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

Feng, Zhishan; Zhao, Li; Wang, Ji; Qiu, Fang-zhou; Zhao, Mengchuan; Wang, Le; Duan, Su-xia; Zhang, Ruiqing; Chen, Chen; Qi, Ju-Ju; Fan, Tao; Li, Guixia

doi:10.1186/s12985-018-1061-0

Cited by 28 publications

(24 citation statements)

References 28 publications

(42 reference statements)

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“…Recently, Feng's group designed locked nucleic acid LNA‐modified primers and developed a multiplex one‐tube nested real‐time RT‐PCR (mOTNRT‐PCR) assay that could detect RSV, hRV and human metapneumovirus (hMPV) simultaneously with higher sensitivity and lower cost compared to the individual RT‐qPCR 74 . This assay was evaluated using 398 clinical samples, and the sensitivity for RSV, hRV, and hMPV was 5 copies per reaction.…”

Section: Multiplex Respiratory Virus Detectionmentioning

confidence: 99%

Recent advances in the detection of respiratory virus infection in humans

Zhang

Wang

Deng

et al. 2020

Journal of Medical Virology

404

363

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Respiratory tract viral infection caused by viruses or bacteria isone of the most common diseases in human worldwide, while those caused by emerging viruses, such as the novel coronavirus, 2019-nCoV that caused the pneumonia outbreak in Wuhan, China most recently, have posed great threats to global public health. Identification of the causative viral pathogens of respiratory tract viral infections is important to select an appropriate treatment, save people's lives, stop the epidemics, and avoid unnecessary use of antibiotics. Conventional diagnostic tests, such as the assays for rapid detection of antiviral antibodies or viral antigens, are widely used in many clinical laboratories. With the development of modern technologies, new diagnostic strategies, including multiplex nucleic acid amplification and microarray-based assays, are emerging. This review summarizes currently available and novel emerging diagnostic methods for the detection of common respiratory viruses, such as influenza virus, human respiratory syncytial virus, coronavirus, human adenovirus, and human rhinovirus. Multiplex assays for simultaneous detection of multiple respiratory viruses are also described. It is anticipated that such data will assist researchers and clinicians to develop appropriate diagnostic strategies for timely and effective detection of respiratory virus infections. K E Y W O R D S adenovirus, coronavirus, diagnostic methods, influenza virus, respiratory syncytial virus, respiratory viral infection, rhinovirus

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Section: Multiplex Respiratory Virus Detectionmentioning

confidence: 99%

Recent advances in the detection of respiratory virus infection in humans

Zhang

Wang

Deng

et al. 2020

Journal of Medical Virology

404

363

View full text Add to dashboard Cite

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“…A wide range of multiplex assays are now available commercially which differ in their use of chemistry, optics and the level of automation. Many test methods have also been described in the literature for potential use as laboratory-developed tests (LDT) (Boivin et al, 2004;Feng et al, 2018;Loens et al, 2012;Létant et al, 2007;Nijhuis et al, 2017). To make an appropriate choice, a careful assessment of performance characteristics as well as costs and benefits associated with the test method is necessary.…”

Section: Discussionmentioning

confidence: 99%

A novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format

Hasan

Mana

Young

et al. 2019

Journal of Virological Methods

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A B S T R A C TCommercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2 × 8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n = 394) with 91%-100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n = 147) previously tested by Qiagen Resplex I &II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected more pathogens (p < 0.05) than Resplex but the rate of pathogen detection was not significantly different from FTDRP21. Respanel is a convenient and inexpensive assay that is more sensitive than Resplex and comparable to FTDRP21 for the detection of common respiratory pathogens.

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“…Given the high sensitivity of ddPCR, Zhao JK et al utilized this technique to evaluate the viral loads of SARS-CoV-2 from upper respiratory tract specimens for the rst time, showing that ddPCR can accurately re ect the viral loads of such specimens, especially nasopharyngeal swabs [18]. Subsequently, Renfei Lu et al used serial dilutions of the same clinical samples to demonstrate that the LoD (limit of detection) of ddPCR is at least 10 times better than that of qRT-PCR [19]. However, the limitation of the ddPCR assay is that it often needs unique supporting reagents, instruments, and professional operators, causing high running costs with moderate throughput.…”

Section: Droplet Digital Pcr (Ddpcr) Is a Third Generation Pcr Based mentioning

confidence: 99%

“…Coronavirus disease 2019 (COVID- 19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2.…”

Section: Introductionmentioning

confidence: 99%

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Zhang

Dai

Wang

et al. 2020

Preprint

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Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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

Cited by 28 publications

References 28 publications

Recent advances in the detection of respiratory virus infection in humans

Recent advances in the detection of respiratory virus infection in humans

A novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

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