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Volume 28 Issue 2
April 2013
    Citation: Jikui Deng, Zhuoya Ma, Wenbo Huang, Chengrong Li, Heping Wang, Yuejie Zheng, Rong Zhou, Yi-Wei Tang. Respiratory Virus Multiplex RT-PCR Assay Sensitivities and Influence Factors in Hospitalized Children with Lower Respiratory Tract Infections .VIROLOGICA SINICA, 2013, 28(2) : 97-102.  http://dx.doi.org/10.1007/s12250-013-3312-y
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    Respiratory Virus Multiplex RT-PCR Assay Sensitivities and Influence Factors in Hospitalized Children with Lower Respiratory Tract Infections

    • 1.

      Shenzhen Children's Hospital, Chongqing Medical University, Shenzhen 518026, China

    • 2.

      State Key Laboratory of Respiratory Diseases, Guangzhou Medical University, Guangzhou, Guangdong 510230, China

    • 3.

      Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA

    • Corresponding author: Rong Zhou, zhou3218@yahoo.com
      Yi-Wei Tang, tangy@mskcc.org
    • Received Date: 03 February 2013
      Accepted Date: 18 March 2013
      Available online: 01 April 2013

      Fund Project: Grant Name awarded to the State Key Lab of Respiratory Diseases, Guangzhou Medical College 2007DA780154F0910

    • Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children's Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%-40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011-0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.
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      Respiratory Virus Multiplex RT-PCR Assay Sensitivities and Influence Factors in Hospitalized Children with Lower Respiratory Tract Infections

        Corresponding author: Rong Zhou, zhou3218@yahoo.com
        Corresponding author: Yi-Wei Tang, tangy@mskcc.org
      • 1. Shenzhen Children's Hospital, Chongqing Medical University, Shenzhen 518026, China
      • 2. State Key Laboratory of Respiratory Diseases, Guangzhou Medical University, Guangzhou, Guangdong 510230, China
      • 3. Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
      • Received Date: 03 February 2013
      • Accepted Date: 18 March 2013
      Fund Project:  Grant Name awarded to the State Key Lab of Respiratory Diseases, Guangzhou Medical College 2007DA780154F0910

      Abstract: Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children's Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%-40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011-0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.

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