Evaluation of Three Influenza A and B Real-Time Reverse Transcription-PCR Assays and a New 2009 H1N1 Assay for Detection of Influenza Viruses

Selvaraju, Suresh B.; Selvarangan, Rangaraj

doi:10.1128/jcm.02464-09

Cited by 95 publications

(78 citation statements)

References 14 publications

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“…For human RNA, a commercially available real-time PCR assay specific for ␤2M mRNA was used, because this assay is validated to not detect DNA. Specific PCR assays to detect S. pneumoniae DNA and influenza A RNA were described elsewhere (11,17). MolYsis method I involves a centrifugation step intended to separate the viral supernatant prior to DNase and RNase treatment of the pellet.…”

Section: Resultsmentioning

confidence: 99%

“…5 TCID 50 /ml), adenovirus (2.8 ϫ 10 6 TCID 50 /ml), and influenza A virus (PCR threshold cycle [C T ] ϭ 24) were directly added from cultured stocks (10,11). Then, 5 l of each of bacterial and viral preparation was spiked into 1 ml of CSF or NPA samples and vortexed for 10 s before further processing.…”

Section: Bacterial and Viral Strains Escherichia Coli (American Typementioning

confidence: 99%

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Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

et al. 2016

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Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P < 0.05) and improved the sensitivity of pathogen detection (P < 0.01), with a 20-to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples. Clinical microbiology is one of the most rapidly changing areas of laboratory medicine today due to the introduction of new technologies and automation (1). Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3-6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays. These less common organisms, for which tests are not readily available, are likely responsible for many cases of undiagnosed illness, particularly in critically ill patients and those with compromised immunity (7,8). Therefore, there is increasing interest in the application of novel technologies, such as next-generation sequencing (NGS), for unbiased detection of pathogens in clinical samples.Among the various challenges with the implementation of NGS for routine pathogen detection using metagenomics, the presence of an overwhelming amount of host DNA is one of the most important problems to be addressed. A previous metagenomic study on nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections revealed that up to ϳ95% of raw NGS reads were of human DNA (9). The su...

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Section: Resultsmentioning

confidence: 99%

Section: Bacterial and Viral Strains Escherichia Coli (American Typementioning

confidence: 99%

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

et al. 2016

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“…Thirty nasal swab samples also were collected from one PRRSV-positive swine herd and one PRRSV-negative swine herd, herd D (see Table 4). These serum samples were tested by the HI assay to detect virus-specific antibody to IBVs, and nasal swabs were examined by real-time RT-PCR assays targeting the NS gene of IBV and the M gene of IAV (36). Samples positive for IBV were used for virus isolation in chicken embryonated eggs and MDCK cells.…”

Section: Cellsmentioning

confidence: 99%

Domestic Pigs Are Susceptible to Infection with Influenza B Viruses

Ran

Shen

Lang

et al. 2015

J Virol

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Influenza Influenza viruses are classified as genera A, B, and C, in accordance with the antigenic differences in their nucleoproteins (NP) and matrix 1 (M1) proteins (28). Influenza A (IAV) and B (IBV) viruses can result in severe upper respiratory disease in humans, while influenza C viruses (ICV) cause relatively mild disease (9, 23). Among influenza viruses, IAV and IBV are very similar in terms of genome structure and organization. IBV, along with influenza A(H3N2) and A(H1N1) viruses [including A(H1N1)pdm09 virus], cause seasonal influenza epidemics annually (9, 23). In the United States alone during 1976 to 2007, approximately 3,000 to 49,000 deaths each year have been attributed to these epidemics (42). Some reports indicate that in older children and healthy adults, influenza A(H3N2) virus is responsible for the most severe cases, followed by IBV, while influenza A(H1N1) virus infections tend to manifest as the mildest cases of illness (1,5,23,25). In some seasons, however, IBV may be the predominate strain responsible for influenza activities. This was best exemplified by the 1979-1980 season, in which IBV was the predominant strain circulating in the United States; therefore, it was responsible for influenza outbreaks and excess pneumonia and influenza deaths nationwide (39). Furthermore, IBV has been reported to be associated with central nervous system complications, such as Reye's syndrome and encephalitis in children (1).IBVs continue to circulate worldwide alongside IAVs. Actively circulating IBVs are divided into two genetically and antigenically

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“…The rapid and precise laboratory diagnosis of influenza during pandemic periods is closely related to the control of influenza prevalence and the treatment of critically ill g foot and mouth disease virus; h infectious bronchitis virus; i infectious bursal disease virus; and j control Fig. 3 Results of the assessment of chip sensitivity [9][10][11][12]. The present study explored the possibility of testing for the influenza virus using a microarray.…”

Section: Discussionmentioning

confidence: 99%

“…The chip results were reexamined using American CDC real-time PCR testing method recorded in the literature [9].…”

Section: Validation Of Chips Using Clinical Specimensmentioning

confidence: 99%

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

Tian

et al. 2013

Indian J Microbiol

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In this study, we present a microarray approach for the typing of influenza A and B viruses, and the subtyping of H1 and H3 subtypes. We designed four pairs of specific multiplex RT-PCR primers and eight specific oligonucleotide probes and prepared microarrays to identify the specific subtype of influenza virus. Through amplification and fluorescent marking of the multiplex RT-PCR products on the M gene of influenza A and B viruses and the HA gene of subtypes H1 and H3, the PCR products were hybridized with the microarray, and the results were analyzed using a microarray scanner. The results demonstrate that the chip developed by our research institute can detect influenza A and B viruses specifically and identify the subtypes H1 and H3 at a minimum concentration of 1 9 10 2 copies/lL of viral RNA. We tested 35 clinical samples and our results were identical to other fluorescent methods. The microarray approach developed in this study provides a reliable method for the monitoring and testing of seasonal influenza.

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Evaluation of Three Influenza A and B Real-Time Reverse Transcription-PCR Assays and a New 2009 H1N1 Assay for Detection of Influenza Viruses

Cited by 95 publications

References 14 publications

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

Domestic Pigs Are Susceptible to Infection with Influenza B Viruses

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

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