scholarly journals Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

2015 ◽  
Author(s):  
Alex L Greninger ◽  
Samia N Naccache ◽  
Scot Federman ◽  
Guixia Yu ◽  
Placide Mbala ◽  
...  
Keyword(s):  
Real Time ◽  
Ebola Virus ◽  
Hemorrhagic Fever ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Viral Pathogens ◽  
Web Based ◽  
Lower Titer

We report unbiased metagenomic detection of chikungunya virus (CHIKV), Ebola virus (EBOV), and hepatitis C virus (HCV) from four human blood samples by MinION nanopore sequencing coupled to a newly developed, web-based pipeline for real-time bioinformatics analysis on a computational server or laptop(MetaPORE). At titers ranging from 107-108 copies per milliliter, reads to EBOV from two patients with acute hemorrhagic fever and CHIKV from an asymptomatic blood donor were detected within 4 to 10 minutes of data acquisition, while lower titer HCV virus (1x105 copies per milliliter) was detected within 40 minutes. Analysis of mapped nanopore reads alone, despite an average individual error rate of 24% [range 8-49%], permitted identification of the correct viral strain in all 4 isolates, and 90% of the genome of CHIKV was recovered with >98% accuracy. Using nanopore sequencing, metagenomic detection of viral pathogens directly from clinical samples was performed within an unprecedented <6 hours sample-to-answer turnaround time and in a timeframe amenable for actionable clinical and public health diagnostics.

scholarly journals Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

2015 ◽  
Vol 7 (1) ◽  
Author(s):  
Alexander L. Greninger ◽  
Samia N. Naccache ◽  
Scot Federman ◽  
Guixia Yu ◽  
Placide Mbala ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Viral Pathogens

scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

A point-of-care diagnostic for differentiating Ebola from endemic febrile diseases

2018 ◽  
Vol 10 (471) ◽  
pp. eaat0944 ◽  
Author(s):  
David Sebba ◽  
Alexander G. Lastovich ◽  
Melody Kuroda ◽  
Eric Fallows ◽  
Joshua Johnson ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Clinical Symptoms ◽  
Point Of Care ◽  
Hemorrhagic Fever ◽  
Diagnostic Methods ◽  
Outbreak Detection ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Live Virus ◽  
And Control

Hemorrhagic fever outbreaks such as Ebola are difficult to detect and control because of the lack of low-cost, easily deployable diagnostics and because initial clinical symptoms mimic other endemic diseases such as malaria. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need. Although rapid tests such as lateral flow can be broadly deployed, they are typically not well-suited for differentiating among multiple diseases presenting with similar symptoms. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect and differentiate infection with Ebola virus from other more common febrile diseases. Here, we developed and tested an immunoassay technology that uses surface-enhanced Raman scattering (SERS) tags to simultaneously detect antigens from Ebola, Lassa, and malaria within a single blood sample. Results are provided in <30 min for individual or batched samples. Using 190 clinical samples collected from the 2014 West African Ebola outbreak, along with 163 malaria positives and 233 negative controls, we demonstrated Ebola detection with 90.0% sensitivity and 97.9% specificity and malaria detection with 100.0% sensitivity and 99.6% specificity. These results, along with corresponding live virus and nonhuman primate testing of an Ebola, Lassa, and malaria 3-plex assay, indicate the potential of the SERS technology as an important tool for outbreak detection and clinical triage in low-resource settings.

scholarly journals A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Stefan Moritz Neuenschwander ◽  
Miguel Angel Terrazos Miani ◽  
Heiko Amlang ◽  
Carmen Perroulaz ◽  
Pascal Bittel ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Sequence Data ◽  
Automated Analysis ◽  
Amplicon Sequencing ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Taxonomic Identification ◽  
Consensus Analysis

ABSTRACT Amplicon sequencing of the 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations, with the most significant being the inability to resolve mixed amplicons when closely related species are coamplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read next-generation sequencing (NGS) technologies could solve the mixed amplicon issue but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling a flexible number of samples per run and an adjustable sequencing time. Here, we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (long read consensus analysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN’s behavior when dealing with samples with known cross-contamination levels. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single-base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability, and availability of amplicon sequencing in diagnostic settings.

scholarly journals Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014

2015 ◽  
Vol 53 (6) ◽  
pp. 1915-1920 ◽  
Author(s):  
Jian Zhuge ◽  
Eric Vail ◽  
Jeffrey L. Bush ◽  
Lauren Singelakis ◽  
Weihua Huang ◽  
...  
Keyword(s):  
New York ◽  
Real Time ◽  
Reverse Transcription ◽  
New York State ◽  
Respiratory Illness ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
York State ◽  
Reverse Transcription Pcr ◽  
Enterovirus D68

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5′ untranslated region (5′-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.

scholarly journals Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala

2018 ◽  
Vol 30 (4) ◽  
pp. 523-529 ◽  
Author(s):  
Lyndal S. Hulse ◽  
Danica Hickey ◽  
Jessica M. Mitchell ◽  
Kenneth W. Beagley ◽  
William Ellis ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Pathogens ◽  
Simultaneous Detection ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Phascolarctos Cinereus ◽  
In The Wild ◽  
Labeled Probe ◽  
Free Ranging

Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.

scholarly journals Real-time PCR for detecting circulating dengue virus in the Guangdong Province of China in 2006

