirus ( MrNV) and extra small virus (XSV) and immunological changes in experimentally injected Macrobrachium rosenbergii

Macrobrachium rosenbergii was experimentally challenged with Macrobrachium rosenbergii nodavirus ( MrNV) and extra small virus (XSV) to study the clearance of these viruses and consequent changes in various immunological parameters. The healthy animals were injected MrNV and XSV intramuscularly and various organ samples such as gill tissue, head soft tissue, pleopods and intestine were collected at different time intervals of 3, 5, 10, 15, 25, 50, 75 and 100 d post-infection (p.i.) to study the viral clearance. Tissue tropism and clearing of MrNV and XSV were confirmed by RT-PCR, nested RT-PCR and bioassay. These 2 viruses failed to cause mortality or clinical signs of disease in injected adult prawns during the experimental period of 100 days. The result of RT-PCR analysis revealed that all the organs showed positive for both viruses by single step RT-PCR on 3, 5 and 10 d p.i., positive by nested RT-PCR on 15 and 20 d p.i. and all the organs became negative at 25 d p.i. onwards. The viral inoculum prepared from the tissue of MrNV and XSV-injected M. rosenbergii at 3, 5, 10, 15 and 20 d p.i. caused 100% mortality in post-larvae of M. rosenbergii at 9, 8, 7, 10 and 10 d p.i., respectively whereas the inoculum prepared at 25, 50 and 100 d p.i. failed to cause significant mortality in post-larvae of prawn. Immunological parameters such as proPO, superoxide anion, SOD, THC, clotting time and oxyhemocyanin were determined in MrNV and XSV-injected prawns and significant differences in some of the immunological parameters were found in the early days p.i. and became insignificant in the later days p.i.

Evaluation of Pharmacokinetics of Bioreducible Gene Delivery Vectors by Real-time PCR

To investigate pharmacokinetics of reversibly stabilized DNA nanoparticles (rSDN) using a single-step lysis RT-PCR. rSDN were prepared by coating bioreducible polycation/DNA polyplexes with multivalent N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. Targeted polyplexes were formulated by linking cyclic RGD ligand (c(RGDyK)) to the HPMA surface layer of rSDN. The pharmacokinetic parameters in tumor-bearing mice were analyzed by PKAnalyst. The pharmacokinetics of naked plasmid DNA, simple DNA polyplexes, rSDN, and RGD-targeted rSDN exhibited two-compartment model characteristics with area under the blood concentration-time curve (AUC) increasing from 1,102 ng x ml(-1) x min(-1) for DNA to 3,501 ng x ml(-1) x min(-1) for rSDN. Non-compartment model analysis revealed increase in mean retention time (MRT) from 4.5 min for naked DNA to 22.9 min for rSDN. RT-PCR is a sensitive and convenient method suitable for analyzing pharmacokinetics and biodistribution of DNA polyplexes. Surface stabilization of DNA polyplexes can significantly extend their MRT and AUC compared to naked DNA. DNA degradation in rSDN in blood circulation, due to a combined effect of disulfide reduction and competitive reactions with charged molecules in the blood, contributes to DNA elimination.

Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by analysis of patient-specific genomic DNA fusion junctions

To evaluate current detection methods for FIP1L1-PDGFRA in hypereosinophilic syndrome (HES), we developed a means to rapidly amplify genomic break points. We screened 202 cases and detected genomic junctions in all samples previously identified as RT-PCR positive (n=43). Genomic fusions were amplified by single step PCR in all cases whereas only 22 (51%) were single step RT-PCR positive. Importantly, FIP1L1-PDGFRA was detected in two cases that initially tested negative by RT-PCR or fluorescence in situ hybridization. Absolute quantitation of the fusion by real-time PCR from genomic DNA (gDNA) using patient-specific primer/probe combinations at presentation (n=13) revealed a 40-fold variation between patients (range, 0.027-1.1 FIP1L1-PDGFRA copies/haploid genome). In follow up samples, quantitative analysis of gDNA gave 1-2 log greater sensitivity than RQ-PCR of cDNA. Minimal residual disease assessment using gDNA showed that 11 of 13 patients achieved complete molecular response to imatinib within a median of 9 months (range, 3-17) of starting treatment, with a sensitivity of detection of up to 1 in 10(5). One case relapsed with an acquired D842V mutation. We conclude that detection of FIP1L1-PDGFRA from gDNA is a useful adjunct to standard diagnostic procedures and enables more sensitive follow up of positive cases after treatment.

Diagnosis of Enteroviral-associated-Meningitis by RT-PCR for the First Time in Iraq

Introduction: A protocol for testing cerebrospinal fluid specimens using RT-PCR for the diagnosis of central nervous system infections was developed 2 and used to test 20 CSF specimens.Objectives: A-Evaluation of efficiency of In-house prepared Extraction reagent. BAmplified of Enteroviral-RNA genome by single-step RT-PCR. Methods: 20cerebrospinal fluid specimens were included. RNA was extracted from 250μl by method Chomcynki, 1993. EV-RT-PCR was performed for all specimens studied.Results: We successfully isolated total RNA from 20 CSF specimens of AM cases. Ten of 20 specimens were positive by RT-PCR.
 Conclusions: Single-step extraction method was allowed isolation of RNA in 4hr. and provided both high yield and purity of un-degraded RNA preparation. EV-RTPCR detection test is suitable for clinical diagnosis of EV-related meningitis and may improve the management of EV-related neurologic syndromes.

