Reverse Transcription-multiplex PCR Research Articles

A reverse transcription-multiplex PCR strategy devised for concomitant detection and differentiation of foot and mouth disease virus serotypes O, A and Asia 1 in India

Serotype identification occupies the central part of foot and mouth disease (FMD) diagnosis workflow and vaccination decision tree. In this study, a reverse transcription-multiplex PCR (RT-mPCR) strategy wherein three assays with unique combinations of serotype specific primers targeting the VP1 region was developed to differentiate FMD virus serotypes O, A and Asia 1 based on differential size of the PCR amplicons on agarose gel. Their diagnostic performance relative to the mPCR assay in use in India was evaluated on 169 clinical samples and 210 cell culture grown virus isolates. The relative diagnostic sensitivity was found to be 99.69%, 98.78% and 99.08% for primer combinations 1, 2 and 3, respectively. These assays proved their worth by detecting serotype in three FMD suspected specimens that went undiagnosed in the existing mPCR and also by identifying multiple serotypes in the same sample. Their detection limits varied from log10 2 to log10 4 viral RNA dilution and from 100 to 0.1 TCID50 virus depending on the serotype. The validated novel mPCR assays show promise to be included in the routine diagnostic tool-box to augment the efficiency of diagnosis of FMD virus serotypes that display extreme genetic diversity and a tendency of transboundary dispersal.

Detection of viable Salmonella Typhi by reverse transcription-multiplex polymerase chain reaction

The detection of viable (clinically significant) Salmonella enterica subsp. enterica serovar Typhi in food is essential from clinical, epidemiological, and infection control perspective. In this study, a method for detection of viable S. Typhi was developed based on reverse transcription-multiplex PCR (RT-MPCR). Three primer pairs used in RT-MPCR were invA-f/invA-r, ivaB-f/ivaB-r and fliC-d-f/fliC-d-r that were specific to invA gene, ivaB region and fliC-d gene, respectively. Of all S. enterica serovars, only S. Typhi had all target DNA that was able to be amplified by all 3 primer pairs. When RNA extracted from S. Typhi was subjected to RT-MPCR, 3 DNA products with the sizes of 284, 599 and 763 were observed. This method was shown to specifically detect S. Typhi with the detection limit of 50 CFU in pure culture. Moreover, RT-MPCR was shown to be able to detect S. Typhi and to discriminate between viable and nonviable cells in food.

Nicotine excites corticotropin-releasing hormone mRNA-expressing neuron in the hypothalamic paraventricular nucleus in vitro in rats.

Nicotine is known to modulate the activity of the hypothalamic-pituitary-adrenal axis by stimulating corticotropin-releasing hormone (CRH) release from the hypothalamic paraventricular nucleus (PVN). However, the mechanism by which nicotine affects the hypothalamic-pituitary-adrenal axis by modulating PVN CRH neuronal activity is currently unclear. Here, we examined the effects of nicotine on PVN CRH-mRNA-expressing neurons in vitro in rats by whole-cell patch-clamp recordings, biocytin staining, and single-cell reverse transcription-multiplex PCR techniques. Of the 146 PVN putative parvocellular neurons, 17.1% (25/146) coexpressed GAPDH mRNA and CRH mRNA. Under current-clamp recording conditions, application of nicotine (1 μM) induced excitation in 92% (23/25) PVN CRH-mRNA-expressing neurons, which showed a significant increase in the spike firing rate accompanied by a depolarization of the membrane potential. Nicotine induced an increase in the spike firing rate of PVN CRH-mRNA-expressing neurons in a concentration-dependent manner. The half-effective concentration (EC50) of nicotine for increasing the spike firing rate of PVN CRH-mRNA-expressing neurons was 1.6 μM. Extracellular application of ionotropic glutamate receptor antagonist kynurenic acid (1 mM) abolished the nicotine-induced excitation of PVN CRH-mRNA-expressing neurons. Moreover, application of nicotine induced a significant increase in the spontaneous excitatory postsynaptic currents frequency, but without significantly altering the spontaneous excitatory postsynaptic currents amplitude of the CRH-mRNA-expressing neurons. Biocytin staining confirmed that the nicotine-sensitive CRH-mRNA-expressing neurons were located in the PVN parvocellular division. These results indicated that extracellular administration of nicotine indirectly excited PVN CRH-mRNA-expressing neurons, suggesting that nicotine modulated PVN CRH secretion by enhancement of both the presynaptic action potential drive and quantal glutamate release.

