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82171915.Pdf View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector International Journal of Infectious Diseases 49 (2016) 80–86 Contents lists available at ScienceDirect International Journal of Infectious Diseases jou rnal homepage: www.elsevier.com/locate/ijid Simultaneous detection of 13 viruses involved in meningoencephalitis using a newly developed multiplex PCR Mag-array system a,1 a,1 a a a a a Xiaodan Shi , Rui Wu , Ming Shi , Linfu Zhou , Mengli Wu , Yining Yang , Xinyue An , a a b b, a, Wen Dai , Liang Tian , Chen Zhang , Xuejun Ma **, Gang Zhao * a Department of Neurology, Xijing Hospital, The Fourth Military Medical University, Changle-Xi Road 167, Xi’an 710032, China b Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No.155, Changbai Road, Changping District, Beijing 100050, China A R T I C L E I N F O S U M M A R Y Article history: Background: The early detection and identification of pathogens in central nervous system viral Received 14 December 2015 infections associated with neurological disease increases the survival rate. However, the limitations of Received in revised form 27 April 2016 current diagnostic methods contribute to a lack of proper diagnosis in 62% of patients. Therefore, a robust Accepted 19 May 2016 method for detecting multiple viruses in a single reaction with high specificity, throughput, and speed is required. Keywords: Methods: A multiplex PCR Mag-Array (MPMA) system was developed that integrates three strategies: MPMA system chimeric primer design, temperature switch PCR, and MagPlex-TAG techniques. The MPMA was used to Meningoencephalitis viruses amplify 13 target viral sequences simultaneously, with plasmids containing specific viral sequences as Cerebrospinal fluid standard samples. To evaluate its clinical performance, 177 cerebrospinal fluid (CSF) samples were tested. Results: The MPMA system presented high specificity and efficiency in detecting a control panel of 13 plasmids. Among 177 CSF samples, consistent results were achieved for 19 samples pre-tested using a commercial kit. Viral pathogens were found in 28/138 undiagnosed samples, with herpes simplex viruses (HSV-1 and HSV-2) being predominant. The 20 non-infectious samples revealed negative results. Compared to sequencing methods, sensitivity for detecting HSV-1 and HSV-2 was 100% and 98.78%, respectively, and specificity was 100% and 98.22%, respectively. Conclusions: A robust MPMA system that can simultaneously and reliably detect 13 meningoencephali- tis-associated viruses with high specificity, throughput, and speed has been developed. ß 2016 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). progresses to a life-threatening stage for which few curative 1. Introduction 4 methods are available. The common viral pathogens causing central nervous system (CNS) infections are human enterovirus Although the annual incidence of encephalitis and meningitis is (HEV), herpes simplex virus type 1 (HSV-1), herpes simplex virus estimated to be 0.07 to 12.6 cases per 100 000 individuals type 2 (HSV-2), cytomegalovirus (CMV), varicella zoster virus worldwide, severe disability has been reported in up to 56% of 1–3 (VZV), and Epstein–Barr virus (EBV). Mumps virus (MuV) and survivors and mortality rates range between 7% and 18%. Early 5,6 measles virus (MeV) are undergoing a worldwide resurgence. identification of the causative viral pathogen is important in order Among HEV, subtypes such as coxsackie A virus type 16 (CA16), to develop an effective treatment plan before the disease enterovirus type 71 (EV71), and echoviruses (ECHO) are frequently 7,8 found to be involved in CNS infection worldwide. In addition to these, JC virus (JCV) is a major viral cause of progressive multifocal * Corresponding author. Tel.: +86 2984775368. leukoencephalopathy that is rarely included in routine detection ** Corresponding author. Tel.: +86 1058900810. 9 systems, and Japanese encephalitis virus (JEV) is a major cause of E-mail addresses: [email protected] (X. Ma), [email protected] viral encephalitis in geographical areas such as Asia and the (G. Zhao). 