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      Rapid typing of influenza viruses using super high-speed quantitative real-time PCR


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          ► A super high speed qRT-PCR (SHRT-PCR) was developed. RT reaction and PCR (40 cycles) can be completed in less than 20 min. ► Despite the high speed, the sensitivity and specificity of the SHRT-PCR are comparable to conventional qRT-PCR system. ► The SHRT-PCR can be used to test clinical samples.


          The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2 h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18 s/cycle; 40 cycles in less than 20 min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10 −1 plaque-forming unit/reaction for viruses in culture supernatants during 20 min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.

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          Triple-reassortant swine influenza A (H1) in humans in the United States, 2005-2009.

          Triple-reassortant swine influenza A (H1) viruses--containing genes from avian, human, and swine influenza viruses--emerged and became enzootic among pig herds in North America during the late 1990s. We report the clinical features of the first 11 sporadic cases of infection of humans with triple-reassortant swine influenza A (H1) viruses reported to the Centers for Disease Control and Prevention, occurring from December 2005 through February 2009, until just before the current epidemic of swine-origin influenza A (H1N1) among humans. These data were obtained from routine national influenza surveillance reports and from joint case investigations by public and animal health agencies. The median age of the 11 patients was 10 years (range, 16 months to 48 years), and 4 had underlying health conditions. Nine of the patients had had exposure to pigs, five through direct contact and four through visits to a location where pigs were present but without contact. In another patient, human-to-human transmission was suspected. The range of the incubation period, from the last known exposure to the onset of symptoms, was 3 to 9 days. Among the 10 patients with known clinical symptoms, symptoms included fever (in 90%), cough (in 100%), headache (in 60%), and diarrhea (in 30%). Complete blood counts were available for four patients, revealing leukopenia in two, lymphopenia in one, and thrombocytopenia in another. Four patients were hospitalized, two of whom underwent invasive mechanical ventilation. Four patients received oseltamivir, and all 11 recovered from their illness. From December 2005 until just before the current human epidemic of swine-origin influenza viruses, there was sporadic infection with triple-reassortant swine influenza A (H1) viruses in persons with exposure to pigs in the United States. Although all the patients recovered, severe illness of the lower respiratory tract and unusual influenza signs such as diarrhea were observed in some patients, including those who had been previously healthy. 2009 Massachusetts Medical Society
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            Simultaneous detection of influenza viruses A and B using real-time quantitative PCR.

            Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.
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              Early diagnosis of SARS Coronavirus infection by real time RT-PCR

              Background: A novel coronavirus was recently identified as the aetiological agent of Severe Acute Respiratory Syndrome (SARS). Molecular assays currently available for detection of SARS-coronavirus (SARS-Cov) have low sensitivity during the early stage of the illness. Objective: To develop and evaluate a sensitive diagnostic test for SARS by optimizing the viral RNA extraction methods and by applying real-time quantitative RT-PCR technology. Study design: 50 nasopharyngeal aspirate (NPA) samples collected from days 1–3 of disease onset from SARS patients in whom SARS CoV infections was subsequently serologically confirmed and 30 negative control samples were studied. Samples were tested by: (1) our first generation conventional RT-PCR assay with a routine RNA extraction method (Lancet 361 (2003) 1319), (2) our first generation conventional RT-PCR assay with a modified RNA extraction method, (3) a real-time quantitative RT-PCR assay with a modified RNA extraction method. Results: Of 50 NPA specimens collected during the first 3 days of illness, 11 (22%) were positive in our first generation RT-PCR assay. With a modification in the RNA extraction protocol, 22 (44%) samples were positive in the conventional RT-PCR assay. By combining the modified RNA extraction method and real-time quantitative PCR technology, 40 (80%) of these samples were positive in the real-time RT-PCR assay. No positive signal was observed in the negative controls. Conclusion: By optimizing RNA extraction methods and applying quantitative real time RT-PCR technologies, the sensitivity of tests for early diagnosis of SARS can be greatly enhanced.

                Author and article information

                J Virol Methods
                J. Virol. Methods
                Journal of Virological Methods
                Published by Elsevier B.V.
                22 August 2011
                December 2011
                22 August 2011
                : 178
                : 1
                : 75-81
                [a ]Department of Molecular Medical Research, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan
                [b ]Department of Microbiology, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-Cho, Shinjuku-ku, Tokyo 169-0073, Japan
                [c ]Trust Medical company limited, 1044 Asazuma-cho, Kasai, Hyogo 679-0105, Japan
                [d ]Department of Laboratory Medicine, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113-0021, Japan
                [e ]Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, N18 W9, Kita-ku, Sapporo 060-0818, Japan
                Author notes
                [* ]Corresponding author. Tel.: +81 3 5316 3299; fax: +81 3 5316 3173. shibasaki-ft@ 123456igakuken.or.jp
                Copyright © 2011 Published by Elsevier B.V.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 28 June 2011
                : 10 August 2011
                : 16 August 2011

                Microbiology & Virology
                influenza virus,typing,super high-speed quantitative real-time pcr
                Microbiology & Virology
                influenza virus, typing, super high-speed quantitative real-time pcr


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