Development and validation of a commercial real-time NASBA assay for the rapid confirmation of influenza A H5N1 virus in clinical samples

Abstract The Hologic Panther Fusion® Open Access™ functionality allows implementation of laboratory-developed tests (LDTs), with fully automated sample extraction, real-time PCR, and result interpretation. We report the development and validation of a multiplex LDT for norovirus G1, norovirus G2, and rotavirus from stool samples on this system. The LDT was optimized for primer and probe sequences, salt concentration, and PCR annealing temperature. Reproducibility of the PCR and extraction process was assessed. Performance of the multiplex LDT assay was evaluated with external quality assessment (EQA) samples and compared to a commercial multiplex assay (Allplex™ GI-Virus Assay, Seegene) in clinical samples. Salt concentrations and annealing/extension temperature were optimized to 4 mM MgCl2, 70 mM KCl, 20 mM Tris, and 60 °C, respectively. The user-prepared part of the LDT PCR mix (containing salts, probes, and primers) was stable for ≥ 11 days onboard the instrument. We observed reproducible results of PCR and the extraction process. The LDT had a sensitivity comparable to or greater than the commercial Allplex™ assay and showed excellent linearity. Forty-five EQA samples yielded the expected result with the LDT. There was 100% concordance between LDT and Allplex™ results in 160 clinical samples. Results from the suspension and direct swab stool sample preparation methods were highly concordant in the LDT. We report the successful development and validation of a multiplex PCR LDT for detection of norovirus G1, norovirus G2, and rotavirus from stool samples on the Panther Fusion® system.

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Novel real-time PCR-based patho- and phylotyping of potentially zoonotic avian influenza A subtype H5 viruses at risk of incursion into Europe in 2017

Eurosurveillance ◽

10.2807/1560-7917.es.2017.22.1.30435 ◽

2017 ◽

Vol 22 (1) ◽

Cited By ~ 9

Author(s):

Mahmoud M Naguib ◽

Annika Graaf ◽

Andrea Fortin ◽

Christine Luttermann ◽

Ulrich Wernery ◽

...

Keyword(s):

Avian Influenza ◽

Real Time ◽

Influenza A ◽

Wild Bird ◽

Reaction Times ◽

Highly Pathogenic ◽

Bird Populations ◽

Western Africa ◽

Speed Up ◽

Development And Validation

Since November 2016, Europe witnesses another wave of incursion of highly pathogenic avian influenza (HPAI) A(H5) viruses of the Asian origin goose/Guangdong (gs/GD) lineage. Infections with H5 viruses of clade 2.3.4.4b affect wild bird and poultry populations. H5 viruses of clades 2.2, 2.3.1.2c and 2.3.4.4a were detected previously in Europe in 2006, 2010 and 2014. Clades 2.2.1.2 and 2.3.2.1.c are endemic in Egypt and Western Africa, respectively and have caused human fatalities. Evidence exists of their co-circulation in the Middle East. Subtype H5 viruses of low pathogenicity (LPAI) are endemic in migratory wild bird populations. They potentially mutate into highly pathogenic phenotypes following transmission into poultry holdings. However, to date only the gs/GD H5 lineage had an impact on human health. Rapid and specific diagnosis marks the cornerstone for control and eradication of HPAI virus incursions. We present the development and validation of five real-time RT-PCR assays (RT-qPCR) that allow sequencing-independent pathotype and clade-specific distinction of major gs/GD HPAI H5 virus clades and of Eurasian LPAI viruses currently circulating. Together with an influenza A virus-generic RT-qPCR, the assays significantly speed up time-to-diagnosis and reduce reaction times in a OneHealth approach of curbing the spread of gs/GD HPAI viruses.

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Sensitive quantitative and rapid immunochromatographic diagnosis of clinical samples by scanning electron microscopy - preparing for future outbreaks

10.1101/2020.05.20.20106864 ◽

2020 ◽

Author(s):

Hideya Kawasaki ◽

Hiromi Suzuki ◽

Masato Maekawa ◽

Takahiko Hariyama

Keyword(s):