2008 ◽  
Vol 57 (12) ◽  
pp. 1547-1552 ◽  
Author(s):  
Zhijun Bai ◽  
Licheng Liu ◽  
Zeng Tu ◽  
Lisi Yao ◽  
Jianwei Liu ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Serum Samples ◽  
Serological Tests ◽  
Wide Range ◽  
Pcr Assays

Dengue virus (DENV) causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome. We developed four real-time quantitative PCR assays for each serotype of DENV based on computational analysis. These assays had high sensitivity and specificity without cross-reactivity for the four serotypes. To evaluate the performance of these assays in detecting and typing the virus in clinical samples, we analysed 64 serum samples from Guangdong during 2006. The results showed that 71 % of those samples were positive by the DEN-1 assay. The DENV assay results, in agreement with the serological tests and sequencing analysis, showed that the pathogen resulting in the DF explosion in Guangdong in 2006 belonged to DEN-1. Compared to the serological assays, the real-time PCR assays that we developed were much more sensitive in the 1–3 days after onset of the symptoms.

scholarly journals Development of a 5′-Nuclease Real-Time PCR Assay Targeting fliP for the Rapid Identification of Burkholderia mallei in Clinical Samples

2006 ◽  
Vol 52 (2) ◽  
pp. 307-310 ◽  
Author(s):  
Herbert Tomaso ◽  
Holger C Scholz ◽  
Sascha Al Dahouk ◽  
Meike Eickhoff ◽  
Thomas M Treu ◽  
...  
Keyword(s):  
Real Time ◽  
United Arab Emirates ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Turnaround Time ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Burkholderia Mallei ◽  
Pcr Assay ◽  
Bacterial Dna

Abstract Background: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. Methods: We developed a B. mallei-specific 5′-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. Results: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. Conclusions: Compared with conventional PCR, our rapid 5′-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

scholarly journals Utility of a Sequence-Independent, Single-Primer-Amplification (SISPA) and Nanopore Sequencing Approach for Detection and Characterization of Tick-Borne Viral Pathogens

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 203
Author(s):  
Annika Brinkmann ◽  
Steven Uddin ◽  
Eva Krause ◽  
Rebecca Surtees ◽  
Ender Dinçer ◽  
...  
Keyword(s):  
Virus Detection ◽  
Hemorrhagic Fever ◽  
Nanopore Sequencing ◽  
Viral Pathogens ◽  
Viral Loads ◽  
Hemorrhagic Fever Virus ◽  
Base Calling ◽  
Next Generation Sequencing Ngs ◽  
Virus Genomes ◽  
Single Primer

Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks.

scholarly journals 149. Extraction-free RT-PCR to Detect SARS-CoV-2 Variants of Concern

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S89-S91
Author(s):  
Brian L Harry ◽  
Yue Qiu ◽  
Ling Lu ◽  
Mara Couto-Rodriguez ◽  
Dorottya Nagy-Szakal ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Research Support ◽  
Pcr Assay ◽  
Synthetic Controls ◽  
Novel Coronavirus

Abstract Background SARS-CoV-2 variants of concern (VOC) have challenged real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods for the diagnosis of COVID-19. Methods The CDC 2019-Novel Coronavirus real-time RT-PCR panel was modified to create a single-plex extraction-free proxy RT-PCR assay, VOCFast™. This assay uses the nucleocapsid N1 as well as novel primer/probe pairs to target VOC mutations in the Orf1a and spike (S) genes. For analytical validation of VOCFast, synthetic controls for the Wuhan, alpha/B.1.1.7, beta/B.1.351, and gamma/P.1 strains were tested at various concentrations. Clinical validation was performed using patient anterior nares swab and saliva specimens collected in the Denver, CO area between Nov 2020 and Feb 2021 or in March 2021. Orthogonal next-generation sequencing (NGS) was also performed. Results Similar N1 quantification cycle (Cq) values corresponding to viral load were observed for all strains, suggesting that VOC mutations do not affect performance of the N1 primer/probe. Orf1a-mut and S1-mut primer/probes generated a stable high Cq value for the Wuhan strain. Conversely, Orf1a-mut Cq values were inversely correlated with viral load for all VOC. The S1-mut Cq was inversely correlated with viral load of the alpha strain, but did not reliably amplify beta/gamma VOC. The limit of detection was 8 copies/uL. The first set of COVID-19 patient specimens revealed no amplification using Orf1a-mut whereas 53% of specimens collected in Mar 2021 demonstrated amplification by Orf-1a. Orthogonal testing by the SARS-CoV-2 NGS Assay and COVID-DX software demonstrated that 12/12 alpha strains, 2/2 beta/gamma strains, and 33/33 Wuhan strains were correctly identified by VOCFast. Detection of VOC in clinical specimens and validation by NGS Conclusion The combination of the N1, Orf1a-mut, and S1-mut primers/probes in VOCFast can distinguish the Wuhan, alpha, and beta/gamma strains and it consistent with NGS results. Testing of clinical samples revealed that VOC emerged in Denver, CO in March 2021. Future work to discriminate beta, gamma, and emerging VOC is ongoing. In summary, VOCFast is an extraction-free RT-PCR assay for nasal swab and saliva specimens that can identify VOC with a turnaround time suitable for clinical testing. Disclosures Brian L. Harry, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder) Mara Couto-Rodriguez, MS, Biotia (Employee) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder) Shi-Long Lu, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder)

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