Surveillance of dengue and chikungunya infection in Dong Thap, Vietnam: A 13-month study.

To establish a surveillance in Dong Thap, at the border with Cambodia by assessing the presence of DENV serotypes and CHIKV among patients hospitalized at Dong Thap general hospital. Cross-sectional descriptive analysis was conducted on a cohort of 131 patients hospitalized with acute fever and symptoms compatible with dengue or chikungunya. The study was conducted from January 2012 to February 2013. The full clinical picture was established as well as serological and molecular detection. Serological analysis was sequentially performed on blood samples collected on admission and an average of seven days after admission. The detection of IgM antibody to DENV was performed by IgM capture ELISA and the detection of DENV and CHIKV RNA was done by reverse-transcription multiplex PCR. 101 patients out of 131 (77%) were confirmed with dengue. All four dengue serotypes were detected with a predominance of DENV2 and DENV4. No chikungunya infection was detected although reported in neighboring Cambodia. A differential efficiency of serological dengue detection was observed. Efficiency was 29% upon admission and 53% after seven days on the same patients. 30 patients out of 131 (23%) were negative with both DENV and CHIKV. Dengue is at risk of being underestimated and chikungunya is not systematically detected. Changes in detection and surveillance procedures are therefore discussed to increase efficiency of dengue detection and continue the monitoring the emergence of CHIKV in Dong Thap province and in Vietnam.

Survey of viral infections in spontaneous grapevines from natural environments in Sicily

The presence of nine of the most economical important grapevine viruses was surveyed in eight spontaneous grapevine populations from natural areas of Sicily. Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1, 2 and 3 (GLRaV-1, −2 and −3), Grapevine virus A and B (GVA, GVB), Grapevine rupestris stem pitting-associated virus (GRSPaV) and Grapevine fleck virus (GFKV) infections were assessed by molecular and serological methods in 73 vines collected in summer 2013 and winter 2013–2014. Reverse transcription-multiplex PCR detected nine GRSPaV- and one GFLV-infected plants in five and one grapevine populations, respectively, while no ArMV, GLRaV-1, GLRaV-2, GLRaV-3, GVA, GVB, GFKV infections were detected. ELISA tests on dormant wood confirmed the result of the molecular detection. The relationship of Sicilian wild grapevine isolates with isolates from grapevines of different geographic origin was investigated through sequencing of the complete coat protein (CP) region of GRSPaV, and partial CP and homing protein (HP) domains of GFLV. Pairwise comparison among the Sicilian GRSPaV CP sequences showed identity scores ranging from 81.67 % to 99.87 % and from 92.66 % to 100 % at nucleotide (nt) and amino acid level (aa), respectively. For GFLV, the CP sequence showed identity ranges from 84.28 % to 90.48 % (nt) and from 89.39 % to 97.11 % (aa); higher variability was obtained analysing the HP domain with identity scores ranging from 68.42 to 93.28 % (nt) and from 58.02 % to 93.23 % (aa). According to phylogenetic analyses, GRSPaV and GFLV isolates from Sicilian wild grapevines clustered with isolates from cultivated grapevines without any correlation between isolate distribution and their geographic origin.

913 Detection of RSV Types A & B and Influenza Virus Types A & B in CAP by Reverse Transcription-Multiplex PCR

Respiratory syncytial virus (RSV) type A and B, influenza A and B cause about 80% of viral lower respiratory tract infections. Multiplex RT-PCR has a significant advantage in that it...

