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Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses

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Abstract

Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5′ untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie®II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie®II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

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Abbreviations

HRVs:

Human rhinoviruses

RT-GEAR:

Reverse transcription genome exponential amplification reaction

RT-LAMP:

Reverse transcription loop-mediated isothermal amplification

UTR:

Untranslated region

HEV:

Human enterovirus

CVA:

Coxsackievirus A

CVB:

Coxsackievirus B

Echo:

Echovirus

PV:

Poliovirus

FluA H3:

Influenza A virus H3

FluB:

Influenza B virus

PIV:

Parainfluenza virus

ADV:

Adenovirus

RSV:

Respiratory syncytial virus

HMPV:

Human metapneumovirus

COPD:

Chronic obstructive pulmonary disease

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Acknowledgements

We acknowledge the Laboratory of Virology, Capital Institute of Pediatrics, for providing nasopharyngeal aspirates, the National Laboratory for Poliomyelitis for providing the control viruses and clinical specimens, and the Chinese National Influenza Center for providing respiratory swabs used in this study. This work was supported by the China Mega-Project for Infectious Disease (2016ZX10004001-002, 2016ZX10004-001), Beijing Municipal Science & Technology Commission project (D151100002115003) and Guangzhou Municipal Science & Technology Commission project (2015B2150820).

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    Authors and Affiliations

    1. Key Laboratory for Medical Virology, National Health and Family Planning Commission, Chinese Center for Disease Control and Prevention, National Institute for Viral Disease Control and Prevention, No.155 Changbai Road, Changping, Beijing, 102206, China

      Li Guan, Hang-Yu Zhou, Kai Nie, Xin-Na Li, Dan Zhang, Juan Song & Xue-Jun Ma

    2. Laboratory of Virology, Beijing Key Laboratory of Etiology of Viral Diseases in Children, Capital Institute of Pediatrics, 2 Yabao Road, Beijing, 100020, China

      Lin-Qing Zhao & Yuan Qian

    3. School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, Guangdong, 510182, China

      Dan Zhang

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    1. Li Guan
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    Correspondence to Yuan Qian or Xue-Jun Ma.

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    Funding

    This study was funded by the China Mega-Project for Infectious Disease (2016ZX10004001-002, 2016ZX10004-001), Beijing Municipal Science & Technology Commission project (D151100002115003) and Guangzhou Municipal Science & Technology Commission project (2015B2150820).

    Conflict of interest

    All authors declare that they have no competing interests.

    Ethical approval

    All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

    Informed consent

    All aspects of the study were performed in accordance with national ethics regulations and appraised by the Institutional Review Boards of the Center for Disease Control and Prevention of China. Children’s parents were apprised of the study’s purpose and of their right to keep information confidential. Written informed consent was obtained from parents or caregivers.

    Additional information

    L. Guan, L.-Q. Zhao and H.-Y. Zhou contributed equally to this work.

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    Guan, L., Zhao, LQ., Zhou, HY. et al. Reverse transcription genome exponential amplification reaction assay for rapid and universal detection of human rhinoviruses. Arch Virol 161, 1891–1898 (2016). https://doi.org/10.1007/s00705-016-2858-z

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