10 1 Western Pacific. Xiaodan Shi and Rui Wu contributed equally to this study. http://dx.doi.org/10.1016/j.ijid.2016.05.023 1201-9712/ß 2016 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). X. Shi et al. / International Journal of Infectious Diseases 49 (2016) 80–86 81 Traditional identification techniques such as pathogen culture In this study, a multiplex PCR Mag-array (MPMA) system was and antigen or antibody detection have limited capacity in their developed, based on the Luminex-200 system in combination with clinical application, owing to low sensitivity and specificity and MagPlex-TAG technology (xTAG); this system detects 13 viruses 11,12 high time requirements. Advanced molecular tools have aided simultaneously: HSV-1, HSV-2, CMV, VZV, EBV, MuV, MeV, JCV, the development of commercial products that offer much- JEV, CA16, EV71, ECHO, and HEV. 1 enhanced sensitivity and diagnostic turnaround times. However, these products have been designed to target only a few virus types, 2. Materials and methods greatly limiting their clinical application. Additionally, despite improvements in sensitivity over previous methods, large volumes 2.1. Plasmids and CSF samples of cerebrospinal fluid (CSF) are still required by currently available commercial products, which can be quite challenging for the Fragments of the specific genes from 13 selected viruses (HSV- patient due to the side effects of lumbar puncture. Therefore, there 1, HSV-2, CMV, VZV, EBV, MuV, MeV, JCV, JEV, CA16, EV71, ECHO, is an urgent need for a highly sensitive, highly specific, and high- and HEV) were synthesized by Sangon Biotech (Shanghai, China) throughput pathogen detection method. (Table 1) and subcloned into the pcDNA3.1 plasmid (Thermo Fisher 15 The Luminex-200 MagPlex-TAG beads array system (Luminex Scientific, Waltham, MA, USA). Each recombinant pcDNA3.1 Corp., Austin, TX, USA) is capable of simultaneously detecting plasmid was used as a standard plasmid for subsequent experi- 100 different PCR products and is thus ideal for high-throughput ments. A blank pcDNA3.1 plasmid was used as a vehicle plasmid 13 screening. For example, the Luminex-200 xTAG respiratory control (VPC). pathogen panel can simultaneously detect 14 selected viruses To further confirm the feasibility of the MPMA system, 177 CSF 14 involved in respiratory viral infections. Unfortunately, similar specimens were collected (138 undiagnosed specimens from products are not yet available for the detection of multiple viruses patients with meningoencephalitis symptoms, 19 samples pre- associated with CNS infection diseases. tested using commercial kits, and 20 specimens from patients Table 1 a Primers and probes 0 0 Name Sequence (5 !3 ) Gene target Conc. (nM) Tm GC% Length b HSV-1-F AGGTGACACTATAGAATATTTGATTTAAGAGTGTTGAATGTATGAAGCGCGAACTGACGA 81L 120 59.67 55.56 189 HSV-1-R GTACGACTCACTATAGGGAGGTGGGCGTGGCACTATC 120 60.51 66.67 HSV-1-P TTTGATTTAAGAGTGTTGAATGTA b HSV-2-F AGGTGACACTATAGAATATATTAGAGTTTGAGAATAAGTAGTTATCGGAGGCCCTTTGTTGC UL23 40 60.39 55 163 HSV-2-R GTACGACTCACTATAGGGAGCCGATCCGTCCTTGTTTTC 40 59.55 55 HSV-2-P TATTAGAGTTTGAGAATAAGTAGT b VZV-F AGGTGACACTATAGAATATTGTGTAGTTAAGAGTTGTTTAATGCAGGGAATTGGAGCGGTT QCMD 120 60.68 57.89 222 VZV-R GTACGACTCACTATAGGGAACTTGGTCGGATAGGGTGGT 120 60.25 55 VZV-P TTGTGTAGTTAAGAGTTGTTTAAT b CMV-F AGGTGACACTATAGAATAGATAGATTTAGAATGAATTAAGTGTCTACTACACGTCAGCGTTCG UL83 160 59.87 52.38 202 CMV-R GTACGACTCACTATAGGGAACACCTTGACGTACTGGTCAC 160 59.93 52.38 CMV-P GATAGATTTAGAATGAATTAAGTG b EBV-F AGGTGACACTATAGAATAGTGTTATAGAAGTTAAATGTTAAGTTCGTTGCCCACAGTCAGG LMP-2A 120 60.23 57.89 165 EBV-R GTACGACTCACTATAGGGAGGTTTTGCGGAGACAAGCAG 120 60.04 55 EBV-P GTGTTATAGAAGTTAAATGTTAAG MeV-F AGGTGACACTATAGAATAGTTTGTTGAAAGTGTAAAGTATATTCAGCAACTGCATGGTAGCTT KJ36545 120 60.27 47.62 250 MeV-R GTACGACTCACTATAGGGACTATCACTGGGTCATCCGTCG 120 60 57.14 MeV-P GTTTGTTGAAAGTGTAAAGTATAT MuV-F AGGTGACACTATAGAATATAGATTAGTTGATAAGTGTGTAATTCGGAGAACAAGCCAGATACC KF481689 100 59.52 52.38 202 MuV-R GTACGACTCACTATAGGGACTAGGACTGTACCGACTCCCA 100 60.06 57.14 MuV-P TAGATTAGTTGATAAGTGTGTAAT JCV-F AGGTGACACTATAGAATAAGAGAGTTTGTGTATATGTATAAATCCATGTCCAGAGTCTTCTGC argno 120 59.45 52.38 202 JCV-R GTACGACTCACTATAGGGAGCCATTCATGAGAGGATTGTGC 120 59.97 50 JCV-P AGAGAGTTTGTGTATATGTATAAA JEV-F AGGTGACACTATAGAATATTAAGAAGAATTGTATATGAGAGTTCATGGCAGTGTCCTACGTG GP1 120 59.75 55 183 JEV-R GTACGACTCACTATAGGGATTTCACATCCAGCCTCCGAC 120 60.04 55 JEV-P TTAAGAAGAATTGTATATGAGAGT b ECHO-F AGGTGACACTATAGAATAAGAGAGTTTGTGTATATGTATAAACCCTAAAAGTTATGAGGATTTCGCA VP1 120 59.41 40 131 ECHO-R GTACGACTCACTATAGGGAATTACCCTCCGTCCAGAATACG 120 59.37 40 ECHO-P AGAGAGTTTGTGTATATGTATAAA b EV71-F AGGTGACACTATAGAATATAGATTAGTTGATAAGTGTGTAATGAGCCCTCACACACGCTCTAC VP1 100 60.9 60 152 EV71-R GTACGACTCACTATAGGGACGCAGCTCGTCCTTGACAT 100 60.45 57.89 EV71-P TAGATTAGTTGATAAGTGTGTAAT CA16-F AGGTGACACTATAGAATAGTTTGTTGAAAGTGTAAAGTATATCCAGCCTGATATTTGTCGAAGC TW-3126-98 polyprotein 90 59.71 50 157 CA16-R GTACGACTCACTATAGGGACACAGTCACCAGGTTCCGAG 90 60.32 60 CA16-P
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