Detection Of Influenza Virus Type Research Articles

A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A.

IntroductionInfluenza A is known as a public health concern worldwide. In this study, a novel one-step real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was designed and optimized for the detection of influenza A viruses.Material and methodsThe primers and probe were designed based on the analysis of 90 matrix nucleotide sequence data of influenza type A subtypes from the GenBank database of the National Center for Biotechnology Information (NCBI). The influenza virus A/Tehran/5652/2010 (H1N1 pdm09) was used as a reference. The rtRT-PCR assay was optimized, compared with that of the World Health Organization (WHO), and its analytical sensitivity, specificity and reproducibility were evaluated. In total, 64 nasopharyngeal swabs from patients with influenza-like illness (ILI) and 41 samples without ILI symptoms were tested for the virus, using conventional cell culture, direct immunofluorescence antibody (DFA) methods, and one-step rtRT-PCR with the designed primer set and probe and the WHO’s.ResultsThe optimized assay results were similar to the WHO’s. The optimized assay results were similar to WHO’s, with non-significant differences for 10–103 copies of viral RNA/reaction (p > 0.05). It detected 10 copies of viral RNA/reaction with high reproducibility and no cross reactivity with other respiratory viruses. A specific cytopathic effect was observed in 6/64 (9.37%) of the ILI group using conventional culture and DFA staining methods; however, it was not seen in non-ILI. Also, the results of our assay and the WHO’s were similar to those of viral isolation and DFA staining.ConclusionsGiven the high specificity, sensitivity and reproducibility of this novel assay, it can serve as a reliable diagnostic tool for the detection of influenza A viruses in clinical specimens and lab experiments.

Detection of respiratory tract pathogens with molecular biology methods.

This paper describes the use in routine diagnosis of virological kit, which was designed to identify the 15 most common respiratory viruses in clinical specimens of nasopharyngeal aspirates, swabs, and bronchoalveolar lavage. It is a one-step multiplex RT-PCR system for the detection of influenza virus type A and type B, human respiratory syncytial virus type A, B; human adenovirus, human metapneumovirus, human coronaviruses 229E/NL63 and OC43, human parainfluenza type 1, 2, 3, human rhinovirus type A, B, human enterovirus, and bocavirus 1, 2, 3, 4. The article presents research conducted on the basis of swabs collected from patients who came to the Ear, Nose, and Throat Emergency Care Unit at the Department of Otolaryngology, Military Medical Institute in Warsaw, in February 2013. Due to the nature of work in an laryngological emergency ward, the material was collected only from those patients who reported problems associated with rhinitis or any dysfunction of the upper respiratory tract. The study shows that patients who came to seek laryngological assistance were usually infected with viruses having affinity for the airway epithelium.

A novel highly sensitive and specific one-step real time PCR assay for the detection of influenza virus type A

A novel highly sensitive and specific one-step real time PCR assay for the detection of influenza virus type A

Simultaneous detection of influenza virus type B and influenza A virus subtypes H1N1, H3N2, and H5N1 using multiplex real-time RT-PCR

Use of multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza type B virus and influenza A virus subtypes H5N1, H3N2, and H1N1 has been described. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2) copies per microliter or 10(-3)-10(-2) TCID50/L for each subtype, as well as a high reproducibility with coefficient of variation (CV) ranging from 0.27% to 4.20%. The assays can be performed commendably on various models of real-time PCR instruments; including ABI7500, ROCH 2.0, and Mx3005p. In an analysis of 436 clinical samples from patients during the year 2009, this detection method has successfully identified 261 positive samples, as compared to only 189 positive samples using the conventional cell culture systems, and at the same time further differentiated them as 35 type B, 21 subtype H1N1, and 205 subtype H3N2. The results indicate that the multiplex real-time RT-PCR method is a potential tool for rapid screening of influenza virus from a large pool of clinical samples during flu pandemics and facilitates early influenza virus identification in most public health laboratories around the world.

DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7

Endemic of avian influenza virus (AIV) in Asia and epizootics in some European regions have caused considerable public concern on a possible pandemic of AIV. A rapid method for virus detection and effective surveillance in wild avian, poultry production as well as in humans is required. In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification of RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal and tracheal swab specimens was completed in less than 2 h with 94% accuracy.

Simultaneous detection of different respiratory virus by a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting assay

Cell culture and immunofluorescence (IF) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections, but these assays have a low sensitivity and are time consuming. We developed a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting (mRT-PCR–FT-RDB) assay for the simultaneous detection of influenza virus type A including H5 subtype and H9 subtype, influenza virus type B, parainfluenza virus types 1 and 3, respiratory syncytial virus, human rhinovirus, and human coxsackievirus. In comparison with viral culture and IF assay as the gold standard method, the mRT-PCR–FT-RDB assay gave a sensitivity and a specificity of 100% and 98%. The high sensitivity and specificity, the rapid result turnaround time, and the reduced expense of the mRT-PCR–FT-RDB assay compared with viral culture and IF assay suggest that this assay would be a significant improvement over traditional ones for the detection of respiratory viruses in a clinical laboratory.

