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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.

Thanh TT, Anh NT, Tham NT, Van HM, Sabanathan S, Qui PT, Ngan TT, Van TT, Nguyet LA, Ny NT, Thanh le TM, Chai OK, Perera D, Viet do C, Khanh TH, Ha do Q, Tuan HM, Wong KT, Hung NT, Chau NV, Thwaites G, van Doorn HR, Van Tan L - Virol. J. (2015)

Bottom Line: In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome.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 %).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.

View Article: PubMed Central - PubMed

Affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam. thanhtt@oucru.org.

ABSTRACT

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.

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Related in: MedlinePlus

Linearity of the multiplex RT-PCR assay using 10-fold dilution series of plasmid DNA containing the PCR templates of EV (a) and EV-A71 (b), Cp = crossing point
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Fig1: Linearity of the multiplex RT-PCR assay using 10-fold dilution series of plasmid DNA containing the PCR templates of EV (a) and EV-A71 (b), Cp = crossing point

Mentions: The linearity of the multiplex assay was calculated from amplification of serial ten-fold dilutions of plasmid DNA and EV-A71 RNA. The linear regression of plasmid DNA and viral RNA was 0.993 and 0.995, and 0.996 and 0.995 for the EV and EV-A71, respectively (Figs. 1 and 2).Fig. 1


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.

Thanh TT, Anh NT, Tham NT, Van HM, Sabanathan S, Qui PT, Ngan TT, Van TT, Nguyet LA, Ny NT, Thanh le TM, Chai OK, Perera D, Viet do C, Khanh TH, Ha do Q, Tuan HM, Wong KT, Hung NT, Chau NV, Thwaites G, van Doorn HR, Van Tan L - Virol. J. (2015)

Linearity of the multiplex RT-PCR assay using 10-fold dilution series of plasmid DNA containing the PCR templates of EV (a) and EV-A71 (b), Cp = crossing point
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4464700&req=5

Fig1: Linearity of the multiplex RT-PCR assay using 10-fold dilution series of plasmid DNA containing the PCR templates of EV (a) and EV-A71 (b), Cp = crossing point
Mentions: The linearity of the multiplex assay was calculated from amplification of serial ten-fold dilutions of plasmid DNA and EV-A71 RNA. The linear regression of plasmid DNA and viral RNA was 0.993 and 0.995, and 0.996 and 0.995 for the EV and EV-A71, respectively (Figs. 1 and 2).Fig. 1

Bottom Line: In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome.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 %).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.

View Article: PubMed Central - PubMed

Affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam. thanhtt@oucru.org.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus