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Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease.
J Virol Methods. 2017 10; 248:92-99.JV

Abstract

Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks.

Authors+Show Affiliations

Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Electronic address: Yong.P@chula.ac.th.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

28662914

Citation

Puenpa, Jiratchaya, et al. "Development of Single-step Multiplex Real-time RT-PCR Assays for Rapid Diagnosis of Enterovirus 71, Coxsackievirus A6, and A16 in Patients With Hand, Foot, and Mouth Disease." Journal of Virological Methods, vol. 248, 2017, pp. 92-99.
Puenpa J, Suwannakarn K, Chansaenroj J, et al. Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease. J Virol Methods. 2017;248:92-99.
Puenpa, J., Suwannakarn, K., Chansaenroj, J., Vongpunsawad, S., & Poovorawan, Y. (2017). Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease. Journal of Virological Methods, 248, 92-99. https://doi.org/10.1016/j.jviromet.2017.06.013
Puenpa J, et al. Development of Single-step Multiplex Real-time RT-PCR Assays for Rapid Diagnosis of Enterovirus 71, Coxsackievirus A6, and A16 in Patients With Hand, Foot, and Mouth Disease. J Virol Methods. 2017;248:92-99. PubMed PMID: 28662914.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease. AU - Puenpa,Jiratchaya, AU - Suwannakarn,Kamol, AU - Chansaenroj,Jira, AU - Vongpunsawad,Sompong, AU - Poovorawan,Yong, Y1 - 2017/06/27/ PY - 2017/03/17/received PY - 2017/06/23/revised PY - 2017/06/25/accepted PY - 2017/7/1/pubmed PY - 2018/5/1/medline PY - 2017/7/1/entrez KW - Coxsackievirus A16 KW - Coxsackievirus A6 KW - Diagnosis KW - Enterovirus 71 KW - Multiplex real-time RT-PCR KW - Thailand SP - 92 EP - 99 JF - Journal of virological methods JO - J Virol Methods VL - 248 N2 - Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks. SN - 1879-0984 UR - https://www.unboundmedicine.com/medline/citation/28662914/Development_of_single_step_multiplex_real_time_RT_PCR_assays_for_rapid_diagnosis_of_enterovirus_71_coxsackievirus_A6_and_A16_in_patients_with_hand_foot_and_mouth_disease_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0166-0934(17)30175-1 DB - PRIME DP - Unbound Medicine ER -