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Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT PCR.

Perera D, Podin Y, Akin W, Tan CS, Cardosa MJ - BMC Infect. Dis. (2004)

Bottom Line: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control.When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Health & Community Medicine, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, 94300 Malaysia. davidperera@yahoo.com

ABSTRACT

Background: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.

Methods: Local strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens.

Results: A total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.

Conclusions: Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

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Related in: MedlinePlus

Amplification of CVA16 isolates with primer pair 159S/162A. The top panel shows amplification using primer pair 159S/162A. The bottom panel shows amplification using a pan enterovirus primer set in VP4. Lane 1: HEV71 control; Lane 2: water control; Lane 3: SB3091/SAR/00; Lane 4: SB3279/SAR/00; Lane 5: SB2512/SAR/00; Lane 6: SB3333/SAR/00; Lane 7: SB11444/SAR/03; Lane 8: SB3093/SAR/00; Lane 9: SB2116/SAR/00; Lane 10: SB3283/SAR/00; Lane 11: SB2528/SAR/00; Lane 12: SB2905/SAR/00; Lane 13: SB2486/SAR/00; Lane 14: SB2934/SAR/00. "+" indicates specimens positive using 159S/162A primers. The expected position of the amplicons are indicated by the arrows. MW (bp) marks the lane containing the molecular weight markers in basepairs.
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Figure 1: Amplification of CVA16 isolates with primer pair 159S/162A. The top panel shows amplification using primer pair 159S/162A. The bottom panel shows amplification using a pan enterovirus primer set in VP4. Lane 1: HEV71 control; Lane 2: water control; Lane 3: SB3091/SAR/00; Lane 4: SB3279/SAR/00; Lane 5: SB2512/SAR/00; Lane 6: SB3333/SAR/00; Lane 7: SB11444/SAR/03; Lane 8: SB3093/SAR/00; Lane 9: SB2116/SAR/00; Lane 10: SB3283/SAR/00; Lane 11: SB2528/SAR/00; Lane 12: SB2905/SAR/00; Lane 13: SB2486/SAR/00; Lane 14: SB2934/SAR/00. "+" indicates specimens positive using 159S/162A primers. The expected position of the amplicons are indicated by the arrows. MW (bp) marks the lane containing the molecular weight markers in basepairs.

Mentions: Primer pair 159S/162A occasionally misprimes CVA16 strains and generates amplicons the size expected from HEV71. An example of this is shown in Figure 1 where it can be seen that pan enterovirus primers in VP4 amplified a product in all 12 CVA16 samples and 159S/162A amplified 7 of these. DNA sequencing of the VP4 products confirmed that the samples were CVA16. The PCR products generated with 159S/162A were also sequenced and found to be CVA16, thus confirming that the samples affected did not contain both CVA16 and HEV71.


Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT PCR.

Perera D, Podin Y, Akin W, Tan CS, Cardosa MJ - BMC Infect. Dis. (2004)

Amplification of CVA16 isolates with primer pair 159S/162A. The top panel shows amplification using primer pair 159S/162A. The bottom panel shows amplification using a pan enterovirus primer set in VP4. Lane 1: HEV71 control; Lane 2: water control; Lane 3: SB3091/SAR/00; Lane 4: SB3279/SAR/00; Lane 5: SB2512/SAR/00; Lane 6: SB3333/SAR/00; Lane 7: SB11444/SAR/03; Lane 8: SB3093/SAR/00; Lane 9: SB2116/SAR/00; Lane 10: SB3283/SAR/00; Lane 11: SB2528/SAR/00; Lane 12: SB2905/SAR/00; Lane 13: SB2486/SAR/00; Lane 14: SB2934/SAR/00. "+" indicates specimens positive using 159S/162A primers. The expected position of the amplicons are indicated by the arrows. MW (bp) marks the lane containing the molecular weight markers in basepairs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC415548&req=5

Figure 1: Amplification of CVA16 isolates with primer pair 159S/162A. The top panel shows amplification using primer pair 159S/162A. The bottom panel shows amplification using a pan enterovirus primer set in VP4. Lane 1: HEV71 control; Lane 2: water control; Lane 3: SB3091/SAR/00; Lane 4: SB3279/SAR/00; Lane 5: SB2512/SAR/00; Lane 6: SB3333/SAR/00; Lane 7: SB11444/SAR/03; Lane 8: SB3093/SAR/00; Lane 9: SB2116/SAR/00; Lane 10: SB3283/SAR/00; Lane 11: SB2528/SAR/00; Lane 12: SB2905/SAR/00; Lane 13: SB2486/SAR/00; Lane 14: SB2934/SAR/00. "+" indicates specimens positive using 159S/162A primers. The expected position of the amplicons are indicated by the arrows. MW (bp) marks the lane containing the molecular weight markers in basepairs.
Mentions: Primer pair 159S/162A occasionally misprimes CVA16 strains and generates amplicons the size expected from HEV71. An example of this is shown in Figure 1 where it can be seen that pan enterovirus primers in VP4 amplified a product in all 12 CVA16 samples and 159S/162A amplified 7 of these. DNA sequencing of the VP4 products confirmed that the samples were CVA16. The PCR products generated with 159S/162A were also sequenced and found to be CVA16, thus confirming that the samples affected did not contain both CVA16 and HEV71.

Bottom Line: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control.When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Health & Community Medicine, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, 94300 Malaysia. davidperera@yahoo.com

ABSTRACT

Background: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.

Methods: Local strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens.

Results: A total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.

Conclusions: Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

Show MeSH
Related in: MedlinePlus