The Single-Step Multiplex Reverse Transcription- Polymerase Chain Reaction Assay for Detecting H5 and H7 Avian Influenza A Viruses

Avian influenza (AI) A virus subtypes H5 and H7 cause severe disease in domestic poultry, including chickens and turkeys. Moreover, H5 and H7 AI A viruses can cross the species barrier from poultry to humans. In the present study, we have developed a single-step multiplex reverse transcription-polymerase chain reaction assay (RT-PCR) for detecting H5 and H7 AI A viruses. This assay was applied to the poultry isolates with the aim of establishing a surveillance method to monitor possible transmission to humans. Two subtype-specific primer sets capable of producing PCR products of 157 and 326 base pairs corresponding to AI A virus H5 and H7 subtypes, respectively, were utilized in a one-step and one-tube reaction. The single-step multiplex RT-PCR assay developed in this study was found to be specific for detecting H5 and H7 AI A viruses. No specific amplification bands were detected with total nucleic acids extracted from other influenza hemagglutinin subtypes and other viral pathogens. The sensitivity of this assay was about 10(3) RNA copies/microl. In conclusion, this novel single-step multiplex RT-PCR is a simple assay with high potential for rapid, specific and cost effective laboratory diagnosis of H5 and H7 AI A virus isolates from clinical specimens of poultry.

Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR

Background Molecular diagnosis of Norovirus infection can be a complex multistep process, which requires significant user intervention and expertise, and is not amenable to automation without extensive validation and optimization. Objectives To develop a real-time multiplexed RT-PCR assay with automated sample preparation that requires only a single-step and a single-tube for reverse transcription, amplification, and detection while exceeding the sensitivity of conventional PCR for broad-spectrum Norovirus detection. Study design Limit of detection was assessed against dilutions of clinical specimens. Fifty archived extractions were used to compare TaqMan sensitivity with either a separate RT using random primers or a single-step RT-PCR. The sensitivity of the novel assay was compared with conventional RT-PCR using 100 specimens from gastroenteritis cases. Results Automated extraction reduced RNA recovery by 0.5 logs compared to manual extraction but was more effective at removing PCR inhibitors from stool specimens. The optimized single-step real-time RT-PCR demonstrated no reduction in sensitivity. Together, the sensitivity of the novel assay was 19% higher than manual extraction with conventional RT-PCR. Conclusions A semi-automated and simplified molecular diagnostic protocol for the rapid detection of Norovirus has been achieved. PCR inhibitors are present in human fecal specimens and cause a significant problem for Norovirus detection by RT-PCR.

Real-time reverse transcription-multiplex PCR for simultaneous and specific detection of rfbE and eae genes of Escherichia coli O157:H7

A real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) was developed for detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to rfbE and eae, respectively, were targeted for reverse transcription, amplification, and real-time detection. A single-step RT-PCR kit containing a mixture of reverse transcriptases converted mRNA into cDNA, which was subsequently amplified by Taq polymerase included in the same kit. The real-time detection of amplification products was achieved by incorporating rfbE(O157)- and eae(O157:H7)-specific TaqMan probes in rRT-MPCR. The ability of two sets of primers and probes for specific detection of rfbE(O157) and eae(O157:H7) was initially verified by screening RNA of eight E. coli serotypes possessing different O antigens and eae alleles. These two sets of primers and probes were also tested in a standard real-time PCR (rPCR) using DNA prepared from several E. coli and non-E. coli strains to verify that only rfbE(O157)- and eae(O157:H7)-specific sequences were amplified and detected. The rRT-MPCR was then evaluated for detecting low-level contamination of EHEC O157:H7 in feces. When RNA prepared from bovine feces, which were artificially seeded with EHEC O157:H7 cells and cultured for five hours, was tested in rRT-MPCR as low as 1cfu/g of feces could be detected. The detection range for the two genes in fecal cultures was 5.1 x 10(-1)-5.1 x 10(4) cfu/g of feces. Thus, the described procedure could be applied to rapid detection of very low levels of EHEC O157:H7 using total RNA as a template. Since the presence of rfbE(O157)- and eae(O157:H7)-specific mRNA is dependent on replicating cells, rRT-MPCR could provide important information about the viability of EHEC O157:H7 in feces.

Single-Step Multiplex Reverse Transcription–Polymerase Chain Reaction (RT-PCR) for Influenza A Virus Subtype H5N1 Detection

Influenza A virus subtype H5N1 causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to humans. The aim of this study was to develop a rapid, cost-saving and effective method for influenza A virus subtype H5N1 detection. The selected primer set was used in single-step RT-PCR for simultaneous detection in multiplex format of the 276-, 189-, and 131-bp fragments, corresponding to sequences specific for M, H5 and N1. The amplified DNA fragments were clearly separated by agarose gel electrophoresis. The sensitivity of this assay was about 10(3) copies/microL. Moreover, this method can be applied to detect not only avian but also human influenza A virus subtype H5N1. In conclusion, the highlights of this particular method are its rapidity and cost-effectiveness, thus rendering it feasible and attractive for large-scale screening at times of influenza A virus subtype H5N1 outbreak.