Detection ofAeromonas salmonicidaby Reverse Transcription-Multiplex Polymerase Chain Reaction

Aeromonas salmonicida is one of the major fish pathogens causing economically devastating losses in aquaculture. A. salmonicida subsp. salmonicida is a typical A. salmonicida causing furunculosis, while the other subspecies are atypical strains causing ulcer diseases. PCR-based methods of detecting A. salmonicida suffer from the drawback that they do not distinguish living (pathogenic) from dead cells. In this study, a method of detecting A. salmonicida was developed based on reverse transcription-multiplex PCR (RT-MPCR) using two sets of primers, SV1/SV2 and SF1/SF2, specific to the vapA gene and the fstB gene of A. salmonicida respectively. This method was found to detect A. salmonicida specifically with detection limits of 10 CFU in pure culture and 30 CFU in the presence of tissue debris. It was also found distinguish not only between viable and nonviable cells but also between typical and atypical strains of A. salmonicida. Using RT-MPCR, two DNA fragments, of 542 and 1,258 bp, were amplified from RNA of typical A. salmonicida, whereas only one DNA fragment, of 542 bp, was amplified from the RNA of the atypical ones. The proposed assay was also used successfully to detect A. salmonicida in artificially infected rainbow trout (Oncorhyncus mykiss).

Simultaneous detection of respiratory syncytial virus types A and B and influenza virus types A and B in community-acquired pneumonia by reverse transcription-multiplex PCR

BackgroundRespiratory syncytial virus (RSV) types A and B and influenza A and B cause about 80–90% of viral lower respiratory tract infections. It is impossible to distinguish the cause of viral respiratory infections by their clinical presentation. Multiplex RT-PCR has a significant advantage in that it permits the simultaneous amplification of several viruses in a single reaction facilitating cost-effective diagnosis and perhaps improved clinical management. ObjectivesIn this study, our aim was to determine the prevalence of influenza A and B, and RSV types A and B among children with CAP, by the use of the newly developed rapid, accurate, and pathogen-specific technique of multiplex RT-PCR in order to accomplish the greatest positive effect on patient care and health care costs. Study methodologyThis study is a cross-sectional study involving 24 children admitted to the Children’s Hospital of the Ain Shams University due to severe lower respiratory tract infection (LRTI). Clinical and radiological assessment of all patients was performed followed by the molecular analysis of both respiratory and blood samples of all enrolled patients simultaneously by the use of the newly available reverse transcription-multiplex PCR technique (RT-m PCR). ResultsViral pneumonia was detected in one-third of enrolled patients (8/24), with the predominance of respiratory syncytial virus A (4/8), followed by influenza A virus (3/8) and influenza B virus (1/8) while no cases of respiratory syncytial virus B was detected. The same results were identified in both blood and respiratory specimens. ConclusionReverse transcription-multiplex PCR technique – multiplex has a significant advantage in that it permits the simultaneous amplification of several viruses in a single reaction making it well suited for use in epidemiological studies and to improve etiology-directed clinical management of viral pneumonia.

Identification of Viable <i>Listeria</i> Species Based on Reverse Transcription-Multiplex PCR (RT-MPCR) and Restriction Digestion

A novel method for the identification of viable Listeria species was developed based on reverse transcription-multiplex PCR (RT-MPCR) and restriction digestion. The targets for RT-MPCR were iap mRNAs whose genes are common to all Liseria species. A set of five primers was used in this study. Two of them were genus specific, and the other three were specific to L. monocytogenase, L. innocua, and L. grayi respectively. By RT-MPCR, L. monocytogenese, L. innocua, L. grayi, and a group of Listeria species, including L. ivanovii, L. welshimeri, and L. seeligeri, were specifically identified. To differentiate the latter three Listeria species, RT-MPCR products were subjected to digestion with HpaI and ScaI. The sensitivity of RT-MPCR in detecting Listeria species was determined to be 50 CFU/mL. RT-MPCR was found to discriminate between viable and nonviable cells and to detect viable Listeria species in a food model.

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.

Analysis of Chromosome Translocations Involving MML by Hybridization with an Oligonucleotide Microarray

Identification of chromosome rearrangements is of importance for exact diagnosis, risk assessment, and therapy in blood malignancies. A new method was proposed for rapid and accurate identification of leukemia forms caused by chromosome rearrangements involving MLL (11q23). The method combines reverse transcription-multiplex PCR and hybridization with an oligonucleotide microarray. The microarray was designed to detect the five most common MLL rearrangements: t(4;11) MLL/AF4, t(9;11) MLL/AF9, t(11;19) MLL/ELL, t(11;19) MLL/ENL, and dup(11) MLL/MLL. With clinical specimens, the method was shown to efficiently identify the chromosome translocations in leukemia patients.