MRPrimerV: a database of PCR primers for RNA virus detection

Kim, Hyerin; Kang, NaNa; An, Kyuhyeon; Kim, Doyun; Koo, Jae Hyung; Kim, Min‐Soo

doi:10.1093/nar/gkw1095

Cited by 17 publications

(18 citation statements)

References 18 publications

(17 reference statements)

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“…By amplification of the 16S rRNA gene, we restricted identification in this study to bacteria excluding the identification of viruses or fungi. To broaden the detection, less biased methods such as Multiple Displacement Amplification (MDA) [55] to amplify whole genomes or the use of additional primer pairs designed to identify fungi or viruses of interest can be integrated [98, 99]. There is also the option to include a pre-treatment step using chemical agents, such as propidium monoazide, which prevents DNA from dead cells to be amplified, ensuring amplification of DNA from viable bacteria [100].…”

Section: Discussionmentioning

confidence: 99%

Rapid metagenomics analysis of EMS vehicles for monitoring pathogen load using nanopore DNA sequencing

et al. 2019

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Pathogen monitoring, detection and removal are essential to public health and outbreak management. Systems are in place for monitoring the microbial load of hospitals and public health facilities with strategies to mitigate pathogen spread. However, no such strategies are in place for ambulances, which are tasked with transporting at-risk individuals in immunocompromised states. As standard culturing techniques require a laboratory setting, and are time consuming and labour intensive, our approach was designed to be portable, inexpensive and easy to use based on the MinION third-generation sequencing platform from Oxford Nanopore Technologies. We developed a transferable sampling-to-analysis pipeline to characterize the microbial community in emergency medical service vehicles. Our approach identified over sixty-eight organisms in ambulances to the genera level, with a proportion of these being connected with health-care associated infections, such as Clostridium spp . and Staphylococcus spp . We also monitored the microbiome of different locations across three ambulances over time, and examined the dynamic community of microorganisms found in emergency medical service vehicles. Observed differences identified hot spots, which may require heightened monitoring and extensive cleaning. Through metagenomics analysis it is also possible to identify how microorganisms spread between patients and colonize an ambulance over time. The sequencing results aid in the development of practices to mitigate disease spread, while also providing a useful tool for outbreak prediction through ongoing analysis of the ambulance microbiome to identify new and emerging pathogens. Overall, this pipeline allows for the tracking and monitoring of pathogenic microorganisms of epidemiological interest, including those related to health-care associated infections.

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Section: Discussionmentioning

confidence: 99%

Rapid metagenomics analysis of EMS vehicles for monitoring pathogen load using nanopore DNA sequencing

et al. 2019

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“…Assay 1, RSV, HRV, and HMPV; assay 2, PIV1, PIV2, and PIV3; assay 3, EV, FluA, and FluB; assay 4, HCoV229E, HCoVOC43 and HCoVNL63; assay 5, ADV, HBoV, and Rnasep. All the sequences of LNA‐outer primer, inner primer, and probe were obtained either from the reported literature 4,8–22 or slightly modified by removing a few bases of reported inner primers. All the outer primers were modified by LNA.…”

Section: Methodsmentioning

confidence: 99%

Development and evaluation of a panel of multiplex one‐tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions

Zhao

et al. 2020

Journal of Medical Virology

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Multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one-tube nested real-time PCR (mOTNRT-PCR) assay consisting of five separate internally controlled RT-qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT-PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT-PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real-time PCR (RT-qPCR) assay. The analytical sensitivities of mOTNRT-PCR panel ranged from 2 to 20 copies/reaction, and no cross-reaction with common respiratory viruses was observed. The coefficients of variation of intra-assay and inter-assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT-PCR assay panel provides a more sensitive and high-throughput method for the detection of 14 respiratory viruses.

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“…Inner primer and probe sequences were derived from previously published RT-qPCR assays targeting nucleoprotein gene for RSV [ 8 ], the 5′untranslated region for HRV [ 9 ] and the nucleoprotein gene for HMPV [ 10 ]. The outer primer sequences of RSV, HRV and HMPV were derived from previously published [ 8 – 10 , 14 , 26 , 27 ] with base modifications by LNA [ 17 ]. LNA modification can increase maximum annealing temperature of primers [ 28 ].…”

Section: Methodsmentioning

confidence: 99%

A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus

Feng

Zhao

Wang

et al. 2018

Virol J

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BackgroundRespiratory syncytial virus (RSV), human Rhinovirus (HRV) and human Metapneumo Virus (HMPV) are important viral pathogens causing acute respiratory tract infections in the hospitalized patients. Sensitive and accurate detection of RSV, HRV and HMPV is necessary for clinical diagnosis and treatment.ResultsA locked nucleic acid (LNA)-based multiplex closed one-tube nested real-time RT-PCR (mOTNRT-PCR) assay was developed for simultaneous detection of RSV, HRV and HMPV. The sensitivity, specificity, reproducibility and clinical performance of mOTNRT-PCR were evaluated and compared with individual real time PCR (RT-qPCR) assay using clinical samples. The analytical sensitivity of mOTNRT-PCR assay was 5 copies/reaction for RSV, HRV and HMPV, respectively, and no cross-reaction with other common respiratory viruses was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.51 to 3.67%. Of 398 nasopharyngeal aspirates samples tested, 109 (27.39%), 150 (37.69%) and 44 (11.06%) were positive for RSV, HRV and HMPV, respectively, whereas 95 (23.87%), 137 (34.42%) and 38 (9.55%) were positive for RSV, HRV and HMPV, respectively, by individual RT-qPCR assay. Thirty three samples that were positive by mOTNRT-PCR but negative by RT-qPCR were confirmed as true positives by sequencing using reported traditional two-step nested PCR assay.ConclusionmOTNRT-PCR assay reveals extremely higher sensitivity than that of RT-qPCR assay for detecting RSV, HRV and HMPV in clinical settings.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-1061-0) contains supplementary material, which is available to authorized users.

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MRPrimerV: a database of PCR primers for RNA virus detection

Cited by 17 publications

References 18 publications

Rapid metagenomics analysis of EMS vehicles for monitoring pathogen load using nanopore DNA sequencing

Rapid metagenomics analysis of EMS vehicles for monitoring pathogen load using nanopore DNA sequencing

Development and evaluation of a panel of multiplex one‐tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions

A multiplex one-tube nested real time RT-PCR assay for simultaneous detection of respiratory syncytial virus, human rhinovirus and human metapneumovirus

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