Lateral-flow immunoassay as a diagnostic test for influenza type A and B in children

Background The diagnosis of influenza remains difficult toestablish because of its similar symptoms to those of respiratoryinfection caused by other viruses. The “gold standard” for thediagnosis of influenza is viral culture, which takes time to gainthe result and is expensive as well. A simple, rapid, and easilyused tool for detection of influenza virus type A and B is needed.Objective To assess the accuracy of lateral-flow immunoassay withQuick Vue Influenza A+B ® in detecting influenza virus of typeA and B.Methods This was an observational study designed for diagnostictest. The subjects were children aged 0-14 years old presentingwith acute respiratory infection in primary Health Care Jetis ,Godean I, Godean II and Prof. Dr. Sardjito Hospital Yogyakarta,from October 2005 to May 2007. Specimens were collected fromboth the anterior nares and the throat by physicians for lateral-flow immunoassay with Quick Vue Influenza A+B ® and viralculture as gold standard. Lateral-flow immunoassay was done ineach study centre, nasal specimen was placed in an extractionreagent tube and sent to NAMRU II laboratory.Results There were 255 children enrolled in this study. Lateral-flow immunoassay by Quick Vue Influenza A+B ® has sensitivity70% (CI95% 6;83%), specificity 93% (CI95% 90;97%), positivepredictive value 68% (CI95% 54;82%), negative predictive value94% (CI95% 91;97%), positive likelihood ratio 10,56 (CI95%6,14;18,19) and negative likelihood ratio 0,32 (CI95% 0,21; 0,51).Conclusion Lateral-flow immunoassay (Quick Vue InfluenzaA+B ® ), nasal swab specimen is not accurate to detect influenzavirus A and B in children.

Development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type A including H5 and H9 subtypes

Avian influenza viruses (AIVs) are endemic in wild birds and, if transmitted to poultry, can cause serious economic losses. The aim of this study was to develop a multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of influenza virus type A, including H5 and H9 subtypes. The selected primers and various labeled TaqMan reporter probes corresponding to matrix, H5, and H9 genes were used in a multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. The results showed that the multiplex real-time RT-PCR assay can be applied to detect RNA of influenza virus type A including H5 and H9 subtypes with a high specificity and a sensitivity of 10 copies per reaction. As a result of its short turnaround times and a high specificity and sensitivity, the assay is very suitable for large-scale screening during AIV outbreaks.

Commercial Immunoassay Kits for the Detection of Influenza Virus Type A: Evaluation of Their Use with Poultry

Five antigen capture immunoassay test kits, Directigen Flu A (Becton Dickinson), QuickVue Influenza test kit (Quidel), FLU OIA (ThermoBiostar), Zstat Flu (ZymeTx, Inc.) and NOW FLU A Test (Binax) were used to detect avian influenza virus (AIV) in clinical specimens as per manufacturers' protocols. Each kit was shown to be specific for AIV propagated in embryonating chicken eggs (ECE); other respiratory viruses of poultry tested gave negative results. The Directigen Flu A kit proved to be 10-fold more sensitive than the other kits, capable of detecting 10(4.7) mean embryo lethal dose (ELD50)/ml in allantoic fluid; this is more sensitive than the hemagglutination test using chicken erythrocytes. None of the kits proved to be sufficiently sensitive to reliably detect AIV in oropharyngeal and cloacal swabs collected from chickens experimentally infected with AIV subtype H6N2. In two different experiments, individual swabs and pools of five or six swabs were tested. By virus isolation, 39 individual oropharyngeal swabs tested positive for AIV, but Directigen and Flu OIA only detected 2/39 and NOW FLU A 1/39. Zstat and QuickVue did not detect any. Five individual cloacal swabs positive by virus isolation were negative with all five kits. In a second experiment using pools of five swabs, 26 swab pools were positive by virus isolation and 5/26 were positive by Directigen, the only kit to provide any positive results. Five cloacal swab pools were also positive by virus isolation and 1/5 was positive by Directigen; all other test kits were negative. All of these experiments were performed using the H6N2 subtype of AIV. The results are disappointing, as the kits have proven to be insensitive for detecting AIV when compared with the gold standard, virus isolation. This limits their use in diagnostic field investigations. Individual or groups of chickens could be assumed to be positive for AIV if positive by any of the kits, but a negative result with any of the kits would not prove that birds were AIV free.

Evaluation of a single-step multiplex RT-PCR for influenza virus type and subtype detection in respiratory samples.

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza virus type and subtypes was evaluated for its use in clinical specimens. One part of each specimen was tested using an established standard culture and/or monoclonal antibody-based immunofluorescence assay method, and the other part was tested by the multiplex RT-PCR method for the presence or absence of the influenza virus, and its type and subtype. Sixty-seven specimens were examined. The results revealed a strong agreement between the data obtained by the established method and those obtained by the multiplex RT-PCR.