Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease

Virology Journal

Le Van Tan³  H Rogier van Doorn¹  Guy Thwaites¹  Nguyen Van Vinh Chau⁶  Nguyen Thanh Hung⁷  Kum Thong Wong⁵  Ha Manh Tuan⁴  Do Quang Ha³  Truong Huu Khanh⁷  Do Chau Viet⁴  David Perera²  Ong Kien Chai⁵  Le Thi My Thanh⁶  Nguyen Thi Han Ny³  Lam Anh Nguyet³  Tran Thi My Van⁶  Tran Thuy Ngan³  Phan Tu Qui⁶  Saraswathy Sabanathan³  Hoang Minh Tu Van⁴  Nguyen Thi Tham³  Nguyen To Anh³  Tran Tan Thanh³ 

[1] Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK;Institute of Health and Community Medicine, Universiti Malaysia Sarawak, Sarawak, Malaysia;Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam;Children Hospital Number Two, Ho Chi Minh City, Vietnam;Facuty of Medicine, University of Malaya, Lumpur, Malaysia;Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam;Children Hospital Number One, Ho Chi Minh City, Vietnam

关键词: Diagnosis;    Real-time RT-PCR;    Enterovirus A71;    Enteroviruses;    Hand foot and mouth disease;   

Others  :  1224929
DOI  :  10.1186/s12985-015-0316-2

 received in 2015-03-20, accepted in 2015-05-30,  发布年份 2015

【 摘 要 】

Background

Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response.

Methods

We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.

Results

The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.

Conclusion

We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.

【 授权许可】

   
2015 Thanh et al.

【 图 表 】

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