Electron Microscopy ◽

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Influenza A ◽

Clinical Samples ◽

Chain Reaction ◽

Detection Ability ◽

Polymerase Chain ◽

Scanning Electron

AbstractBackgroundAs pathogens such as influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can easily cause pandemics, rapid diagnostic tests are crucial for implementing efficient quarantine measures, providing effective treatments to patients, and preventing or containing a pandemic infection. Here, we developed the immunochromatography-NanoSuit® method, an improved immunochromatography method combined with a conventional scanning electron microscope (SEM), which enables observation of immunocomplexes labeled with a colloidal metal.Methods and FindingsThe detection ability of our method is comparable to that of real-time reverse transcription-polymerase chain reaction and the detection time is approximately 15 min. Our new immunochromatography-NanoSuit® method suppresses cellulose deformity and makes it possible to easily focus and acquire high-resolution images of gold/platinum labeled immunocomplexes of viruses such as influenza A, without the need for conductive treatment as with conventional SEM. Electron microscopy (EM)-based diagnosis of influenza A exhibited 94% clinical sensitivity (29/31) (95% confidence interval [95%CI]: 78.58–99.21%) and 100% clinical specificity (95%CI: 97.80–100%). EM-based diagnosis was significantly more sensitive (71.2%) than macroscopic diagnosis (14.3%), especially in the lower influenza A-RNA copy number group. The detection ability of our method is comparable to that of real-time reverse transcription-polymerase chain reaction.ConclusionsThis simple and highly sensitive quantitative analysis method involving immunochromatography can be utilized to diagnose various infections in humans and livestock, including highly infectious diseases such as COVID-19.

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Development and validation with clinical samples of internally controlled multiplex real-time PCR for diagnosis of BKV and JCV infection in associated pathologies

Comparative Immunology Microbiology and Infectious Diseases ◽

10.1016/j.cimid.2011.12.010 ◽

2012 ◽

Vol 35 (2) ◽

pp. 173-179 ◽

Cited By ~ 3

Author(s):

Ana Bárcena-Panero ◽

Juan E. Echevarría ◽

María Pilar Romero-Gómez ◽

Enrique Royuela ◽

Ana Castellanos ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Development And Validation

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Real-time molecular beacon NASBA for rapid and sensitive detection of norovirus GII in clinical samples

Canadian Journal of Microbiology ◽

10.1139/w09-105 ◽

2009 ◽

Vol 55 (12) ◽

pp. 1375-1380 ◽

Cited By ~ 15

Author(s):

Safaa Lamhoujeb ◽

Hugues Charest ◽

Ismail Fliss ◽

Solange Ngazoa ◽

Julie Jean

Keyword(s):

Real Time ◽

Molecular Beacon ◽

Clinical Samples ◽

Rt Pcr ◽

Reading Frame ◽

Viral Loads ◽

Reverse Transcription Pcr ◽

Highly Sensitive ◽

Nasba Assay ◽

Norovirus Gii

To improve the sensitivity and efficiency of the real-time nucleic acid sequence based amplification (NASBA) assay targeting the open reading frame 1–2 (ORF1–ORF2) junction of the norovirus (NoV) genome, a selection of clinical samples were analyzed. The assay results were compared with those of TaqMan and conventional reverse transcription PCR (RT-PCR) and a commercial enzyme-linked immunoassay (ELISA) for the specific detection of GII NoV in 96 fecal samples. Based on end-point dilution, the two real-time assays had similar sensitivities (0.01 particle detectable units), two log10cycles greater than that of conventional RT-PCR. GII NoV was detected in 88.54% of the samples by real-time NASBA, in 86.46% by TaqMan RT-PCR, in 81.25% by conventional RT-PCR, and in 65.7% by ELISA. The two real-time assays were in agreement for 88.5% of the samples. These results demonstrate that real-time NASBA with a molecular beacon probe is highly sensitive, accurate, and specific for NoV detection in clinical samples. Applying this technique to samples with complex matrix and low viral loads, such as food and environmental samples, could be useful for the detection of NoVs and will improve the prevention of NoV outbreaks.

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A rapid and label-free platform for virus capture and identification from clinical samples

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910113117 ◽

2019 ◽

Vol 117 (2) ◽

pp. 895-901 ◽

Cited By ~ 25

Author(s):

Yin-Ting Yeh ◽

Kristen Gulino ◽

YuHe Zhang ◽

Aswathy Sabestien ◽

Tsui-Wen Chou ◽

...

Keyword(s):

Influenza Virus ◽

Real Time ◽

Influenza A ◽

Respiratory Infections ◽

Surface Enhanced Raman Spectroscopy ◽

Clinical Samples ◽

Label Free ◽

Influenza A Viruses ◽

Virus Identification ◽

Surface Enhanced Raman

Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 102 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.

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